The Multiple Roles of Antibiotics and Antibiotic Resistance in Nature | 1 the MULTIPLE ROLES of ANTIBIOTICS and ANTIBIOTIC RESISTANCE in NATURE

THETHE MULTIPLEMULTIPLE ROLESROLES OFOF ANTIBIOTICSANTIBIOTICS ANDAND ANTIBIOTICANTIBIOTIC RESISTANCERESISTANCE ININ NATURENATURE

TopicTopic EditorEditor FionaFiona WalshWalsh

MICROBIOLOGY FRONTIERS COPYRIGHT STATEMENT ABOUT FRONTIERS © Copyright 2007-2015 Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering Frontiers Media SA. All rights reserved. approach to the world of academia, radically improving the way scholarly research is managed. All content included on this site, such as The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share text, graphics, logos, button icons, images, and generate knowledge. Frontiers provides immediate and permanent online open access to all video/audio clips, downloads, data compilations and software, is the property its publications, but this alone is not enough to realize our grand goals. of or is licensed to Frontiers Media SA (“Frontiers”) or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their FRONTIERS JOURNAL SERIES respective authors, subject to a license granted to Frontiers. The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online The compilation of articles constituting journals, promising a paradigm shift from the current review, selection and dissemination this e-book, wherever published, as well as the compilation of all other content on processes in academic publishing. this site, is the exclusive property of All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service Frontiers. For the conditions for downloading and copying of e-books from to the scholarly community. At the same time, the Frontiers Journal Series operates on a revo- Frontiers’ website, please see the Terms lutionary invention, the tiered publishing system, initially addressing specific communities of for Website Use. If purchasing Frontiers e-books from other websites or sources, scholars, and gradually climbing up to broader public understanding, thus serving the interests the conditions of the website concerned of the lay society, too. apply. Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission. DEDICATION TO QUALITY Individual articles may be downloaded Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interac- and reproduced in accordance with the principles of the CC-BY licence subject to tions between authors and review editors, who include some of the world’s best academicians. any copyright or other notices. They may Research must be certified by peers before entering a stream of knowledge that may eventually not be re-sold as an e-book. reach the public - and shape society; therefore, Frontiers only applies the most rigorous and As author or other contributor you grant a CC-BY licence to others to reproduce unbiased reviews. your articles, including any graphics and Frontiers revolutionizes research publishing by freely delivering the most outstanding research, third-party materials supplied by you, in accordance with the Conditions for evaluated with no bias from both the academic and social point of view. Website Use and subject to any copyright By applying the most advanced information technologies, Frontiers is catapulting scholarly notices which you include in connection with your articles and materials. publishing into a new generation. All copyright, and all rights therein, are protected by national and international copyright laws. The above represents a summary only. WHAT ARE FRONTIERS RESEARCH TOPICS? For the full conditions see the Conditions Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are for Authors and the Conditions for Website Use. collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot ISSN 1664-8714 research area! ISBN 978-2-88919-525-1 Find out more on how to host your own Frontiers Research Topic or contribute to one as an DOI 10.3389/978-2-88919-525-1 author by contacting the Frontiers Editorial Office: [email protected]

Frontiers in Microbiology April 2015 | The Multiple Roles of Antibiotics and Antibiotic Resistance in Nature | 1 THE MULTIPLE ROLES OF ANTIBIOTICS AND ANTIBIOTIC RESISTANCE IN NATURE

Topic Editor: Fiona Walsh, National University of Ireland Maynooth, Ireland

Antibiotics and antibiotic resistance have most commonly been viewed in the context of human use and effects. However, both have co-existed in nature for millennia. Recently the roles of antibiotics and antibiotic resistance genes have started to be discussed in terms of functions other than bacterial inhibition and protection. This special topic will focus on both the traditional role of antibiotics as warfare mechanisms and their alternative roles and uses within nature such as antibiotics Antibiotic susceptibility testing of bacteria as signals or communication mechanisms, isolated from soil antibiotic selection at low concentrations, the non-specific role of resistance mechanisms in nature: e.g. efflux pumps, evolution of antibiotic resistance and the role of persisters in natural antibiotic resistance.

Frontiers in Microbiology April 2015 | The Multiple Roles of Antibiotics and Antibiotic Resistance in Nature | 2 Table of Contents

04 The Multiple Roles of Antibiotics and Antibiotic Resistance in Nature Fiona Walsh 05 Investigating Antibiotic Resistance in Non-Clinical Environments Fiona Walsh 10 A Brief Multi-Disciplinary Review on Antimicrobial Resistance in Medicine and Its Linkage to the Global Environmental Microbiota L. Cantas, Syed Q. A. Shah, L. M. Cavaco, C. M. Manaia, F. Walsh, M. Popowska, H. Garelick, H. Bürgmann and H. Sørum 24 Antibiotic Resistance Shaping Multi-Level Population Biology of Bacteria Fernando Baquero, Ana P. Tedim and Teresa M. Coque 39 Antibiotics as Selectors and Accelerators of Diversity in the Mechanisms of Resistance: From the Resistome to Genetic Plasticity in the β-Lactamases World Juan-Carlos Galán, Fernando González-Candelas, Jean-Marc Rolain and Rafael Cantón 56 Evolutionary Consequences of Antibiotic Use for the Resistome, Mobilome, and Microbial Pangenome Michael R. Gillings 66 “Stormy Waters Ahead”: Global Emergence of Carbapenemases Gopi Patel and Robert A. Bonomo 83 The Multifaceted Roles of Antibiotics and Antibiotic Resistance in Nature Saswati Sengupta, Madhab K. Chattopadhyay and Hans-Peter Grossart 96 Broad-Host-Range IncP-1 Plasmids and Their Resistance Potential Magdalena Popowska and Agata Krawczyk-Balska 104 RND Multidrug Efflux Pumps: What are they Good for? Carolina Alvarez-Ortega, Jorge Olivares and José L. Martínez 115 Extracellular DNA-Induced Antimicrobial Peptide Resistance Mechanisms in Pseudomonas Aeruginosa Shawn Lewenza 121 Concentration-Dependent Activity of Antibiotics in Natural Environments Steve P. Bernier and Michael G. Surette

Frontiers in Microbiology April 2015 | The Multiple Roles of Antibiotics and Antibiotic Resistance in Nature | 3 EDITORIAL published: 27 August 2013 doi: 10.3389/fmicb.2013.00255

The multiple roles of antibiotics and antibiotic resistance in nature

Fiona Walsh*

Agroscope Changins Wädenswil, Wädenswil, Switzerland *Correspondence: ﬁ[email protected] Edited by: Rustam I. Aminov, University of the West Indies, Jamaica

Keywords: antibiotic resistance, environment, clinical pathogens, soil, plasmids, intrinsic factor

There have been many calls for more information about the nat- and diversification of antibiotic resistance mechanisms, in partic- ural resistome and these have also highlighted the importance of ular using the β-lactamasesasmodels(Galán et al., 2013; Patel understanding the environmental resistome in the preservation of and Bonomo, 2013; Popowska and Krawczyk-Balska, 2013). The antibiotics for the treatment of infections. However, to date there β-lactamases constitute the most widespread mechanism of resis- have been few studies which have investigated the roles of antibi- tance, at least among pathogenic bacteria, with more than 1000 otics and resistances outside of the clinical environment. This lack enzymes identified in the literature. We present some examples of data also highlights the difficulties faced by microbiologists in of the alternative functions for the multi-drug resistance mecha- designingtheseexperimentstoproducemeaningfuldata.Antibiotics nisms of efflux, that range from bacterial interactions with plant and antibiotic resistance have most commonly been viewed in the or animal hosts, to the detoxification of metabolic intermediates context of human use and effects. However, both have co-existed in or the maintenance of cellular homeostasis and also the poten- nature for millennia. Recently the roles of antibiotics and antibiotic tial role of extracellular DNA in antibiotic resistance and virulence resistance genes have started to be discussed in terms of functions (Alvarez-Ortega et al., 2013). Bacterial responses to antibiotics other than bacterial inhibition and protection. This special topic may be concentration dependent and so we discuss the different has focused on both the traditional role of antibiotics as warfare types of interactions mediated by antibiotics and non-antibiotic mechanisms and their alternative roles and uses within nature. metabolites as a function of their concentrations and speculate on The research topic starts with an introduction into antimicro- how these may amplify the overall antibiotic resistance/tolerance bial resistance in medicine, its linkage to the global environmental and the spread of antibiotic resistance determinants in a con- microbiota and the many different roles of antibiotics and antibi- text of poly-microbial community (Bernier and Surette, 2013; otic resistance in nature, providing the background to the topic Lewenza, 2013; Sengupta et al., 2013). The use of antibiotics (Cantas et al., 2013). The following chapter discusses the idea that may also be regarded as pollution. Thus, the widespread use and the understanding of antibiotic resistance implies expanding our abuse of antibiotic therapy has evolutionary and ecological con- knowledge on multi-level population biology of bacteria (Baquero sequences, some of which are only just beginning to be examined et al., 2013). This brings with it inherent problems of design- (Gillings, 2013). ing experimental procedures and standards that can be used in These papers thoroughly review the many different aspects of many different microbiomes from human to soil and is discussed antibiotic resistance and the roles of antibiotics in nature and link further by Walsh (Walsh, 2013). A number of models are pro- these to the emerging antibiotic resistances of particular impor- posed to study and understand the biological impact of selection tance to the treatment of infectious diseases.

REFERENCES global environmental microbiota. Patel, G., and Bonomo, R. A. (2013). Received: 07 August 2013; accepted: 08 Alvarez-Ortega, C., Olivares, J., and Front. Microbiol. 4:96. doi: 10.3389/ “Stormy waters ahead”: global August 2013; published online: 27 August Martínez, J. L. (2013). RND mul- fmicb.2013.00096 emergence of carbapenemases. 2013. tidrug efﬂux pumps: what are they Galán, J. C., González-Candelas, F., Front. Microbiol. 4:48. doi: Citation: Walsh F (2013) The multiple good for. Front. Microbiol. 4:7. doi: Rolain, J. M., and Cantón, R. (2013). 10.3389/fmicb.2013.00048 roles of antibiotics and antibiotic resis- 10.3389/fmicb.2013.00007 Antibiotics as selectors and accel- Popowska, M., and Krawczyk-Balska, tance in nature. Front. Microbiol. 4:255. Baquero,F.,Tedim,A.P..,andCoque, erators of diversity in the mecha- A. (2013). Broad-host-range doi: 10.3389/fmicb.2013.00255 T. M. (2013). Antibiotic resistance nisms of resistance: from the resis- IncP-1 plasmids and their resis- This article was submitted to shaping multi-level population biol- tome to genetic plasticity in the β- tance potential. Front. Microbiol. Antimicrobials, Resistance and ogy of bacteria. Front. Microbiol. lactamases world. Front. Microbiol. 4:44. doi: 10.3389/fmicb.2013. Chemotherapy, a section of the journal 4:15. doi: 10.3389/fmicb.2013.00015 4:9. doi: 10.3389/fmicb.2013.00009 00044 Frontiers in Microbiology. Bernier, S. P., and Surette, M. G. (2013). Gillings, M. R. (2013). Evolutionary Sengupta, S., Chattopadhyay, M. K., Copyright © 2013 Walsh. This is an open- Concentration-dependent activity consequences of antibiotic use and Grossart, H. P. (2013). The access article distributed under the terms of of antibiotics in natural environ- for the resistome, mobilome and multifaceted roles of antibiotics the Creative Commons Attribution License ments. Front. Microbiol. 4:20. doi: microbial pangenome. Front. and antibiotic resistance in nature. (CC BY). The use, distribution or reproduc- 10.3389/fmicb.2013.00020 Microbiol. 4:4. doi: 10.3389/fmicb. Front. Microbiol. 4:47. doi: 10.3389/ tion in other forums is permitted, provided Cantas, L., Shah, S. Q. A., Cavaco, 2013.00004 fmicb.2013.00047 the original author(s) or licensor are cred- L. M., Manaia, C. M., Walsh, F., Lewenza, S. (2013). Extracellular DNA- Walsh, F. (2013). Investigating antibi- ited and that the original publication in this Popowska, M., et al. (2013). A induced antimicrobial peptide resis- otic resistance in non-clinical journal is cited, in accordance with accepted brief multi-disciplinary review tance mechanisms in Pseudomonas environments. Front. Microbiol. academic practice. No use, distribution or on antimicrobial resistance in aeruginosa. Front. Microbiol. 4:21. 4:19. doi: 10.3389/fmicb.2013. reproduction is permitted which does not medicine and its linkage to the doi: 10.3389/fmicb.2013.00021 00019 comply with these terms.

www.frontiersin.org August 2013 | Volume 4 | Article 255 | 4 MINI REVIEW ARTICLE published: 15 February 2013 doi: 10.3389/fmicb.2013.00019 Investigating antibiotic resistance in non-clinical environments

Fiona Walsh* Department of Bacteriology, Research Station Agroscope Changins-Wädenswil, Federal Department of Economic Affairs, Education and Research, Wädenswil, Switzerland

Edited by: There have been many calls for more information about the natural resistome and Jose L. Martinez, Centro Nacional de these have also highlighted the importance of understanding the soil resistome in the Biotecnología, Spain preservation of antibiotics for the treatment of infections. However, to date there have Reviewed by: been few studies which have investigated the culturable soil resistome, which highlights the Jian-Hua Liu, South China Agricultural University, China difficulties faced by microbiologists in designing these experiments to produce meaningful Fernando Baquero, Ramón y Cajal data.TheWorld Health Organization definition of resistance is the most fitting to non-clinical Institute for Health Research, Spain environmental studies: antimicrobial resistance is resistance of a microorganism to an *Correspondence: antimicrobial medicine to which it was previously sensitive. The ideal investigation of Fiona Walsh, Department of non-clinical environments for antibiotic resistance of clinical relevance would be using Bacteriology, Research Station Agroscope Changins-Wädenswil, standardized guidelines and breakpoints. This review outlines different definitions and Federal Department of Economic methodologies used to understand antibiotic resistance and suggests how this can be Affairs, Education and Research, performed outside of the clinical environment. Wädenswil, Switzerland. e-mail: fiona.walsh@agroscope. Keywords: susceptibility testing, soil, definition of resistance, resistome, guidelines admin.ch

The World Health Organization World Health Day 2011 high- for the treatment of infections (Pruden et al., 2006; American lighted the problems of antibiotic resistance under the title Academy of Microbiology, 2009; Aminov, 2009; Rosenblatt- “Antibiotic resistance: no action today, no cure tomorrow”. Every Farrell, 2009). However, to date there have been few studies which year 25,000 people in the European Union die because of a seri- have investigated the culturable soil resistome, in antibiotic pro- ous resistant bacterial infection, mostly acquired in health care ducing bacteria Streptomyces, and an isolated cave microbiome settings1. The search for antibiotics, understanding their mech- (D’Costa et al., 2006; Bhullar et al., 2012). Numerous studies have anisms of action and the development and spread of antibiotic been performed using polymerase chain reaction (PCR) or quanti- resistance has resulted in the development of many industries tative PCR (qPCR) to screen the environment for known resistance and novel research areas for over 100 years. However, the num- genes or more recently metagenomics (Volkmann et al., 2004; ber of antibiotics coming to the market over the past 30 years Chen et al., 2007; Knapp et al., 2008; Lata et al., 2009; Zhang et al., has dramatically declined as many pharmaceutical companies and 2011; McGarvey et al., 2012; Popowska et al., 2012). Functional biotech firms abandoned the search for antibiotics in favor of other metagenomics have also been used to identify novel resistance pharmaceuticals (Spellberg et al., 2004). Antibiotic resistance has mechanisms in soil (Allen et al., 2009; Donato et al., 2010; Torres- developed over time from resistance to single classes of antibi- Cortés et al., 2011). The identification of antibiotic resistance otics to multi-drug resistance and extreme drug resistance. Until hotspots and the understanding of the evolution and ecology of recently, antibiotics and antibiotic resistance were thought of in antibiotic resistance in the environment are novel areas of research. terms of treatments of infections and the prevention of successful There have been more reviews written on this topic that research treatment, respectively. The mechanisms of action and resistance papers to date, which highlights the difficulties faced by micro- have been studied almost exclusively in pathogenic bacteria. It biologists in designing these experiments to produce meaningful is only in recent years that research in antibiotic resistance has data. We are faced with some fundamental difficulties in assessing focused on the environment from which the antibiotics were ini- and analyzing the environmental antibiotic resistome. tially extracted: soil microorganisms and the soil ecosystem. With Antibiotic resistance and antibiotic breakpoints have been an every decreasing supply of novel antibiotics and increasing defined within the context of their medical functions. Clini- resistance the emphasis has turned to defining the natural antibi- cal breakpoints define bacteria as susceptible, intermediate, or otic resistome and understanding the ecology and evolution of resistant to an antibiotic and are calculated using several fac- antibiotic resistance in the non-clinical environment. These recent tors, including clinical results from studies, wild type minimum research focuses are thought to bring answers to help prevent a inhibitory concentration (MIC) distributions for the relevant bac- return to the pre-antibiotic era. terial species, antibiotic dosing and pharmacokinetic (PK) and There have been many calls for more information about the nat- pharmacodynamics (PD) measurements (Clinical and Labora- ural resistome and these have also highlighted the importance of tory Standards Institute [CLSI], 2006; European Committee on understanding the soil resistome in the preservation of antibiotics Antimicrobial Susceptibility Testing [EUCAST], 2011). This is used as a guide for the clinician to decide how to treat the patient, 1http://www.who.int/world-health-day/2011/en/ with antibiotic resistance meaning treatment failure. But what

www.frontiersin.org February 2013 | Volume 4 | Article 19 | 5

“fmicb-04-00019” — 2013/2/13 — 16:00 — page1—#1 Walsh Resistance outside clinic

does antibiotic resistance mean out-with the human health or natural ecology and evolution of resistance. The idea is that by infection treatment context? Weneed to also define what an antibi- learning more about how resistance has developed over time we otic is in the natural environment, as this too is a term adapted for can understand antibiotic resistance evolution and spread within use in terms of treating bacterial infections. patients and also help to predict the future evolution of resis- The soil may be a reservoir of resistance genes, which are already tance to existing and novel antibiotics. Our use of antibiotics has present in human pathogens or which may emerge to increase the changed the course of antibiotic resistance ecology and evolu- current arsenal of antibiotic resistance mechanisms in pathogens. tion. The environment does not exist in a separate world to that Most antibiotics used in human medicine have been isolated from of humans. There is a constant flow to and from soil, especially soil microorganisms. Therefore, soil is thought of as a potential in urban and agricultural environments. Human activities such reservoir of antibiotic resistance genes. The presence of antibiotics as using antibiotics in the treatment of human and animal dis- in soil is believed to have promoted the development of highly eases or in agriculture, but also pollution and climate change have specific antibiotic resistance mechanisms in antibiotic producing altered the soil environment. If the soil is a reservoir of antibi- and non-producing bacteria (D’Costa et al., 2006). This belief is otic resistance mechanisms, we need to identify how our actions based on studies, which have identified resistance genes such as and climatic change will also affect the soil resistome. The topic blaCTX-M, qnrA, and blaNDM as originating in the environmen- of ecology and evolution of resistance in the environment will be tal bacteria Kluyvera spp., Shewanella algae, and Erythrobacter discussed in other papers within this special topic review and will litoralis, respectively (Oliver et al., 2001; Nordmann and Poirel, not be addressed in this review. 2005; Zheng et al., 2011). These genes are clinically relevant resis- We frequently refer to bacteria as being resistant to antibiotics, tance genes and are currently causing difficulties in the treatment but rarely do we consider what that means. Even the most resis- of bacterial infections. The origins of other plasmid mediated tant bacterium can be inhibited or killed by a sufficiently high resistance genes are still unknown. Anthropocentrism has led to concentration of antibiotic; patients, however, would not be able the view that genes such as blaCTX−M, qnrA, and blaNDM have to tolerate the high concentration required in some cases (Hawkey, evolved in nature as antibiotic resistance genes. However, the true 1998). In order to study the antibiotic resistome, we need to know functions of these genes remain to be characterized. By approach- what antibiotic and antibiotic resistance means in terms of soil ing antibiotic resistance in nature not from an anthropocentric bacteria. Antibiotic action and resistance up until a few years ago viewpoint, but from bacterial evolution and ecological stand- has been studied almost exclusively in terms of human or ani- points will we be able to identify the origins and evolution of such mal pathogens. We can separate the study of the soil antibiotic genes. resistome into two different contexts: While soil may be a reservoir of antibiotic resistance genes, 1. Clinical relevance: antibiotic resistance of relevance to but not all resistance mechanisms are necessarily a threat to the pathogens continued use of antibiotics in all pathogens. Intrinsic resistance 2. Natural relevance: ecology and evolution of antibiotic resistance is a characteristic of almost all isolates of the bacterial species (Leclercq et al., 2011). Intrinsic resistance occurs when the antimi- These separations ensure that when we study antibiotic resis- crobial activity of the drug is clinically insufficient or antimicrobial tance in clinical terms that we have a definition of antibiotics and resistance is innate, rendering it clinically ineffective (Leclercq antibiotic resistance, as they are used in medicine. I will focus on et al., 2011). The commonly believed theory of the role of the the clinical relevance for the remainder of this review. The term soil resistome is that antibiotic production and resistance co-exist antibiotic was described by Waksman (1973) as a description of in soil bacteria, as demonstrated by studies of antibiotic biosyn- a use, a laboratory effect or an activity of a chemical compound. thetic pathways and genome analysis (Cases and de Lorenzo, 2005; Davies and Davies (2010) defined an antibiotic as any class of D’Costa et al., 2007). The theory is that without the resistance organic molecule that inhibits or kills microbes by specific inter- gene the antibiotic producing bacteria would self-destruct, on actions with bacterial targets, without any consideration of the production of the antibiotic. However, Davies and Davies (2010) source of the particular compound or class. Antibiotic resistance pointed out that this theory remains to be proven. In order to from a clinical viewpoint has been defined by the European Agency understand the importance of soil as a potential reservoir of antibi- for the evaluation of medicinal products, as microbiologically otic resistance mechanisms we need to investigate these theories. resistant or clinically resistant2. The soil may be a reservoir of antibiotic resistance genes, but we Microbiological resistance: need to ask which resistance mechanisms are relevant to clinical “Resistant microorganisms from a microbiological point of antibiotic use? view are those that possess any kind of resistance mechanism or The human pathogen is not the ancestral home of antibi- resistance gene.” This definition is quantified using MIC data and otic resistance as they have developed to infect humans, not to breakpoints for the antibiotics. live in soil with antibiotic producers. Therefore, if soil is the Clinical resistance: natural reservoir of antibiotic resistance the most important resis- “The classification of a bacteria as susceptible or resistant tance mechanisms are those that can transfer from soil bacteria depends on whether an infection with the bacterium responds to pathogenic bacteria. Thus, we need to study soil bacteria as to therapy.” a reservoir of antibiotic resistance with relevance to the clinical use of antibiotics. Another reason to study the soil resistome is to 2http://www.ema.europa.eu/docs/en_GB/document_library/Report/2009/10/ understand the roles of antibiotics and resistance in nature: the WC500005166.pdf

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 19 | 6

“fmicb-04-00019” — 2013/2/13 — 16:00 — page2—#2 Walsh Resistance outside clinic

Clinical resistance is a complex concept in which the type of susceptibility testing of bacteria in non-clinical environments is infecting bacterium, its location in the body, the distribution of the required. The culture-based studies of antibiotic resistance in soil antibiotic in the body and its concentration at the site of infection, bacteria to date have defined antibiotic resistance as growth at and the immune status of the patient all interact. The difficulty 20 mg/L (D’Costa et al., 2007; Bhullar et al., 2012). This arbitrary arises when we try to apply these definitions to soil bacteria or non- definition is based on the use of 20 mg/L as the breakpoint concen- pathogenic bacteria, where no breakpoints exist. If we identify a tration in the initial soil resistome study of Streptomyces species novel resistance mechanism in a soil bacteria, will this resistance (D’Costa et al., 2007). This definition is used for all bacteria and gene cause resistance in a human pathogen? The antibiotic resis- all classes of antibiotics. tance definition of most relevance in both clinical and non-clinical The clinical breakpoint of an antibiotic is determined by com- environments is that of the WHO: bining the relevant factors in setting breakpoints for antimicrobial Antimicrobial resistance is resistance of a microorganism to an agents and consist of, as defined by the European Committee on antimicrobial medicine to which it was previously sensitive. Antimicrobial Susceptibility Testing: In order to define antibiotic resistance in non-clinical environments we need to address the specific context and define what 1. Available formulations is meant by sensitive. In terms of clinically relevant antibiotic 2. Standard and maximum dosing resistances, we can define resistance in all contexts as bacteria con- 3. Clinical indications and target organisms taining a known resistance gene or those, which are no longer 4. MIC distributions for individual species inhibited at the site of infection. These bacteria would then be 5. Pharmacokinetic (PK) data in humans considered resistant to the respective antibiotics. Defining an 6. Pharmacodynamic (PD) data antibiotic sensitive bacteria is a more complex task, especially with 7. Information from modeling processes respect to bacteria inhabiting different environments. A bacteria 8. Clinical data relating outcome to MIC values defined as sensitive may be so in soil, but in the presence of clin- 9. Information on resistance mechanisms, the clinical significance ically relevant antibiotic concentrations, may be highly mutable of the resistance mechanisms, and the MICs for organisms or have inducible resistance mechanisms. Thus, a sensitive bacte- expressing the resistance mechanisms ria is one, which would not be readily selected in the presence of higher concentrations of antibiotic than those concentrations in However, where no PK/PD data for antibiotics with a particular species have been generated the breakpoints should be based the environment. If an environmental species in a particular place 3 increases its MIC to a certain antibiotic along a limited period on epidemiological cut-off (ECOFF) values for the antibiotics . of time it can be considered that it has become “more” resistant In the case of clinical infections these are limited to antibiotics or less susceptible. However, defining resistance by the presence used to treat the infection. In a recent evaluation of Pasteurella of known resistance genes is limiting the search for resistance to multocida by EUCAST the ECOFF values were determined using those already characterized. Thus, we propose that such resistance approximately 250 isolates for the tests and were estimated by genes be separated into pR-genes, potential resistance genes or visual inspection or statistically calculated (Turnidge et al., 2006). pre-resistance genes and aR-genes, genes known to produce an Ideally greater than 250 isolates would be obtained from multiantibiotic resistance phenotype in bacteria capable of survival and ple centers or countries in order to establish a worldwide ECOFF integration into the human or animal microbiotia. values. Each country could then survey their own multi-center How do we decide which resistances are relevant? In clinical sites in order to identify edaphic influences on the soil resistome. practice we know that Pseudomonas aeruginosa is intrinsically Although the culturable bacteria represent less than 1% of the resistant to ampicillin due to chromosomally mediated AmpC, total bacterial population, within this 1% remains a large number efflux and impermeability. Therefore, clinical Pseudomonas aerug- of bacterial species, for which there are no antibiotic breakpoint inosa are not tested for susceptibility to ampicillin as it is not values. The possibility of culturing many more organisms than this used to treat Pseudomonas aeruginosa infections. However, if soil 1% will be provided by the advances in “culturomics” and com- is described as a potential reservoir of resistance genes, which bining metagenomics data with concurrent sequencing (Lagier can move from the chromosome to mobile elements and then et al., 2012; VanInsberghe et al., 2013). In order to define a bac- to pathogens we need to test all of the bacteria, not just the teria as resistant we need to test the MIC distribution within pathogens or antibiotic producers. If we identify a penicillin resis- the population and identify the breakpoint. Therefore, in terms tant Pedobacter species, does this mean that Pedobacter contains a of defining antibiotic breakpoints for bacteria from non-clinical potential novel resistance gene or, like Pseudomonas aeruginosa it environments the ECOFF would be the most appropriate. In order is mediated by intrinsic resistance or that it is only or importance to create breakpoints for these environments we would need to col- if it can be expressed in pathogenic bacteria? lect and test at least 250 isolates from different locations, in order to Novel resistance genes may be identified using functional have a representative sample. With breakpoints we can set standard metagenomics; total soil DNA is extracted, digested, and ligated guidelines for susceptibility testing of antibiotics in non-clinical into a vector. The vector is transferred into a bacterial host, e.g., environments. Susceptibility testing is the gold standard of antibi- Escherichia coli and the functional characteristics are measured otic resistance testing used throughout the world in hospitals. using clinical breakpoints (Handelsman, 2004). However, in hos- It is a relatively cheap and easy technique with little need for pitals antibiotic resistance is generally tested using MIC or disk diffusion assays. A definition of antibiotic resistance for antibiotic 3http://www.eucast.org/

www.frontiersin.org February 2013 | Volume 4 | Article 19 | 7

“fmicb-04-00019” — 2013/2/13 — 16:00 — page3—#3 Walsh Resistance outside clinic

sophisticated or expensive equipment. Therefore, using sus- the bacterial community can be analyzed for antibiotic resistance ceptibility testing would enable the comparison of non-clinical genes without the need to culture these organisms and can be data with clinical data. However, this is limited to culturable used to detect as yet unknown antibiotic resistance genes. The bacteria. gene’s function is expressed as the antibiotic resistance pheno- Non-culture-based techniques are required to investigate the type in the host bacteria. However, the disadvantage lies with entire bacterial community. The bacteria that can be grown in the fact that the host bacteria is frequently Escherichia coli, which the laboratory are only a small fraction of the total diversity that may not be capable of expressing all of the resistance genes, exists in nature. Approximately only 1% of bacteria on Earth can e.g., strA. be readily cultivated in vitro (Staley and Konopka, 1985; Amann Antibiotic resistance genes and plasmids have been identified et al., 2001). Therefore, non-culture-based tools such as PCR and in pristine and agricultural ecosystems using PCR, functional metagenomics are required to capture the non-culturable section metagenomics and pyrosequencing tools (Pruden et al., 2006; of the non-clinical antibiotic resistome. However, one disadvan- Allen et al., 2009; Heuer and Smalla, 2012). Both novel resistance tage of these tools are that they are limited to screening for known genes and bifunctional genes were identified in soil from apple resistance genes and mechanisms and identification of only the orchards (Donato et al., 2010). The revolution in high-throughput resistance gene rather than being able to investigate the process sequencing has brought unique opportunities and challenges to involved in resistance, as with culturable bacteria. PCR and quan- the field of environmental microbiology. Since its introduction titative PCR have been frequently used to determine the presence in 2005, the number of metagenomic libraries in the database of resistance genes in nature and the effects of agriculture on the has increased year by year and following the quantum leaps of emergence and spread of resistance. The most frequently used next generation sequencing techniques (Schmieder and Edwards, methods to determine the presence of antibiotic resistance in the 2012). Dependent on the sequencing platform, up to 500 Gb environment have been PCR detection, microarray detection or of sequencing data can be generated per single run (Shokralla real-time PCR detection of known resistance genes (Chen et al., et al., 2012). 2007; Koike et al., 2007; Peak et al., 2007; Walsh and Rogers, The complete characterization of the soil antibiotic resistome 2008; Borjesson et al., 2009; Walsh et al., 2010). These techniques requires both culture and non-culture-based approaches. Using are limited to detecting genes, which are known and have been defined guidelines and definitions the scientific community can sequenced. More recently functional metagenomics have iden- standardize the methodologies required. This has proved very tified novel resistance genes and antibiotic biosynthesis genes effective in the surveillance of antibiotic resistance, emerging present in environmental bacteria (Donato et al., 2010; Torres- trends, and novel resistance mechanisms in clinical bacteria. The Cortés et al., 2011). The direct applications of sequencing have to advantage of identifying the bacteria associated with the resis- date been mostly focused on the elucidation of species variations tance mechanism, if it is a novel mechanism, is that the entire within the environment (Kohler et al., 2005; Janssen, 2006; Lauber genetic cascade or pathway leading to resistance can be studied in et al., 2009; Shange et al., 2012). depth. However, culture-based techniques alone will only describe The application of functional metagenomics on soil DNA a small fraction of the bacteria present in non-clinical environ- have identified novel resistance genes and antibiotic biosyn- ments. A combination of culture-based and non-culture-based thesis genes present in environmental bacteria (Donato et al., standardized techniques will provide a wealth of information on 2010; Torres-Cortés et al., 2011). Functional metagenomics is the the composition of the non-clinical antibiotic resistome. The genomic study, without culturing, of a population of microor- WHO definition of resistance is the most fitting to non-clinical ganisms (Handelsman, 2004). This approach has been applied to environmental studies. The ideal investigation of non-clinical the study of the antibiotic resistome present in environmental environments for antibiotic resistance of clinical relevance would samples (Riesenfeld et al., 2004; Donato et al., 2010; Torres- be using standardized guidelines and breakpoints as outlined in Cortés et al., 2011). The advantages of metagenomics are that this review.

REFERENCES Aminov, R. I. (2009). The role of Cases, I., and de Lorenzo, V. (2005). for Bacteria that Grow Aerobically: Allen, H. K., Moe, L. A., Rodbum- antibiotics and antibiotic resistance Promoters in the environment: tran- Approved Standard, 7th Edn (CLSI rer, J., Gaarder, A., and Handelsman, in nature. Environ. Microbiol. 11, scriptional regulation in its natu- Document M7-A7). Wayne: Clinical J. (2009). Functional metagenomics 2970–2988. ral context. Nat. Rev. Microbiol.3, and Laboratory Standards Institute. reveals diverse beta-lactamases in a Bhullar, K., Waglechner, N., Pawlowski, 105–118. Davies, J., and Davies, D. (2010). Ori- remote Alaskan soil. ISME J. 3, A., Koteva, K., Banks, E. D., Johnston, Chen, J., Yu, Z. T., Michel, F. C., gins and evolution of antibiotic resis- 243–251. M. D., et al. (2012). Antibiotic resis- Wittum, T., and Morrison, M. tance. Microbiol. Mol. Biol. Rev. 74, Amann, R., Fuchs, B. M., and tance is prevalent in an isolated cave (2007). Development and appli- 417–433. Behrens, S. (2001). The iden- microbiome. PLoS ONE 7:e34953. cation of real-time PCR assays D’Costa,V.M., Griffiths, E., and Wright, tification of microorganisms by doi: 10.1371/journal.pone.0034953 for quantification of erm genes G. D. (2007). Expanding the soil ?uorescence in situ hybridisa- Borjesson, S., Dienues, O., Jarnheimer, conferring resistance to macrolides- resistome: exploring environmental tion. Curr. Opin. Biotechnol. 12, P. A., Olsen, B., Matussek, A., and lincosamides-streptogramin B in diversity. Curr. Opin. Microbiol. 10, 231–236. Lindgren, P. E. (2009). Quantifica- livestock manure and manure man- 481–489. American Academy of Microbiology. tion of genes encoding resistance to agement systems. Appl. Environ. D’Costa, V. M., McGrann, K. M., (2009). Antibiotic Resistance: An Eco- aminoglycosides, beta-lactams and Microbiol. 73, 4407–4416. Hughes, D. W., and Wright, logical Perspective on an Old Problem tetracyclines in wastewater environ- Clinical and Laboratory Standards G. D. (2006). Sampling the (Washington: American Academy of ments by real-time PCR. Int. J. Envi- Institute. (2006). Methods for Dilu- antibiotic resistome. Science 311, Microbiology). ron. Health Res. 19, 219–230. tion Antimicrobial Susceptibility Tests 374–377.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 19 | 8

“fmicb-04-00019” — 2013/2/13 — 16:00 — page4—#4 Walsh Resistance outside clinic

Donato, J. J., Moe, L. A., Converse, for the rapid detection of vanA gene antibiotic resistance genes. Environ. Waksman, S. A. (1973). History of the B. J., Smart, K. D., Berklein, F. C., in surface waters and aquatic macro- Microbiol. 6, 981–989. word “antibiotic”. J. Hist. Med. Allied McManus, P.S., et al. (2010). Metage- phyte by molecular beacon probe. Rosenblatt-Farrell, N. (2009). Antibi- Sci. 28, 284–286. nomic analysis of apple orchard soil Environ. Sci. Technol. 43, 3343–3348. otics in the environment. Environ. Walsh, F., and Rogers, T. R. (2008). reveals antibiotic resistance genes Lauber, C. L., Hamady, M., Knight, Health Perspect. 117, A248–A250. Detection of blaVIM-2 carbapene- encoding predicted bifunctional pro- R., and Fierer, N. (2009). Schmieder, R., and Edwards, R. (2012). mase in Pseudomonas aeruginosa in teins. Appl. Environ. Microbiol. 13, Pyrosequencing-based assessment Insights into antibiotic resistance Ireland. J. Antimicrob. Chemother. 61, 4396–4401. of soil pH as a predictor of soil through metagenomic approaches. 219–220. European Committee on Antimicro- bacterial community structure at Future Microbiol. 7, 73–89. Walsh, F., Cooke, N. M., Smith, S. G., bial Susceptibility Testing. (2011). the continental scale. Appl. Environ. Shange, R. S., Ankumah, R. O., Ibekwe, Moran, G. P., Cooke, F. J., Ivens, Clinical Breakpoints. Available at: Microbiol. 75, 5111–5120. A. M., Zabawa, R., and Dowd, A., et al. (2010). Comparison of http://www.eucast.org/clinical_ Leclercq, R., Cantón, R., Brown, S. E. (2012). Distinct soil bac- two DNA microarrays for detection breakpoints D. F., Giske, C. G., Heisig, P., terial communities revealed under of plasmid-mediated antimicrobial Handelsman, J. (2004). Metagenomics: Macgowan, A. P., et al. (2011). a diversely managed agroecosys- resistance and virulence factor genes application of genomics to uncul- EUCAST expert rules in antimi- tem. PLoS ONE 7:e40338. doi: in clinical isolates of Enterobacte- tured microorganisms. Microbiol. crobial susceptibility testing. Clin. 10.1371/journal.pone.0040338 riaceae and non-Enterobacteriaceae. Mol. Biol. Rev. 68, 669–685. Microbiol. Infect. doi: 10.1111/j.1469- Shokralla, S., Spall, J. L., Gibson, J. Int. J. Antimicrob. Agents 35, 593–598. Hawkey, P. (1998). The origins and 0691.2011.03703.x [Epub ahead of F., and Hajibabaei, M. (2012). Next- Zhang, T., Zhang, X. X., and Ye, molecular basis of antibiotic resis- print]. generation sequencing technologies L. (2011). Plasmid metagenome tance. BMJ 317, 657–659. McGarvey, K. M., Queitsch, K., and for environmental DNA research. reveals high levels of antibiotic resis- Heuer, H., and Smalla, K. (2012). Fields, S. (2012). Wide variation in Mol. Ecol. 21, 1794–1805. tance genes and mobile genetic ele- Plasmids foster diversification and antibiotic resistance proteins iden- Spellberg, B., Powers, J. H., Brass, E. ments in activated sludge. PLoS adaptation of bacterial populations in tified by functional metagenomic P., Miller, L. G., and Edwards, J. ONE 6:e26041. doi: 10.1371/jour- soil. FEMS Microbiol. Rev. 36, 1083– screening of a soil DNA library. Appl. E. Jr. (2004). Trends in antimicro- nal.pone.0026041 1104. Environ. Microbiol. 78, 1708–1714. bial drug development: implications Zheng, B., Tan, S., Gao, J., Han, H., Janssen, P. H. (2006). Identifying Nordmann, P., and Poirel, L. (2005). for the future. Clin. Infect. Dis. 38, Liu, J., Lu, G., et al. (2011). An unex- the dominant soil bacterial taxa in Emergence of plasmid-mediated 1279–1286. pected similarity between antibiotic- libraries of 16S rRNA and 15S rRNA resistance to quinolones in Enter- Staley, J. T., and Konopka, A. (1985). resistant NDM-1 and beta-lactamase genes. Appl. Environ. Microbiol. 72, obacteriaceae. J. Antimicrob. Measurement of in situ activities of II from Erythrobacter litoralis. Protein 1719–1728. Chemother. 56, 463–469. nonphotosynthetic microorganisms Cell 2, 250–258. Knapp, C. W., Engemann, C. A., Han- Oliver, A., Pérez-Díaz, J. C., Coque, in aquatic and terrestrial habitats. son, M. L., Keen, P. L., Hall, K. J., and T. M., Bacquero, F., and Cantón, Annu.Rev.Microbiol. 39, 321–346. Graham, D. W. (2008). Indirect evi- R. (2001). Nucleotide sequence Torres-Cortés, G., Millán, V., Ramírez- dence of transposon-mediated selec- and characterization of a novel Saad, H. C., Nisa-Martínez, R., tion of antibiotic resistance genes in cefotaxime-hydrolyzing beta- Toro, N., and Martínez-Abarca, F. Conflict of Interest Statement: The aquatic systems at low-level oxyte- lactamase (CTX-M-10) isolated in (2011). Characterization of novel author declares that the research was tracycline exposures. Environ. Sci. Spain. Antimicrob. Agents Chemother. antibiotic resistance genes identi- conducted in the absence of any com- Technol. 42, 5348–5353. 45, 616–620. fied by functional metagenomics on mercial or financial relationships that Kohler, F., Hamelin, J., Gillet, F., Peak, N., Knapp, C. W., Yang, R. K., soil samples. Environ. Microbiol. 13, could be construed as a potential con- Gobat, J.-M., and Buttler, A. (2005). Hanfelt, M. M., Smith, M. S., Aga, 1101–1114. flict of interest. Soil microbial community changes D. S., et al. (2007). Abundance of Turnidge, J., Kahlmeter, G., and Kro- in wooded mountain pastures due six tetracycline resistance genes in nvall, G. (2006). Statistical charac- Received: 29 October 2012; accepted: to simulated effects of cattle grazing. wastewater lagoons at cattle feedlots terisation of bacterial wild-type MIC 27 January 2013; published online: 15 Plant Soil 278, 327–340. with different antibiotic use strate- value distributions and the deter- February 2013. Koike, S., Krapac, I. G., Oliver, H. D., gies. Environ. Microbiol. 9, 143–151. mination of epidemiological cut-off Citation: Walsh F (2013) Investigating Yannarell, A. C., Chee-Sanford, J. C., Popowska, M., Rzeczycka, M., Miernik, values. Clin. Microbiol. Infect. 12, antibiotic resistance in non-clinical envi- Aminov, R. I., et al. (2007). Moni- A., Krawczyk-Balska, A., Walsh, 418–425. ronments. Front. Microbio. 4:19. doi: toring and source tracking of tetra- F., and Duffy, B. (2012). Influ- VanInsberghe, D., Hartmann, M., Gor- 10.3389/fmicb.2013.00019 cycline resistance genes in lagoons ence of soil use on prevalence of don, S. R., and Mohn, W. W. (2013). This article was submitted to Fron- and groundwater adjacent to swine tetracycline, streptomycin, and ery- Isolation of a substantial proportion tiers in Antimicrobials, Resistance and production facilities over a 3-year thromycin resistance and associated of bacterial forest soil communities as Chemotherapy, a specialty of Frontiers in period. Appl. Environ. Microbiol. 73, resistance genes. Antimicrob. Agents detected via pyrotag sequencing. doi: Microbiology. 4813–4823. Chemother. 56, 1434–1443. 10.1128/AEM.03112-12 [Epub ahead Copyright © 2013 Walsh. This is an open- Lagier, J. C., Armougom, F., Million, Pruden, A., Pei, R. T., Storteboom, H., to print]. access article distributed under the terms M., Hugon, P., Pagnier, I., Robert, C., and Carlson, K. H. (2006). Antibiotic Volkmann, H., Schwartz, T., Bischoff, of the Creative Commons Attribution et al. (2012). Microbial culturomics: resistance genes as emerging contam- P., Kirchen, S., and Obst, U. License, which permits use, distribution paradigm shift in the human gut inants: studies in northern Colorado. (2004). Detection of clinically rel- and reproduction in other forums, pro- microbiome study. Clin. Microbiol. Environ. Sci. Technol. 40, 7445–7450. evant antibiotic-resistance genes in vided the original authors and source Infect. 18, 1185–1193. Riesenfeld, C. S., Goodman, R. M., and municipal wastewater using real-time are credited and subject to any copy- Lata, P., Ram, S., Agrawal, M., and Handelsman, J. (2004). Uncultured PCR (TaqMan). J. Microbiol. Methods right notices concerning any third-party Shanker, R. (2009). Real time PCR soil bacteria are a reservoir of new 56, 277–286. graphics etc.

www.frontiersin.org February 2013 | Volume 4 | Article 19 | 9

“fmicb-04-00019” — 2013/2/13 — 16:00 — page5—#5 REVIEW ARTICLE published: 14 May 2013 doi: 10.3389/fmicb.2013.00096 A brief multi-disciplinary review on antimicrobial resistance in medicine and its linkage to the global environmental microbiota

L. Cantas 1*, Syed Q. A. Shah 1, L. M. Cavaco 2, C. M. Manaia 3, F. Wa l s h 4, M. Popowska 5, H. Garelick 6, H. Bürgmann 7 and H. Sørum 1

1 Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, Oslo, Norway 2 Research Group for Microbial Genomics and Antimicrobial Resistance, National Food Institute, Technical University of Denmark, Kgs Lyngby, Denmark 3 Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia, Centro Regional do Porto da Universidade Católica Portuguesa, Rua Dr. António Bernardino Almeida, Porto, Portugal 4 Bacteriology, Agroscope Changins-Wädenswil, Wädenswil, Switzerland 5 Department of Applied Microbiology, Institute of Microbiology, University of Warsaw, Warsaw, Poland 6 Department of Natural Sciences, Middlesex University, London, UK 7 Department of Surface Waters - Research and Management, Eawag, Swiss Federal Institute for Aquatic Science and Technology, Kastanienbaum, Switzerland

Edited by: The discovery and introduction of antimicrobial agents to clinical medicine was Stefania Stefani, University one of the greatest medical triumphs of the 20th century that revolutionized the of Catania, Italy treatment of bacterial infections. However, the gradual emergence of populations of Reviewed by: antimicrobial-resistant pathogenic bacteria resulting from use, misuse, and abuse of Aixin Yan, The University of Hong Kong, Hong Kong antimicrobials has today become a major global health concern. Antimicrobial resistance Ingeborg M. Van Geijlswijk, (AMR) genes have been suggested to originate from environmental bacteria, as clinically Utrecht University, Netherlands relevant resistance genes have been detected on the chromosome of environmental *Correspondence: bacteria. As only a few new antimicrobials have been developed in the last decade, the L. Cantas, Department of Food further evolution of resistance poses a serious threat to public health. Urgent measures Safety and Infection Biology, Section for Microbiology, Immunology and are required not only to minimize the use of antimicrobials for prophylactic and therapeutic Parasitology, Norwegian School of purposes but also to look for alternative strategies for the control of bacterial infections. Veterinary Science, PO Box 8146 This review examines the global picture of antimicrobial resistance, factors that favor its Dep. NO-0033 Oslo, Norway. spread, strategies, and limitations for its control and the need for continuous training of e-mail: [email protected] all stake-holders i.e., medical, veterinary, public health, and other relevant professionals as well as human consumers, in the appropriate use of antimicrobial drugs.

Keywords: antimicrobial resistance, human and veterinary medicine, environment, soil, wastewater, resistance genes

BACKGROUND antimicrobials have been extremely important corner stones of Arguably one of the greatest examples of serendipity in science modern medicine since the last half of the previous century. was the discovery of natural antimicrobials between Alexander Antimicrobial drugs have saved millions of people from life- Fleming and Ernest Duchesne. Although Fleming generally holds threatening bacterial infections and eased patients’ suffering. the reputation of the discovery of penicillin in 1928, a French Today, the treatment of bacterial infections is once again becom- medical student, Ernest Duchesne (1874–1912), originally dis- ing increasingly complicated because microorganisms are devel- covered the antimicrobial properties of Penicillium earlier, in oping resistance to antimicrobial agents worldwide (Pouillard, 1896. He observed Arab stable boys that kept their saddles in 2002; Levy and Marshall, 2004; Alanis, 2005; Pallett and Hand, a dark and damp room to encourage mold to grow on them, 2010). which they said helped heal saddle sores. Curious, Duchesne A causal relationship has been demonstrated between the prepared a suspension from the mold and injected it into dis- increased use of antimicrobials in both human and veteri- eased guinea pigs along with a lethal dose of virulent typhoid nary medicine, the greater movement of people, as well as bacilli and still all animals remained healthy. His work, how- domestic and wild animals, the increased industrialization ever, was ignored because of his young age and unknown status and the increased prevalence of antimicrobial-resistant bacte- (Pouillard, 2002). In a way, with the success of the natural antibi- ria (Holmberg et al., 1987; Cheng et al., 2012). Antimicrobial otic penicillin and the synthetic antimicrobial sulfonamides in resistance (AMR) was identified in pathogenic bacteria concur- the first half of the 20th century, the modern antimicrobial rev- rently with the development of the first commercial antibiotic olution began. Since then, new natural antimicrobial compounds produced by microorganisms, penicillin (Abraham and Chain, were discovered and many semi-synthetic and synthetic antimi- 1940). Although the first concerns about drug-resistant bacteria crobial drugs were created to combat bacterial infections. Thus, appeared in hospitals, where most antimicrobials were being used

www.frontiersin.org May2013|Volume4|Article96| 10 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

(Levy, 1998), the importance of AMR was already recognized in different ecological locations (Figure 1), aiming to unify the many 1969 both in humans and veterinary medicine as stated in the important aspects of this problem. Finally we advocate the need Swan Report (Swann, 1969). Further research showed that the for teaching and continuous training of all stake-holders (i.e., origin and spread of AMR is, in fact, a very complex problem. medical, veterinary, public health, and other relevant profession- Hence, there cannot be a single solution for minimizing AMR; als) as well as human consumers of antimicrobial drugs, in the rather a coordinated multi-disciplinary approach will be required appropriate use of antimicrobials. toaddressthisissue(Serrano, 2005; Smith et al., 2009). We must also recognize that wherever antimicrobials are used, AMR will THE HUMAN MEDICINE AND ANTIMICROBIAL RESISTANCE inevitably follow. EMERGENCE OF ANTIMICROBIAL RESISTANCE AND ITS COST The purpose of this review is to highlight the problem of In human medicine AMR is as old as the clinical usage of antimi- resistance to antimicrobials with its consequences, including how crobial compounds. Antimicrobial-resistant pathogens have been the spread of AMR could be limited. We highlight how the observed soon after the introduction of new drugs in hos- numerous useful applications of antimicrobials led to AMR in pitals where antimicrobials are intensively used (Levy, 1998).

FIGURE 1 | Schematic representation of the complexity of the potential genetic links between the microbiotas of the various reservoirs are showed bacterial genetic web of communication between the various by arrows. Thick arrows show major selective pressures for selection of microbiotas that are impacted by the use of antibiotics in a wide antibiotic resistance genes, thin arrows show the signiﬁcant directions of context. The reservoirs where antimicrobials are applied are also suggested gene ﬂow. Future research may document unique arrows that must be as “hot spots” for horizontal gene transfer. The potentially most important integrated in the web drawn.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy May2013|Volume4|Article96| 11 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

Consequently, AMR in the context of human medicine has dom- of defense against the non-Enterobacteriaceae pathogens, such inated the literature for a long time (Figure 2). Over the years, as Pseudomonas aeruginosa and Acinetobacter baumannii (Brown and continuing into the present, almost every known bacterial et al., 1998). However, since the first description of the blaOXA pathogen and numerous human commensals have developed genes, there has been a worldwide increase in the dissemi- resistance to one or more antimicrobials in clinical use (Table 1). nation of new resistance determinants conferring carbapenem Extended-spectrum β-lactamases (ESBL) are the ones most often resistance. For example, the Klebsiella pneumoniae carbapene- encountered in the hospital (intensive care) setting. Methicillin- mase (KPC) type enzymes, Verona integron-encoded metallo-β- resistant Staphylococcus aureus (MRSA) and vancomycin-resistant lactamase (VIM), Imipenemase Metallo-β-lactamase (IMP) and enterococci (VRE) have also been found to have a signifi- New Delhi metallo-β-lactamase (NDM), and the OXA-48 type cant nosocomial ecology (Otter and French, 2010). In addition, of enzymes have been isolated from a number of bacterial gen- ESBL positive bacteria and MRSA infections are increasingly era irrespective of their geographical distribution (Kumarasamy detected in the community. Furthermore, the increase in flu- et al., 2010; Walsh et al., 2011). Carbapenemase resistance mech- oroquinolone resistance due to target-site mutations and the anisms are found among Escherichia coli and Klebsiella isolates in worldwide emergence of plasmid-mediated quinolone resistance hospital settings, and to a lesser extent also in the community, genes may represent a major challenge in future given the crit- thus healthy human carriers begin to be a concern (Nordmann ical importance of this antimicrobial therapy (Cattoir et al., et al., 2012). Furthermore, carbapenemase-producing organisms 2007; Strahilevitz et al., 2009). Carbapenems are the last line have also been isolated from farm animals (Fischer et al., 2012).

FIGURE 2 | The change in number of antimicrobial resistance related Hospital, (hospital∗ OR patient∗ OR clinic∗); Animal, (animal∗ OR veterinary∗ published research papers in different subdisciplines and covering OR livestock∗ OR pig∗ OR cow∗ OR chicken∗ OR poultry); Wastewater, different environments. The data for the graphs were obtained by searching (wastewate∗ OR sewage); Natural water, (wate∗ OR lake OR river OR ocean the ISI web of science for publications with titles matching the query terms OR sea); Soil, (soil∗ OR sediment∗ OR rhizosphere∗ ) (Source: http://apps. (antibioti∗ OR antimicro∗ ) AND resistan∗ AND the following speciﬁc terms: isiknowledge.com/). The search was performed on 06/03/2013.

www.frontiersin.org May2013|Volume4|Article96| 12 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

Table 1 | Antimicrobial resistance detection in some important pathogens soon after arrival of the “magic bullets” into the market.

Year Bacteria Drug resistance Comments References

1948 Staphylococcus aureus Penicillin In British civilian hospitals soon Barber and after the introduction of penicillin Rozwadowska-Dowzenko, 1948 1948 Mycobacterium tuberculosis Streptomycin In the community soon after the Crofton and Mitchison, 1948 clinical usage of this antimicrobial 1950’s–1960’s Escherichia coli, Shigella spp., and Multiple drugs Watanabe, 1963; Olarte, 1983; Salmonella enterica Levy, 2001 1960’s VRE- Enterococcus spp. Multiple drugs Levy and Marshall, 2004; ESBL- E. cloacae, K. pneumoniae, E. coli, Nordmann et al., 2011 MRSA, QR- Enterobacteriaceae MDR- Acinetobacter baumannii, Pseudomonas aeruginosa

VRE, Vancomycin-Resistant Enterococcus; ESBL, Extended-spectrum β-lactamase; MRSA, methicillin/oxacillin-resistant Staphylococcus aureus; QR , Quinolone resistant; MDR, Multi-drug resistant.

HUMAN MOBILITY—THE DIRECT AND INDIRECT IMPACT ON HUMAN human diseases from the mid-1940’s (Gustafson and Bowen, PATHOGENS 1997; McEwen, 2006). Even though some drugs are exclusively The increasing cross-border and cross-continental movements of designed for veterinary use, most belong to the same antimicro- people has a major impact on the spread of multi-resistant bac- bial classes as those used in human medicine with identical or very teria (Linton et al., 1972; Arya and Agarwal, 2011; Cheng et al., similar structures (Swann, 1969; Heuer et al., 2009). 2012). The emergence and global spread of the international clone Annually, large quantities of drugs are administered to ani- 1 of penicillin-resistant Streptococcus pneumoniae (Klugman, mals in the agricultural sector worldwide to secure a sufﬁcient 2002) and the recently occurring New Delhi Metallo-β-lactamase amount of food (meat, eggs, and dairy products) to feed a rapidly (blaNDM-1) producing Enterobacteriaceae,whichinactivatesall growing world human population (Vazquez-Moreno et al., β-lactam antimicrobials, including carbapenems, are good exam- 1990; Roura et al., 1992; Rassow and Schaper, 1996). As data ples. The blaNDM-1 appears to have originated in the Indian sub- collection on antimicrobial use in animals was not harmonized continent and subsequently could be found in North America, the to provide reliable and comparable information, and following a United Kingdom, and other European countries by the movement request from the European Commission the European Medicines of people (Arya and Agarwal, 2011; Walsh et al., 2011). Agency (EMA), the European Surveillance of Antimicrobial The AMR problem remains a growing public health concern Consumption (ESVAC) programme has been created. The because infections caused by resistant bacteria are increasingly ESVAC programme is responsible for collecting, analyzing, and difﬁcult and expensive to treat. The consequences of this problem reporting sales data from European countries and developing are: longer hospital stay, longer time off work, reduced quality of an organized approach for the collection and reporting of data life, greater likelihood of death due to inadequate or delayed treat- on antimicrobial use for animals including annual reporting ment, increases in private insurance coverage and an additional from EU member states (www.ema.europa.eu/ema/index. costs for hospitals when hospital-acquired infections occur in jsp?curl=pages/regulation/document_listing/document_listing_ addition to the increased overall healthcare expenditure (Roberts 000302.jsp). During 2007, in 10 European countries the sale of et al., 2009; Filice et al., 2010; Korczak and Schöffmann, 2010; antimicrobial drugs for therapeutic use as veterinary medicine Wilke, 2010). Thus, in order to calculate the full economic bur- varied from 18 to 188 mg/kg biomass (Grave et al., 2010). den of AMR we have to consider the burden of not having The administration of antimicrobials to food producing antimicrobial treatment options at all, which in the extreme case animals can have other purposes than treatment, such as: would probably cause a breakdown of the entire modern medi- growth promotion (although now totally banned in Europe and cal system (Alanis, 2005; Falagas and Bliziotis, 2007; Pratt, 2010). quinolones for the poultry industry are banned in the USA), pro- In short, everyone will be at risk when antimicrobials become phylaxis, and -metaphylaxis (Anthony et al., 2001; Anderson et al., ineffective and the threat is greatest for young children, the 2003; Casewell et al., 2003; Cabello, 2006). Approximately 70% of elderly, and immune-compromised individuals, such as cancer all the antimicrobials administered in animal farming are used for patients undergoing chemotherapy and organ transplant patients non-therapeutic purposes (Roe and Pillai, 2003). (Tablan et al., 2004). In the European Union (EU) the use of avoparcin was banned in 1997. Furthermore spiramycin, tylosin, and virginiamycin for THE VETERINARY MEDICINE AND AGRICULTURE SECTOR growth promotion were banned from use in 1998. All other CONSUMPTION AND REGULATION OF ANTIMICROBIAL USE IN growth promoters in the feed of food-producing animals were ANIMALS banned in the EU—countries from January 1, 2006 (http:// The antimicrobials, used in veterinary medicine were intro- europa.eu). Data from Denmark showed that animals could be duced soon after they became available for the treatment of produced at a large scale without the use of growth promoters,

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy May2013|Volume4|Article96| 13 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

without adversely affecting the production (Aarestrup et al., 2001; underlying allergic reactions result in an increasing use of semi- Aarestrup, 2005; Hammerum et al., 2007). In the United States synthetic penicillins because of the ineffectiveness of penicillin of America, politicians are discussing the introduction of a sim- against penicillinase producing Staphylococcus pseudintermedius ilar ban on the use of antimicrobials in animal husbandry for (Yoon et al., 2010). Moreover, emerging methicillin-resistant growth promotion (http://www.govtrack.us/congress/bills/109/ Staphylococcus pseudintermedius (MRSP), methicillin-resistant s742). Despite these bans, in some parts of the world, medically Staphylococcus aureus (MRSA), and ESBL producing E. coli dis- important antibiotics are still routinely fed to livestock prophy- playing multidrug resistance has led to increased concern related lactically to increase profits and to ward-off potential bacterial to AMR in companion animal practice (Bannoehr et al., 2007; infections in the stressed and crowded livestock and aquaculture Wieler et al., 2011). Increased antimicrobial resistance devel- environments (Cabello, 2006; Smith et al., 2009; Ndi and Barton, opment and spread in companion animals due to irrational 2012). Because stress lowers the function of the immune system antimicrobial usage, especially overprescribed broad spectrum in animals, antimicrobials are seen as especially useful in intensive antimicrobials without precise diagnostics, inevitably cause (1) confinements of animals (Halverson, 2000). The non-therapeutic animal health problem (increased mortality and morbidity), (2) use of antimicrobials involves low-level exposure through feed economical problem to the owner (more visits-therapies and over long periods—an optimal way in which to enrich resistant prolonged hospitalization), (3) economical problem to the vet- bacterial populations (Sharma et al., 2008; Kohanski et al., 2010; erinarian (possible loss of customers and high costs for hospital Alexander et al., 2011; Gullberg et al., 2011). decontamination), and (4) human health problems (risks of Various monitoring programs around the world have started zoonotic transmission). Because of this threat small animal veteri- monitoring AMR and a range of research activities and inter- narians should prescribe broad spectrum antimicrobials after cul- ventions have shown that antimicrobial usage has a large effect turing and educate pet owners to handle infected-antimicrobial upon selection of AMR in animal production. A rapid and par- treated animals with precaution. allel decrease in resistant Enterococcus faecium from pig and However, emergences of resistance toward antimicrobials poultry has been reported in Denmark after the ban of growth which are critically important for human therapy are the most promoters in livestock (Aarestrup et al., 2001). The Norwegian worrisome. These include the recent emergence of ESBL pro- aquaculture industry has produced over one million tons of ducing and carbapenemase positive Enterobacteriaceae bacteria in farmed fish (http://www.ssb.no/fiskeoppdrett_en/)byusingonly animal production (Horton et al., 2011), the emergence of farm 649 kg of antimicrobials in 2011 (NORM/NORM-VET, 2011). associated MRSA ST398 (the main pig associated clone) (Cuny It is evident from the Danish integrated AMR monitoring and et al., 2010; Kluytmans, 2010; Weese, 2010) and of plasmid- research program (DANMAP) and NORM/NORM-VET Report mediated quinolone resistance in animal isolates and food prod- (NORM/NORM-VET, 2011) that reduction of antimicrobial ucts (Poirel et al., 2005; Nordmann et al., 2011). Unfortunately, usage with strict policies may still be the safest way to control the there are several examples in the literature that show that these development and spread of AMR in this sector in the future. are already widespread in Europe and other parts of the world and have a large impact on human health (Angulo et al., 2004; ANTIMICROBIAL-RESISTANT BACTERIA IN COMPANION ANIMALS Heuer et al., 2009; Forsberg et al., 2012). AND ANIMAL HUSBANDRY Aquaculture (fish, shellfish, and shrimp farming industries) The use of antimicrobials in animal husbandry has for many has developed rapidly in the last decade and has become an years actively selected for bacteria which possess genes capable important food source (FAO, 2010). Fish pathogenic bacteria of conferring AMR (Bastianello et al., 1995; Sundin et al., 1995). often produce devastating infections in fish farms where dense Consequently, this aspect has also seen much attention in the populations of fish are intensively reared. Although modern literature (Figure 2). Despite large differences in methodology, fish farming relies increasingly on vaccination and improved the results of most relevant scientific studies demonstrate that management to avoid infections (Markestad and Grave, 1997; not long after the introduction of antimicrobials in veterinary Midtlyng et al., 2011), still many bacterial infections in fish practice, resistance in pathogenic bacteria, and/or the fecal flora are regularly treated with antimicrobials in medicated feed or was observed (Caprioli et al., 2000; Jean-Louis et al., 2000). In by bath immersion. The most widely used drugs are fluoro- particular an increased emergence of pathogenic bacteria resis- quinolones, florfenicol, oxytetracyclines amoxicillin and sulfon- tant to antimicrobials has occurred in members of the genera amides (Cabello, 2006; Gräslund et al., 2003; Holmström et al., Salmonella, Campylobacter, Listeria, Staphylococcus, Enterococcus, 2003; Primavera, 2006; Soonthornchaikul and Garelick, 2009). and Escherichia coli. Some resistant strains of these genera are By now, most of the fish pathogenic bacteria from fish farms propagated primarily among animals but can subsequently infect with a history of infections have developed AMR (Colquhoun people as zoonotic agents (Levy, 1984; Corpet, 1988; Marshall et al., 2007; Lie, 2008; Sørum, 2008; Farmed Fish Health Report, et al., 1990; Giguêre et al., 2007). 2010; Shah et al., 2012a). Furthermore, in some areas of the In veterinary medicine the use of antimicrobials in compan- world, particularly in South-East Asia, integrated farming is a ion animals such as pets and horses is restricted to therapeutic common practice where organic wastes from poultry and live- purposes only. Companion animals are increasingly treated as stock are widely used in manuring the fish farms (Hoa et al., family members, in the context of applying advanced antimi- 2011; Shah et al., 2012b). It has been reported that antimicrobial crobial treatments to their infectious diseases. For instance, skin residues present in the poultry and livestock waste has pro- infections caused by staphylococci in dogs with or without vided sufficient selection pressure for the selection of AMR genes,

www.frontiersin.org May2013|Volume4|Article96| 14 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

increasing the complexity of transmission of bacteria, and resis- harbor-resistant bacteria (Zhang et al., 2009; Glad et al., 2010; tances between the livestock and aquatic environment (Petersen Lang et al., 2010). et al., 2002; Agersø and Petersen, 2007; Hoa et al., 2011; Shah et al., 2012b). ANTIMICROBIAL RESISTANCE IN THE ENVIRONMENT AMR has been detected in different aquatic environments MICROBIAL COMMUNITIES IN SOIL AND ANTIMICROBIAL and some resistance determinants have been found to originate RESISTANCE from aquatic bacteria. A good example is the recently emerging Research data shows that in diverse soils from various regions of plasmid-mediated quinolone resistance determinants from the the world, there is a wide dispersion of AMR. One explanation qnr family (Ash et al., 2002; Picao et al., 2008)andCTX-Mfrom for this phenomenon is the existence of antimicrobial produc- aquatic Kluyvera spp. (Decousser et al., 2001; Rodriguez et al., ing bacteria in soil. The Actinomycetes, which are common 2004; Ma et al., 2012). In addition, epidemiological and molecular soil bacteria (Streptomyces, Micromonospora, Saccharopolyspora data indicate that some fish pathogens such as Aeromonas are able genus), synthesize over half of all known most clinically rele- to transmit and share AMR determinants with bacteria isolated vant antimicrobials e.g., tetracycline, gentamicin, erythromycin, from humans such as E. coli (Rhodes et al., 2000; Sørum, 2006). streptomycin, vancomycin, and amphotericin. Bacteria of the Similarly, the fish pathogen Yersinia ruckeri have been reported to genus Bacillus also produce antibiotics, e.g., Bacillus brevis which share AMR plasmid and AMR genes with the bacterium causing producing gramicidin (Baltz, 2007). These antimicrobials now human plague (Welch et al., 2007). also reach the environment from human and animal therapeutics, through manure, sewage, agriculture, etc. Many retrospective ANTIMICROBIAL USE IN PLANT AGRICULTURE and prospective studies have been performed to assess the emer- Streptomycin and oxytetracycline are routinely used for the pro- gence and selection of AMR in environmental bacteria. The phylaxis of fire blight disease (causative agent Erwinia amylovora) environment is eventually the largest and most ancient reservoir in apple and pear orchards. Streptomycin use is strictly controlled of potential AMR, constituting the environmental “resistome” within the EU and is only authorized for use on a yearly basis. (Aminov and Mackie, 2007; Allen et al., 2010; D’Costa et al., However, streptomycin use in plant agriculture in the USA has 2011). Under such powerful selection pressure, it is not surpris- been replaced by oxytetracycline, due to streptomycin resistance ing that the soil resistome is so diverse (Knapp et al., 2010). development among E. amylovora in the apple orchards. Oxolinic The best example illustrating this is the tetracycline resistome. acid had been reported to be used in Israel against fire blight and Tetracyclines are an important class of antimicrobials with desir- against rice blight in Japan (Shtienberg et al., 2001). Gentamicin able broad-spectrum activity against numerous pathogens and is used in Mexico and Central America to control Fire Blight and the widespread emergence of resistance has had a massive impact various diseases of vegetable crops (Stockwell and Duffy, 2012). on these drugs (Thaker et al., 2010). Opportunistic pathogens However, the role of antimicrobial use on plants is, knowing the ubiquitous in the soil for example, Pseudomonas aeruginosa, AMR crisis in human medicine, the subject of debate (McManus Acinetobacter spp.,Burkholderiaspp., and Stenotrophomonas spp. et al., 2002). can combine intrinsic resistance to several antimicrobials with a remarkable capacity to acquire new resistance genes (Popowska DISSEMINATION OF ANTIMICROBIAL-RESISTANT BACTERIA et al., 2010). Still, little is known about the diversity, distribution, THROUGH FOOD AND FOOD PRODUCTION and origins of resistance genes, particularly among the as yet non- Resistant bacteria can be transferred from animals and plants cultivable environmental bacteria. In uncultured soil bacteria, to humans in many different ways, which can be catego- identified resistance mechanisms comprise efflux of tetracycline rized into three major modes of transmission: (1) through the and inactivation of aminoglycoside antimicrobials by phosphory- food chain to people (Roe and Pillai, 2003; Soonthornchaikul lation and acetylation (Popowska et al., 2012). In addition, bacte- and Garelick, 2009), (2) through direct or indirect contact ria resistant to macrolides including the new drug telithromycin with livestock industry or animal health workers (Levy et al., have been reported from soil (Riesenfeld et al., 2004a). In a study 1976), (3) through environments which are contaminated by (D’Costa et al., 2006), 480 strains of Streptomyces from soil with manure in agriculture (http://ec.europa.eu/environment/ were screened against 21 antimicrobials. Most strains were found integration/research/newsalert/pdf/279na4.pdf) and aquaculture to be multi-drug resistant to seven or eight antimicrobials on (Petersen et al., 2002; Shah et al., 2012b). The environment con- average, with two strains being resistant to 15 of the 21 drugs. It tains a great variety of bacteria creating an immense pool of AMR was also reported that soil is a reservoir for β-lactamases and these genes that are available for transfer to bacteria that cause human genes, if transferred to pathogens, can then impact human health disease (Riesenfeld et al., 2004b; D’Costa et al., 2006). The real- (Allen et al., 2010). It is supposed that the presence of antibiotics ization of these links sparked the recent interest in the role and in the environment has promoted the acquisition or independent dynamics of environmental AMR (Figure 2). evolution of highly specific resistance elements. These determi- In addition, other sources are available. For instance, wild nants are located mainly on mobile genetic elements such as animals may also be carriers of antimicrobial-resistant bacte- plasmids and conjugative transposons, which ensure their spread ria (Literak et al., 2011). These animals may have close contact by horizontal gene transfer. Conjugative, broad-host-range plas- to human or farming areas and/or waste and become colo- mids play a key role in this process (Martinez, 2009; Stokes nized with resistant strains (Literak et al., 2011; Nkogwe et al., and Gillings, 2011). Numerous studies have demonstrated that 2011). Interestingly, animals in remote areas have been found to the prevalence of such resistance plasmids in soil is very high

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy May2013|Volume4|Article96| 15 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

(Götz et al., 1996). Among the plasmids conferring resistance to sewage treatment plants (Segura et al., 2009; Michael et al., 2013). antimicrobials, representatives of the incompatibility groups P, Although proportion of the antimicrobial compounds are trans- Q, N, and W have been identified. An example of this type of formed and degraded in the environment, the occurrence of mobile genetic elements may be the IncP-1 plasmids (Popowska these micropollutants is reported worldwide, with antimicrobials and Krawczyk-Balska, 2013). Results from the scientific literature of all classes being detected in wastewater habitats in concen- show that plasmids carrying resistance genes have been identi- trations ranging from ng−1 to mgL−1 (Michael et al., 2013). fied in pathogenic bacteria of the genus Escherichia, Salmonella, Simultaneously, urban sewage and wastewater contain AMR bac- Shigella, Klebsiella, Aeromonas, and Pseudomonas,thegenerathat teria and other pollutants, such as pharmaceutical and personal can be found in soil and water (Stokes and Gillings, 2011). hygiene products and heavy metals, whose effects on AMR selec- These plasmids carry determinants for resistance to at least one tion are still not very clear (Graham et al., 2011; Oberlé et al., heavy metal (Ni, Cd, Co, Cu, Hg, Pb, Zn) and antimicrobials 2012; Patra et al., 2012; Novo et al., 2013). Often, wastewater of different groups, i.e., tetracyclines, quinolones, aminoglyco- treatment is not enough to eliminate the antimicrobial residues sides, sulfonamides, β-lactams, and chemotherapeutics (Sen et al., entering the system (Michael et al., 2013). The consequence 2011; Seiler and Berendonk, 2012). Overall these data indicate is that such micropollutants, exerting selective pressures, may that soil bacteria constitute a reservoir of resistance determinants facilitate the selection of AMR bacteria or the acquisition of that can be mobilized into the microbial community including resistance genes by horizontal gene transfer (Martinez, 2009). pathogenic bacteria. Recent studies also indicate a different mech- Indeed, the relevance of wastewater habitats to the dissemina- anism of AMR in soil-derived actinomycetes, by engendering tion of AMR among human pathogens as well as commensal mutations in genes encoding the transcriptional and translational and environmental bacteria is increasingly emphasized (Baquero apparatus that lead to alterations in global metabolism. This ver- et al., 2008; Marshall and Levy, 2011; Czekalski et al., 2012; Rizzo tically selected AMR includes increased production of secondary et al., 2013). Wastewater treatment plants reduce the load of AMR metabolites (Derewacz et al., 2013). Very recently evidence for bacteria, but treated water still carries elevated levels of AMR recent exchange of AMR genes between environmental bacteria bacteria, and may select for strains with high levels of multidrug- and clinical pathogens was presented using a high-throughput resistance (Czekalski et al., 2012). Resistance gene abundance in a functional metagenomic approach (Forsberg et al., 2012). In stream system could be linked to the input of (treated) wastewater this study it was shown that multidrug-resistant soil bacteria and animal husbandry, demonstrating landscape-scale pollution contain resistance gene cassettes against five classes of antimicro- of natural aquatic systems with AMR (Pruden et al., 2012). The bials (β-lactams, aminoglycosides, amphenicols, sulfonamides, currently available literature demonstrates that most of the AMR and tetracyclines) with high nucleotide identity to genes from genetic elements found in clinical isolates are also detected in diverse human pathogens. Therefore, it is important to study this wastewater habitats, even shortly after they have been reported reservoir, which may contribute to the detection of new clini- in hospitals (Szczepanowski et al., 2009; Rizzo et al., 2013). The cally relevant AMR-mechanisms and/or the multidrug-resistant occurrence of the same AMR genetic elements in different habi- pathogens that should be avoided from entering medically impor- tats demonstrates the uniqueness of the resistome, mainly due to tant bacteria (Torres-Cortés et al., 2011). rapid dissemination processes, demonstrating the urgent needs for an integrated approach. ANTIMICROBIAL RESISTANCE IN AQUATIC ENVIRONMENTS Ubiquitous bacteria that can live in the environment and Water is one of the most important habitats for bacteria, hold- are also able to colonize humans are particularly relevant to ing complex microbial communities. Not surprisingly, water the spread of AMR in the environment and the implications also contains AMR bacteria. From natural fresh water systems to human health. Indeed, numerous studies have reported to drinking water, or from sewage to human-engineered water the occurrence of AMR in ubiquitous bacteria isolated from infrastructures, AMR, either intrinsic or acquired, have been wastewater habitats, which are also recognized as opportunistic reported in aquatic environments worldwide (e.g., Goñi-Urriza pathogens, mainly nosocomial agents. AMR bacteria of clinical et al., 2000; Volkmann et al., 2004; Schwartz et al., 2006; Ferreira relevance which may be found in the environment comprise, da Silva et al., 2007; Böckelmann et al., 2009; Vaz-Moreira et al., among others, members of the genera Acinetobacter, Enterococcus, 2011; Falcone-Dias et al., 2012). In this respect, given their Escherichia, Klebsiella, Pseudomonas,andShigella (Blanch et al., characteristics, wastewater habitats are particularly important. 2003; Reinthaler et al., 2003; Ferreira da Silva et al., 2006, 2007; Watkinson et al., 2007; Novo and Manaia, 2010; Czekalski et al., WASTEWATER HABITATS AS RESERVOIRS OF ANTIMICROBIAL 2012). In addition, non-cultivable bacteria may also be important RESISTANCE either for AMR spread or selection. Indeed, over the last years, Among the aquatic environments, wastewater habitats repre- the use of culture independent approaches brought additional sent the most important reservoir of AMR bacteria and genes. insights into the abundance and diversity of resistance genes in This type of water contains human and animal excretions with wastewaters and into the effects of antimicrobials on the bacte- abundant doses of commensal and pathogenic antimicrobial- rial communities (Volkmann et al., 2004; Czekalski et al., 2012; resistant bacteria (Yang et al., 2011; Ye and Zhang, 2011, 2013; Oberlé et al., 2012; Novo et al., 2013). In particular, several stud- Novo et al., 2013). Since antimicrobials are not fully degraded ies presented evidence that in wastewater habitats there is a high in the human and animal body, antimicrobial compounds, their potential for horizontal gene transfer, mediated by plasmids and metabolites and transformation products are abundant in urban facilitated by integrons (Tennstedt et al., 2003; Szczepanowski

www.frontiersin.org May2013|Volume4|Article96| 16 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

et al., 2008; Moura et al., 2010; Zhang et al., 2011). Despite the Table 2 | Degradation rates of various antimicrobials in soil. importance of wastewater as a reservoir for AMR genes, and the Class of Degradation Time References relevance of wastewater treatment to control resistance spread, antimicrobial [%] [d] to date the number of studies that have been published remains relatively low (Figure 2). Macrolides 0–50 5–30 Thiele-Bruhn, 2003* Nevertheless, over the last decades the knowledge in this Sulfonamides 0–50 22–64 Thiele-Bruhn, 2003 area has increased considerably and the importance of wastew- Fluoroquinolones 0–30 56–80 Hektoen et al., 1995; ater treatment systems for the spread of AMR is unequivocally Thiele-Bruhn, 2003 demonstrated. Therefore, it is now possible to address some specific questions which we expect to be a focus of the research in Tetracycline 0–50 10–180 Björklund et al., 1991; this area in the coming years. Examples of these issues are (1) Thiele-Bruhn, 2003 the identification of the conditions that may enhance or mit- Aminoglycosides 0 30 Thiele-Bruhn, 2003 igate the occurrence of horizontal gene transfer and selection β-lactams 0–50 30 Thiele-Bruhn, 2003 of AMR (which pollutants, which concentrations, temperature, Imidasoles 50 14–75 Thiele-Bruhn, 2003 pH, hydraulic residence time of wastewater treatment, etc.); (2) the classification and quantification of risk, e.g., the likelihood Polypeptides 12–90 2–173 Thiele-Bruhn, 2003 that an AMR bacterium or gene from wastewater habitats reach ∗ humans and causes issues for human health; (3) the improvement This reference does not include the modern macrolides with very long of wastewater treatment processes in order to minimize the loads elimination half-lives. For instance, Tulathromycine has an half live (so 50% of antimicrobial-resistant bacteria and genes in the final effluent degraded, not nearly 100%) in soil of 99 days (Pfizer, personal communication (Dodd, 2012). 2013).

THE ANTIMICROBIAL RESISTANCE GENE POOL physicians in reducing antimicrobial overuse should be imple- AMR genes can be differentiated depending on the genetic event mented. Following the analysis more than 500 scientific articles, it that is required for acquiring an AMR phenotype. These include has been suggested that the elimination of non-therapeutic use of genes that are acquired by horizontal gene transfer and genes that antimicrobials in food animals, will lower the burden of AMR in are present in the bacterial genome and that can encode AMR the environment, with consequent benefits to human and animal following gene mutations or activation (Olliver et al., 2005). health (FAAIR Scientific, 2002; Swartz, 2002). AMR features evolve as a consequence of permanent exchange Better management techniques and strict legislation in the of and ever new recombinations of genes, genetic platforms, and use of antimicrobials for therapeutic use in humans and in ani- genetic vectors. Many of these genes are not primarily resistance mals will reduce the risk of development of AMR (Cunha, 2002; genes, but belong to the hidden resistome, the set of genes able to Defoirdt et al., 2011; Midtlyng et al., 2011). For example, the be converted into AMR genes (Baquero et al., 2009). As evidenced prevention of nosocomial transmission of multi-drug resistant by our discussion above, microbial organisms harboring these bacteriaispossiblewithactiveroutinesurveillanceprogramsthat genes are present naturally in all kinds of environments, but also can identify colonized patients. Numerous studies have demon- released into water and soil from organisms, including humans, strated that such a “search and containment” approach and/or where they evolve or increase in abundance under direct selection a “search and destroy” approach in which an attempt is made from exposure to antimicrobials. At the same time, antimicrobials to eliminate carriage of the organism can reduce the incidence (often at low concentrations), disinfectants, and heavy metals are of hospital-acquired infections and be cost-saving (Muto et al., disseminated into the water as well, and may act as selective fac- 2003). tors fostering the evolution of new AMR features (Cantas et al., New management techniques in the animal husbandry, such 2012a,b,c; Cantas et al., unpublished). The rate of degradation as organic farming, need to be thoroughly investigated to ensure of antimicrobials in the environment varies and is dependent on that these are viable alternatives that help to reduce the potential a range of environmental conditions, for example: temperature, for selection of AMR bacteria. Samples from organically farmed available oxygen, pH, presence of alternative sources of organic poultry showed a significantly lower level of AMR in intestinal and inorganic discharges as described in Table 2. bacteria such as E. coli and Campylobacter (Soonthornchaikul et al., 2006). However from organically farmed cattle no signifi- HOW TO SLOW DOWN THE SPREAD AND EVOLUTION OF AMR? cant differences were obtained in microbiological contamination. In this review we have emphasized that the problem of AMR E. coli and S. aureus isolates were found to have significantly lower evolution and dissemination is multifaceted and involves clinical, rates of AMR in organically raised cattle (Sato et al., 2005). More agricultural, technical, and environmental systems. Similarly studies are needed (1) to determine the reasons of antimicrobial strategies to deal with the impending AMR crisis have to take this usage in the farms by veterinarians, (2) to compare and update complexity into account. the recommended treatment protocols for veterinarians through- The overuse of antimicrobials needs to be limited or reduced out different countries, (3) to evaluate the impact of other factors in human medicine, veterinary medicine, agriculture, and aqua- other than AMR development in bacteria: e.g., immune response- culture. Ideally, the use of antimicrobials in agriculture should stress has been indicated to correlate with resistance genetic ele- be eliminated. Intensive programs to educate both patients and ment shuffling among gut microbiota in different animal models,

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy May2013|Volume4|Article96| 17 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

such as: atlantic salmon, zebrafish, neonatal piglets, and cats or contact with the environment. Introduction of antimicrobial (Cantas et al., 2011, 2012a,b,c, 2013). Animal welfare parameters compounds into the aquatic environment via medical therapy, under intensive production such as stress should be investigated agriculture, animal husbandry and companion animals has in future studies with regards to control of resistance development resulted in selective pressures on resident environmental bacteria. in animal husbandry. Development of AMR in environmental bacteria has a great Vaccination and improved hygienic measures are among the impact and may help in explaining how human and animal important cornerstones in controlling infectious diseases and pathogens acquire resistance features. Besides the role of clinical consequently aid in reducing AMR (Potter et al., 2008). The microbiology laboratories with rapid and accurate detection of Norwegian aquaculture may serve as a good example by reduc- a diverse number of pathogens and its drug resistance profiles, tion in the use of antimicrobials from around 50 tons in the late robust routine surveillances in an epidemiological frame-work 1980’s to less than 1000 kg per annum after introduction of effec- covering the whole livestock “food chain” and the environment tive vaccines against devastating fish diseases like furunculosis and need to be taken into consideration. Due to this complexity vibriosis (Midtlyng et al., 2011; NORM/NORM-VET, 2011). the control of AMR has to include numerous actions at diverse The use of pre- and probiotics to improve the health and levels. Future research should focus on finding unknown routes performance of livestock might be a good alternative to growth of transfer of AMR between microbiotas of relevance to the promoters. This is an important biological control aiming to food chain and to all microbiotas of importance for bacterial reduce outbreaks of infectious diseases and which in turn would pathogens when they acquire antibiotic resistance genes laterally. minimize the use of antimicrobials in livestock and aquaculture Ultimately, even a successful integrative approach on all aspects, for therapeutic purposes (Verschuere et al., 2000; Callaway et al., can probably only help to slow down the spread of AMR, not 2008). prevent it. The development of new generations of antimicrobial The issue of dissemination and possible long-term enrich- should therefore receive equal attention. This is summarized ment of AMR and AMR genes in the environment (Knapp and emphasized in a 12-point action plan against the rising et al., 2008) needs to be studied further, with specific regards threats of AMR implemented by the European Commission to the actual risks associated with it. However, taking action is which includes actions in the field of human medicine, animal already possible today. For example, several treatment methods husbandry, veterinary medicine, authorization requirements for waste and wastewater disinfection and removal of microp- for commercialization of human and veterinary drugs and ollutants, including antimicrobials, are available. These include other products, on research, on scientific opinions, and under- various chemical disinfections, UV treatment, and membrane fil- taking also actions on the international level in collabora- tration. Disinfection and DNA degradation of community based tion with the WHO and Codex (http://ec.europa.eu/dgs/health_ and hospital wastewater may be effective means to reduce AMR consumer/does/communication_amr_2011_748_en.pdf, Bush release, although more research is required to fully assess the inac- et al., 2011). tivation of resistance genes (i.e., DNA released from lysed cells The problem of AMR is widespread all over the world, there- that may be available for horizontal gene transfer) by these mea- fore it is not eradicable, but can be managed. Concerted efforts sures (Dodd, 2012). The combined removal of pollutants that between medical doctors, dentists, veterinarians, scientists, fun- are potential selective agents, disinfection, and deactivation of the ders, industry, regulators, and multi-disciplinary approaches are genetic material, may be a useful strategy to reduce the pollution needed to track resistance. Furthermore, global monitoring of of environments with resistance factors. the antimicrobial drug consumption in human and veterinary medicine and AMR, is an essential part of an overall strategy CONCLUSIONS to inform, educate and get commitment of all parties, includ- Inevitably, AMR in medicine has become common place. ing farmers and patients (American Academy of Microbiology, Bacteria have evolved multiple mechanisms for the efficient 2009). All these are important measures for the efficient future evolution and spread of AMR. Meanwhile the new developments use of antimicrobials in medicine. All members of society should of quick and adequate molecular diagnostic techniques for be conscious of their role and take on responsibility for main- the identification and epidemiological surveillance of genetic taining the effectiveness of current and future antimicrobials. We determinants of AMR in different hosts and in the environment believe that future interventions can be successful in minimizing will enhance the number of control options. We have outlined this problem. above a number of potential measures that are enabled by our improved ability to track AMR. However, there seems to be a AUTHOR CONTRIBUTIONS clear need for action and policy changes. This includes drug L. Cantas defined the review theme, established the interdis- licensing, financial incentives, penalties, and ban or restriction ciplinary coordination and the collaborations, designed the on use of certain drugs. Similarly, the prescriber behavior needs manuscript, contributed to the data collection, data analysis, to be altered. Animal health and hygiene needs to be improved. and drafting and writing of the manuscript. Syed Q. A. Shah, In addition, the implementation of microbiological criteria L. M. Cavaco, C. M. Manaia, F. Walsh, M. Popowska: draft- for the detection of certain types of resistant pathogens would ing, writing, and editing the manuscript. H. Garelick and H. be important to control the trade of both food animals and Bürgmann: contributed to data analysis, drafting, and writing the food products. The problem of AMR in human medicine will manuscript. H. Sørum: contributed to manuscript design, data not be solved if nothing is done to limit the constant influx of collection, data analysis, drafting, and writing the manuscript. All resistance genes into the human microbiota via the food chain authors have contributed to, seen and approved the manuscript.

www.frontiersin.org May2013|Volume4|Article96| 18 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

ACKNOWLEDGMENTS AND FUNDING responsible for the use which might be made of the information contained in this publication. The COST Office is not responsible for the external websites referred to in this publication. C. M. Manaia acknowledges National Funds from FCT— Fundação para a Ciência e a Tecnologia through projects PEst- OE/EQB/LA0016/2011 and PTDC/AAC-AMB/113840/2009. F. Walsh acknowledges funding from the Swiss Federal Office for All authors acknowledge DARE TD0803 (Detecting Agriculture, the Swiss Federal Office for the Environment and the Antimicrobial Resistance in Europe) COST Action. (http:// Swiss Expert Committee for Biosafety (SECB). Finally L. Cantas www.cost-dare.eu/). This publication is supported by COST. extend his thanks to MegaVet.no, for helpful support in reviewing Neither the COST Office nor any person acting on its behalf is the manuscript.

REFERENCES resistance genes. FEMS Microbiol. Baquero, F., Martinez, J.-L., and a growing problem for human Aarestrup, F. M. (2005). Veterinary Lett. 271, 147–161. Canton, R. (2008). Antibiotics and and animal health and for the drug usage and antimicrobial resis- Anderson, A. D., Nelson, J. M., antibiotic resistance in water envi- environment. Environ. Microbiol. 8, tance in bacteria of animal origin. Rossiter, S., and Angulo, F. J. ronments. Curr. Opin. Biotechnol. 1137–1144. Basic Clin. Pharmacol. Toxicol. 96, (2003). Public health consequences 19, 260–265. Callaway,T.R.,Edrington,T.S., 271–281. of use of antimicrobial agents in Barber, M., and Rozwadowska- Anderson, R. C., Harvey, R. B., Aarestrup, F. M., Seyfarth, A. M., food animals in the United States. Dowzenko, M. (1948). Infection by Genovese, K. J., Kennedy, C. N., Emborg, H. D., Pedersen, K., Microb. Drug Resist. 9, 373–379. penecillin-resistant staphylococci. et al. (2008). Probiotics, prebi- Hendriksen, R. S., and Bager, F. Angulo, F. J., Nargund, V. N., and Lancet 2, 641–644. otics and competitive exclusion for (2001). Effect of abolishment of Chiller, T. C. (2004). Evidence Bastianello, S. S., Fourie, N., Prozesky, prophylaxis against bacterial dis- the use of antimicrobial agents for of an association between use L.,Nel,P.W.,andKellermann, ease. Anim. Health Res. Rev. 9, growth promotion on occurrence of anti-microbial agents in food T. S. (1995). Cardiomyopathy of 217–225. of antimicrobial resistance in fecal animals and anti-microbial resis- ruminants induced by the litter Cantas, L., Fraser, T. W. K., Fjelldal, P. Enterococci from food animals tance among bacteria isolated from of poultry fed on rations con- G., Mayer, I., and Sørum, H. (2011). in Denmark. Antimicrob. Agents humans and the human health taining the ionophore antibiotic, The culturable intestinal micro- Chemother. 45, 2054–2059. consequences of such resistance. maduramicin.2. macropathology biota of triploid and diploid juve- Abraham, E. P., and Chain, E. (1940). J. Vet. Med. B 51, 374–379. and histopathology. Onderstepoort nile Atlantic salmon (Salmo salar)— An enzyme from bacteria able to Anthony, F., Acar, J., Franklin, A., J. Vet. Res. 62, 5–18. a comparison of composition and destroy penicillin. Nature 146, 837. Gupta, R., Nicholls, T., Tamura, Y., Björklund, H. V., Råbergh, C. M. I., drug resistance. BMC Vet. Res. 7:71. Agersø, Y., and Petersen, A. (2007). The et al. (2001). Antimicrobial resis- and Bylund, G. (1991). Residues of doi: 10.1186/1746-6148-7-71 tetracycline resistance determinant tance: responsible and prudent use oxolinic acid and oxytetracycline in Cantas, L., Le Roux, F., Mazel, D., Tet 39 and the sulphonamide resis- of antimicrobial agents in veteri- fish and sediments from fish farms. and Sørum, H. (2012a). Impact tance gene sulII are common among nary medicine. Rev. Sci. Tech. 20, Aquaculture 97, 85–96. of Antibiotics on the Expression resistant Acinetobacter spp. iso- 829–839. Blanch,A.R.,Caplin,J.L.,Iversen, of the tra Genes and on the Host lated from integrated fish farms in Arya, S. C., and Agarwal, N. (2011). A., Kühn, I., Manero, A., Taylor, Innate Immune Gene Activity Thailand. J. Antimicrob. Chemother. International travel with acquisition H. D., et al. (2003). Comparison during SXT Element Bearing 59, 23–27. of multi-drug resistant Gram neg- of enterococcal populations related Aeromonas salmonicida Infection in Alanis, A. J. (2005). Resistance to ative bacteria containing the New to urban and hospital wastewater Atlantic Salmon (Salmo salar L.). antibiotics: are we in the post- Delhi metallo-beta-lactamase gene, in various climatic and geographic PSN-ICAAC12PS1-B-1330. San antibiotic era? Arch. Med. Res. 36, bla(NDM-1). Travel Med. Infect. European regions. J. Appl. Microbiol. Francisco, CA. 697–705. Dis. 9, 47–48. 94, 994–1002. Cantas, L., Torp, J. R., Alestrøm, P., Alexander, T. W., Yanke, J. L., Reuter, Ash,R.J.,Mauck,B.,andMorgan, Böckelmann,U.,Dorries,H.H., Sørum, H. (2012b). Cultureable gut T.,Topp,E.,Read,R.R.,Selinger,B. M. (2002). Antibiotic resistance of Ayuso-Gabella, M. N., Marçay, M. microbiota diversity in zebrafish. L., et al. (2011). Longitudinal char- gram-negative bacteria in rivers, S., Tandoi, V., Levantesi, C., et al. Zebrafish 9, 26–37. acterization of antimicrobial resis- United States. Emerg. Infect. Dis. 8, (2009). Quantitative PCR monitor- Cantas, L., Midtlyng, P. J., and tance genes in feces shed from cattle 713–716. ing of antibiotic resistance genes Sørum, H. (2012c). Impact of fed different subtherapeutic antibi- Baltz, R. H. (2007). Antimicrobials and bacterial pathogens in three antibiotic treatments on the otics. BMC Microbiol. 11:19. doi: from actinomycetes: back to the European artificial groundwater expression of the R plasmid tra 10.1186/1471-2180-11-19. future. Microbe 2, 125–131. recharge systems. J. Appl. Environ. genes and on the host innate Allen,H.K.,Donato,J.,Wang,H. Bannoehr, J., Ben Zakour, N. L., Waller, Microbiol. 75, 154–163. immune activity during pRAS1 H., Cloud-Hansen, K. A., Davies, J., A. S., Guardabassi, L., Thoday, K. L., Brown, S., Bantar, C., Young, H. K., and bearing Aeromonas hydrophila and Handelsman, J. (2010). Call of van den Broek, A. H., et al. (2007). Amyes, S. G. B. (1998). Limitation infection in zebrafish (Danio the wild: antibiotic resistance genes Population genetic structure of the of Acinetobacter baumannii treat- rerio). BMC Microbiol. 12:37. doi: in natural environments. Nat. Rev. Staphylococcus intermedius group: ment by plasmid-mediated car- 10.1186/1471-2180-12-37 Microbiol. 8, 251–259. insights into agr diversification bapenemase ARI-2. Lancet 351, Cantas, L., Thoresen, I. S., Gjermund, American Academy of Microbiology. and the emergence of methicillin- 186–187. G., Sivertsen, T., Framstad, (2009). “Antibiotic resistance: an resistant strains. J. Bacteriol. 189, Bush, K., Courvalin, P., Dantas, G., T., and Sørum, H. (2013). ecological perspective on an old 8685–8692. Davies, J., Eisenstein, B., Huovinen, “Antibiotikabehandling av problem,” in Report of a colloquium, Baquero, F., Alvarez-Ortega, C., and P., et al. (2011). Tackling antibiotic kolidiaré hos spedgris med mini- 12 to 14 October 2008 (Annecy, Martinez, J. L. (2009). Ecology resistance. Nat. Rev. Microbiol. 9, mal antibiotikaresistensutvikling— France). and evolution of antibiotic resis- 894–896. en modellstudie (in Norwegian),” Aminov, R., and Mackie, R. I. (2007). tance. Environ. Microbiol. Rep.1, Cabello, F. C. (2006). Heavy use of pro- in Husdyrforsøksmøte 2013 - UMB Evolution and ecology of antibiotic 469–476. phylactic antibiotics in aquaculture: (Lillestrøm).

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy May 2013 | Volume 4 | Article 96 | 19 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

Caprioli, A., Busani, L., Martel, antibiotic resistome. Science 311, Como-Sabetti, K., et al. (2010). Gustafson, R. H., and Bowen, R. E. J. L., and Helmuth, R. (2000). 374–377. Excess costs and utilization asso- (1997). Antibiotic use in animal Monitoring of antibiotic resistance Decousser, J. W., Poirel, L., ciated with methicillin resistance agriculture. J. Appl. Microbiol. 83, in bacteria of animal origin: epi- and Nordmann, P. (2001). for patients with Staphylococcus 531–541. demiological and microbiological Characterization of a chromoso- aureus infection. Infect. Control Halverson, M. (2000). The Price We Pay methodologies. Int. J. Antimicrob. mally encoded extended-spectrum Hosp. Epidemiol. 31, 365–373. for Corporate Hogs. Minneapolis, Agents 14, 295–301. class A β-lactamase from Kluyvera Fischer, J., Rodriguez, I., Schmoger, MN: Institute for Agriculture and Casewell, M., Friis, C., Marco, E., cryocrescens. Antimicrob. Agents S., Friese, A., Roesler, U., Helmuth, Trade policy, 32. McMullin, P., and Phillips, I. Chemother. 45, 3595–3598. R., et al. (2012). Escherichia coli Hammerum, A. M., Heuer, O. E., (2003). The European ban on Defoirdt, T., Sorgeloos, P., and Bossier, producing VIM-1 carbapenemase Emborg,H.D.,Bagger-Skjot,L., growth-promoting antibiotics and P. (2011). Alternatives to antibiotics isolated on a pig farm. J. Antimicrob. Jensen, V. F., Rogues, A. M., et al. emerging consequences for human for the control of bacterial disease in Chemother. 67, 1793–1795. (2007). Danish integrated antimi- and animal health. J. Antimicrob. aquaculture. Curr. Opin. Microbiol. Forsberg, K. J., Reyes, A., Wang, B., crobial in resistance monitoring and Chemother. 52, 159–161. 14, 251–258. Selleck, E. M., Sommer, M. O. A., research program. Emerg. Infect. Cattoir, V., Poirel, L., Mazel, D., Soussy, Dodd, M. C. (2012). Potential impacts and Dantas, G. (2012). The shared Dis. 13, 1632–1639. C. J., and Nordmann, P. (2007). of disinfection processes on antibiotic resistome of soil bacteria Hektoen, H., Berge, J. A., Hormazabal, Vibrio splendidus as the source elimination and deactivation of and human pathogens. Science 337, V., and Yndestad, M. (1995). of plasmid-mediated qnrS-Like antibiotic resistance genes during 1107–1111. Persistence of antibacterial agents in quinolone resistance determinants. water and wastewater treatment. Giguêre, S., Prescott, J. F., Baggot, J. marine sediments. Aquaculture 133, Antimicrob. Agents Chemother. 51, J. Environ. Monit. 14, 1754–1771. D., Walker, R. D., and Dowling, P. 175–184. 2650–2651. Derewacz, D. K., Goodwin, C. M. (2007). Antimicrobial Therapy in Heuer, O. E., Kruse, H., Grave, K., Cheng, A. C., Turnidge, J., Collignon, R., McNees, C. R., McLean, Veterinary Medicine.Ames;Oxford: Collignon, P., Karunasagar, I., and P.,Looke,D.,Barton,M.,and J. A., and Bachmann, B. O. Iowa State University Press. Angulo, F. J. (2009). Human health Gottlieb, T. (2012). Control of fluo- (2013). Antimicrobial drug resis- Glad, T.,Bernhardsen, P.,Nielsen, K. consequences of use of antimicro- roquinolone resistance through suc- tance affects broad changes in M., Brusetti, L., Andersen, M., Aars, bial agents in aquaculture. Clin. cessful regulation, Australia. Emerg. metabolomic phenotype in addi- J., et al. (2010). Bacterial diversity Infect. Dis. 49, 1248–1253. Infect. Dis. 18, 1453–1460. tion to secondary metabolism. in faeces from polar bear (Ursus Hoa, P. T. P., Managaki, S., Nakada, N., Colquhoun, D. J., Aarflot, L., and Proc. Natl. Acad. Sci. U.S.A. 110, maritimus)inArcticSvalbard. Takada, H., Shimizu, A., Anh, D. H., Melvold, C. F. (2007). gyrA and 2336–2341. BMC Microbiol. 10:10. doi: 10.1186/ et al. (2011). Antibiotic contamina- parC mutations and associated FAAIR Scientific. (2002). Select find- 1471-2180-10-10 tion and occurrence of antibiotic- quinolone resistance in Vibrio ings and conclusions. Clin. Infect. Goñi-Urriza, M., Capdepuy, M., Arpin, resistant bacteria in aquatic envi- anguillarum serotype O2b strains Dis. 34, S73–S75. C., Raymond, N., Caumette, P., and ronments of northern Vietnam. Sci. isolated from farmed Atlantic Falagas, M. E., and Bliziotis, I. A. Quentin, C. (2000). Impact of an Total Environ. 409, 2894–2901. cod (Gadus morhua)inNorway. (2007). Pandrug-resistant Gram- urban effluent on antibiotic resis- Holmberg, S. D., Solomon, S. L., and Antimicrob. Agents Chemother. 51, negative bacteria: the dawn of tance of riverine Enterobacteriaceae Blake, P. A. (1987). Health and 2597–2599. the post-antibiotic era? Int. J. and Aeromonas spp. J. Appl. Environ. economic impacts of antimicro- Corpet, D. E. (1988). Antibiotic resis- Antimicrob. Agents 29, 630–636. Microb. 66, 125–132. bial resistance. Rev. Infect. Dis. 9, tance from food. N.Engl.J.Med. Falcone-Dias, M. F., Vaz-Moreira, I., Götz, A., Pukall, R., Smit, E., Tietze, 1065–1078. 318, 1206–1207. and Manaia, C. M. (2012). Bottled E., Prager, R., Tschäpe, H., et al. Holmström, K., Gräslund, S., Crofton, J., and Mitchison, D. A. mineral water as a potential source (1996). Detection and characteri- Wahlström, A., Poungshompoo, S., (1948). Streptomycin resistance in of antibiotic resistant bacteria. zation of broad-host range plas- Bengtsson, B. E., and Kautsky, N. pulmonary tuberculosis. Br. Med. J. Water Res. 46, 3612–3622. mids in environmental bacteria by (2003). Antibiotic use in shrimp 2, 1009–1015. FAO. (2010). The State of the PCR. Appl. Environ. Microbiol. 62, farming and implications for envi- Cunha, B. A. (2002). Strategies to World Fisheries and Aquaculture. 2621–2628. ronmental impacts and human control antibiotic resistance. Semin. Rome: United Nations Food and Graham, D. W., Olivares-Rieumont, S., health. Int. J. Food Sci. Technol. 38, Respir. Infect. 17, 250–258. Agriculture Organization. Knapp, C. W., Lima, L., Werner, D., 255–266. Cuny, C., Friedrich, A., Kozytska, Farmed Fish Health Report. (2010). and Bowen, E. (2011). Antibiotic Horton,R.A.,Randall,L.P.,Snary,E. S., Layer, F., Nubel, U., Ohlsen, “Norwegian Veterinary Institute,” in resistance gene abundances asso- L., Cockrem, H., Lotz, S., Wearing, K., et al. (2010). Emergence of The Health Situation in Norwegian ciated with waste discharges to H., et al. (2011). Fecal carriage methicillin-resistant Staphylococcus Aquaculture 2010, eds G. Bornø the Almendares River near Havana, and shedding density of CTX- aureus (MRSA) in different animal andC.Sviland(Oslo:Norwegian Cuba. Environ. Sci. Technol. 45, M extended-spectrum β-lactamase- species. Int. J. Med. Microbiol. 300, Veterinary Institute). 418–424. producing Escherichia coli in cat- 109–117. Ferreira da Silva, M., Tiago, I., Gräslund, S., Holmström, K., and tle, chickens, and pigs: implications Czekalski, N., Berthold, T., Caucci, Verissimo, A., Boaventura, R. A. Wahlström, A. (2003). A field survey for environmental contamination S., Egli, A., and Burgmann, H. R., Nunes, O. C., and Manaia, C. of chemicals and biological prod- and food production. Appl. Environ. (2012). Increased levels of multire- M. (2006). Antibiotic resistance of ucts used in shrimp farming. Mar. Microbiol. 77, 3715–3719. sistant bacteria and resistance genes enterococci and related bacteria Pollut. Bull. 46, 81–90. Jean-Louis,M.,Tardy,F.,Brisabois,A., after wastewater treatment and their in an urban wastewater treatment Grave,K.,Torren-Edo,J.,andMackay, Lailler, R., Coudert, M., Chaslus- dissemination into Lake Geneva, plant. FEMS Microbiol. Ecol. 55, D. (2010). Comparison of the sales Dancla, E. (2000). The French Switzerland. Front. Microbiol. 3:106. 322–329. of veterinary antibacterial agents antibiotic resistance monitoring doi: 10.3389/fmicb.2012.00106 Ferreira da Silva, M., Vaz-Moreira, I., between 10 European countries. programs. Int. J. Antimicrob. Agents D’Costa, V. M., King, C. E., Kalan, Gonzalez-Pajuelo, M., Nunes, O. J. Antimicrob. Chemother. 65, 14, 275–283. L.,Morar,M.,Sung,W.W.L., C., and Manaia, C. M. (2007). 2037–2040. Klugman, K. P. (2002). Bacteriological Schwarz, C., et al. (2011). Antibiotic Antimicrobial resistance patterns in Gullberg, E., Cao, S., Berg, O. G., Ilback, evidence of antibiotic failure in resistance is ancient. Nature 477, Enterobacteriaceae isolated from an C., Sandegren, L., Hughes, D., et al. pneumococcal lower respiratory 457–461. urban wastewater treatment plant. (2011). Selection of resistant bacte- tract infections. Eur. Respir. J. 20, D’Costa, V. M., McGrann, K. M., FEMS Microbiol. Ecol. 60, 166–176. ria at very low antibiotic concentra- 3S–8S. Hughes, D. W., and Wright, Filice,G.A.,Nyman,J.A.,Lexau, tions. PLoS Pathog. 7:e1002158. doi: Kluytmans, J. A. J. W. (2010). G. D. (2006). Sampling the C.,Lees,C.H.,Bockstedt,L.A., 10.1371/journal.ppat.1002158 Methicillin-resistant Staphylococcus

www.frontiersin.org May2013|Volume4|Article96| 20 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

aureus in food products: cause for Lie, Ø. (2008). Improving Farmed Fish Norwegian aquaculture. Aquac. Res. Appl. Microbiol. Biotechnol. 87, concern or case for complacency? Quality and Safety. Woodhead, 42, 28–34. 1157–1166. Clin. Microbiol. Infect. 16, 11–15. Publishing Series in Food Science, Moura, A., Henriques, I., Smalla, K., Oberlé, K., Capdeville, M., Berthe, Knapp, C. W., Dolfing, J., Ehlert, P. Technology and Nutrition No. 162. and Correia, A. (2010). Wastewater T., and Petit, F. (2012). Evidence A., and Graham, D. W. (2010). E-ISBN: 978-1-84569-492-0. bacterial communities bring for a complex relationship between Evidence of increasing antibiotic Linton,K.B.,Lee,P.A.,Rowland, together broad-host range plas- antibiotics and antibiotic-resistant resistance gene abundances in A.J.,Baker,V.N.,Gillespi,W. mids, integrons and a wide diversity Escherichia coli: from medical cen- archived soils since 1940. Environ. A., and Richmond, M. H. (1972). of uncharacterized gene cassettes. ter patients to a receiving envi- Sci. Technol. 44, 580–587. Antibiotic resistance and transmis- Res. Microbiol. 161, 58–66. ronment. Environ. Sci. Technol. 46, Knapp, C. W., Engemann, C. A., sible R-factors in intestinal Coliform Muto,C.A.,Jernigan,J.A.,Ostrowsky, 1859–1868. Hanson,M.L.,Keen,P.L., flora of healthy adults and chil- B. E., Richet, H. M., Jarvis, W. R., Olarte, J. (1983). Antibiotic resistance Hall, K. J., and Graham, D. W. dren in an urban and a rural Boyce, J. M., et al. (2003). SHEA in Mexico. APUA Newslett. 1, 3. (2008). Indirect evidence of community. J. Hyg. (Lond.) 70, guideline for preventing nosocomial Olliver, A., Vallê, M., Chaslus-Dancla, transposon-mediated selection 99–104. transmission of multidrug- E., and Cloeckaert, A. (2005). of antibiotic resistance genes in Literak, I., Petro, R., Dolejska, M., resistant strains of Staphylococcus Overexpression of the multidrug aquatic systems at low-level oxyte- Gruberova, E., Dobiasova, H., Petr, aureus and Enterococcus. Infect. efflux operon acrEF by insertional tracycline exposures. Environ. Sci. J., et al. (2011). Antimicrobial resis- Control Hosp. Epidemiol. 24, activation with IS1 or IS10 elements Technol. 42, 5348–5353. tance in fecal Escherichia coli iso- 362–386. in Salmonella enterica Serovar Kohanski, M. A., DePristo, M. A., lates from healthy urban children Ndi, O., and Barton, M. (2012). Typhimurium DT204 acrB mutants and Collins, J. J. (2010). Sublethal of two age groups in relation to “Antibiotic resistance in animals— selected with fluoroquinolones. antibiotic treatment leads to their antibiotic therapy. Antimicrob. The Australian perspective,” in Antimicrob. Agents Chemother. 49, multidrug resistance via radical- Agents Chemother. 55, 3005–3007. Antimicrobial Resistance in the 289–301. induced mutagenesis. Mol. Cell 37, Ma,J.,Liu,J.H.,Lv,L.,Zong,Z., Environment, eds P. L. Keen and M. Otter,J.A.,andFrench,G.L. 311–320. Sun, Y., Zheng, H., et al. (2012). H. H. M. Montforts (New Jersey, (2010). Molecular epidemiol- Korczak, D., and Schöffmann, C. Characterization of extended- NJ: Wiley-Blackwell), 265–290. ogy of community-associated (2010). Medical and health eco- spectrum β-lactamase genes among Nkogwe, C., Raletobana, J., Stewart- meticillin-resistant Staphylococcus nomic evaluation of prevention- Escherichia coli isolates from duck Johnson, A., Suepaul, S., and aureus in Europe. Lancet Infect. Dis. and control measures related to and environment samples in a duck Adesiyun, A. (2011). Frequency 10, 227–239. MRSA infections or -colonisations farm. Appl. Environ. Microbiol. 78, of detection of Escherichia Pallett, A., and Hand, K. (2010). at hospitals. GMS Health Technol. 3668–3673. coli, Salmonella spp., and Complicated urinary tract infec- Assess. 6, 1–9. Markestad, A., and Grave, K. (1997). Campylobacter spp. in the fae- tions: practical solutions for the Kumarasamy, K. K., Toleman, M. A., Reduction of antibacterial drug use cesofwildrats(Rattusspp.)in treatment of multiresistant Gram- Walsh, T. R., Bagaria, J., Butt, F., in Norwegian fish farming due to Trinidad and Tobago. Vet. Med. negative bacteria. J. Antimicrob. Balakrishnan, R., et al. (2010). vaccination. Dev. Biol. Stand. 90, Int. 2011:686923. doi: 10.4061/ Chemother. 65, 25–33. Emergence of a new antibiotic 365–369. 2011/686923 Patra, S., Das, T. K., Avila, C., Cabello, resistance mechanism in India, Marshall,B.,Petrowski,D.,andLevy, Nordmann, P., Dortet, L., and Poirel, V., Castillio, F., Sarkar, D., et al. Pakistan, and UK: a molecular, S. B. (1990). Inter- and intraspecies L. (2012). Carbapenem resistance (2012). Cadmium tolerance and biological, and epidemiologi- spread of Escherichia coli in a in Enterobacteriaceae: here is antibiotic resistance in Escherichia cal study. Lancet Infect. Dis. 10, farm environment in the absence of the storm! Trends Mol. Med. 18, coli isolated from waste stabiliza- 597–602. antibiotic usage. Proc. Natl. Acad. 263–272. tion ponds. Indian J. Exp. Biol. 50, Lang,K.S.,Anderson,J.M.,Schwarz, Sci. U.S.A. 87, 6609–6613. Nordmann, P., Poirel, L., Toleman, M. 300–307. S., Williamson, L., Handelsman, J., Marshall, B. M., and Levy, S. B. (2011). A., and Walsh, T. R. (2011). Does Petersen, A., Andersen, J. S., Kaewmak, and Singer, R. S. (2010). Novel flor- Food animals and antimicrobials: broad-spectrum β-lactam resistance T., Somsiri, T., and Dalsgaard, fenicol and chloramphenicol resis- impacts on human health. Clin. due to NDM-1 herald the end of A. (2002). Impact of integrated tance gene discovered in Alaskan Microbiol. Rev. 24, 718–733. the antibiotic era for treatment of fish farming on antimicrobial soil by using functional metage- Martinez, J. L. (2009). Environmental infections caused by Gram-negative resistance in a pond environ- nomics. Appl. Environ. Microbiol. pollution by antibiotics and by bacteria? J. Antimicrob. Chemother. ment. Appl. Environ. Microbiol. 68, 76, 5321–5326. antibiotic resistance determinants. 66, 689–692. 6036–6042. Levy, S. B. (1984). “Antibiotic resis- Environ. Pollut. 157, 2893–2902. NORM/NORM-VET. (2011). Usage of Picao, R. C., Poirel, L., Demarta, A., tant bacteria in food of man and McEwen, S. A. (2006). Antibiotic use Antimicrobial Agents and Occurrence Silva, C. S. F., Corvaglia, A. R., animals,” in Antimicrobials Ans in animal agriculture: what have we of Antimicrobial Resistance in Petrini, O., et al. (2008). Plasmid- Agriculture,edM.Woodbine learned and where are we going? Norway. Tromsø/Oslo 2012. mediated quinolone resistance in (London: Butterworths), 525–531. Anim. Biotechnol. 17, 239–250. ISSN:1502-2307 (print)/1890- Aeromonas allosaccharophila recov- Levy, S. B. (1998). The challange of McManus, P. S., Stockwell, V. O., 9965 (electronic). (Tromsø/Oslo: ered from a Swiss lake. J. Antimicrob. antibiotic resistance. Sci. Am. 278, Sundin,G.W.,andJones,A.L. Norwegian Veterinary Institute). Chemother. 62, 948–950. 46–53. (2002). Antibiotic use in plant Available online at: http://www. Poirel, L., Liard, A., Rodriguez- Levy, S. B. (2001). Antibiotic resis- agriculture. Annu. Rev. Phytopathol. vetinst.no/eng/Publications/Norm- Martinez,J.M.,andNordmann,P. tance: consequences of inaction. 40, 443–465. Norm-Vet-Report/Norm-Norm-Vet- (2005). Vibrionaceae as a possible Clin. Infect. Dis. 33, 124–129. Michael, I., Rizzo, L., McArdell, C. S., rapporten-2010 source of Qnr-like quinolone resis- Levy, S. B., FitzGerald, G. B., and Manaia, C. M., Merlin, C., Schwartz, Novo, A., André, S., Viana, P., Nunes, tance determinants. J. Antimicrob. Macone, A. B. (1976). Changes in T., et al. (2013). Urban wastewa- O. C., and Manaia, C. M. (2013). Chemother. 56, 1118–1121. intestinal flora of farm personnel ter treatment plants as hotspots for Antibiotic resistance, antimicrobial Popowska, M., and Krawczyk-Balska, after introduction of a tetracycline- the release of antibiotics in the envi- residues and bacterial community A. (2013). Broad-Host-Range IncP- supplemented feed on a farm. N. ronment: a review. Water Res. 47, composition in urban wastewater. 1 plasmids and their resistance Engl.J.Med.295, 583–588. 957–995. Water Res. 47, 1875–1887. potential. Front. Microbiol. 4:44. doi: Levy, S. B., and Marshall, B. (2004). Midtlyng,P.J.,Grave,K.,andHorsberg, Novo, A., and Manaia, C. M. (2010). 10.3389/fmicb.2013.00044 Antibacterial resistance worldwide: T. E. (2011). What has been done Factors influencing antibiotic Popowska, M., Miernik, A., Rzeczycka, causes, challenges and responses. to minimize the use of antibac- resistance burden in municipal M., and Lopaciuk, A. (2010). Nat. Med. 10, 122–129. terial and antiparasitic drugs in wastewater treatment plants. The impact of environmental

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy May2013|Volume4|Article96| 21 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

contamination with antibiotics for antibiotic resistant bacteria and in FAO Fisheries technical paper 469 Sørum, H. (2008). “Antibiotic resis- on levels of resistance in soil genes spread into the environment: (Rome; United Nations), 89. tance associated with veterinary bacteria. J. Environ. Qual. 39, areview.Sci. Total Environ. 447, Available online at: ftp://ftp.fao.org/ drug use in fish farms,” in Improving 1679–1687. 345–360. docrep/fao/009/a0282e/a0282e00. Farmed Fish Quality and Safety, Popowska, M., Rzeczycka, M., Miernik, Roberts, R. R., Hota, B., Ahmad, I., pdf ed ø. Lie (Cambridge: WoodHead A., Krawczyk-balska, A., Walsh, Scott, R. D., Foster, S. D., Abbasi, Shah, S. Q. A., Karatas, S., Nilsen, Publishing Limit), 157–182. F., and Duffy, B. (2012). Influence F., et al. (2009). Hospital and soci- H., Steinum, T. M., Colquhoun, Stockwell, V. O., and Duffy, B. of soil use on prevalence of etal costs of antimicrobial-resistant D. J., and Sørum, H. (2012a). (2012). Use of antibiotics in tetracycline, streptomycin, and ery- infections in a Chicago teaching Characterization and expression of plant agriculture. Rev. Sci. Tech. 31, thromycin resistance and associated hospital: implications for antibiotic the gyrA gene from quinolone resis- 199–210. resistance genes. Antimicrob. Agents stewardship. Clin. Infect. Dis. 49, tant Yersinia ruckeri strains iso- Stokes,H.W.,andGillings,M.R. Chemother. 56, 1434–1443. 1175–1184. lated from Atlantic salmon (Salmo (2011). Gene flow, mobile genetic Potter, A., Gerdts, V., and Littel-Van Rodriguez, M. M., Power, P., Radice, salar L.) in Norway. Aquaculture elements and the recruitment of den Hurk, S. V. (2008). Veterinary M., Vay, C., Famiglietti, A., Galleni, 350–353, 37–41. antibiotic resistance genes into vaccines: alternatives to antibiotics? M., et al. (2004). Chromosome- Shah, S. Q. A., Colquhoun, D. J., Gram negative pathogens. FEMS Anim. Health Res. Rev. 9, 187–199. encoded CTX-M-3 from Kluyvera Nikuli, H. L., and Sørum, H. Microbiol. Rev. 35, 790–819. Pouillard, J. (2002). A forgotten dis- ascorbata: a possible origin of (2012b). Prevalence of antibiotic Strahilevitz, J., Jacoby, G., Hooper, covery: doctor of medicine Ernest plasmid-borne CTX-M-1-derived resistance genes in the bacterial D. C., and Robicsek, A. (2009). Duchesne, s thesis (1874-1912) cefotaximases. Antimicrob. Agents flora of integrated fish farming Plasmid-mediated quinolone resis- [Article in French]. Hist.Sci.Med. Chemother. 48, 4895–4897. environments of Pakistan and tance: a multifaceted threat. Clin. 36, 11–20. Roe, M. T., and Pillai, S. D. (2003). Tanzania. Environ. Sci. Technol. 46, Microbiol. Rev. 22, 664–689. Pratt, R. (2010). Preparation for a post Monitoring and identifying antibi- 8672–8679. Sundin, G. W., Monks, D. E., and antibiotic era must start now. Nurs. otic resistance mechanisms in bac- Sharma, R., Munns, K., Alexander, Bender, C. L. (1995). Distribution Times 106, 26. teria. Poult. Sci. 82, 622–626. T., Entz, T., Mirzaagha, P., Yanke, of the streptomycin-resistance Primavera, J. H. (2006). Overcoming Roura, E., Homedes, J., and Klasing, K. L. J., et al. (2008). Diversity and transposon Tn5393 among the impacts of aquaculture on the C. (1992). Prevention of immuno- distribution of commensal fecal phylloplane and soil bacte- coastal zone. Ocean Coast. Manage. logic stress contributes to the Escherichia coli bacteria in beef cat- ria from managed agricultural 49, 531–545. growth-permitting ability of dietary tle administered selected subthera- habitats. Can. J. Microbiol. 41, Pruden, A., Arabi, M., and Storteboom, antibiotics in chicks. J. Nutr. 122, peutic antimicrobials in a feedlot 792–799. H. N. (2012). Correlation between 2383–2390. setting. Appl. Environ. Microbiol. 74, Swann, M. M. (1969). Use of Antibiotics upstream human activities and Sato, K., Bartlett, P. C., and Saeed, 6178–6186. in Animal Husbandry and Veterinary riverine antibiotic resistance M. A. (2005). Antimicrobial sus- Shtienberg, D., Zilberstaine, M., Medicine (Swann Report).London: genes. Environ. Sci. Technol. 46, ceptibility of Escherichia coli isolates Oppenheim, D., Herzog, Z., HMSO. 11541-11549. from dairy farms using organic ver- Manulis, S., Shwartz, H., et al. Swartz, M. N. (2002). Human diseases Rassow, D., and Schaper, H. (1996). sus conventional production meth- (2001). Efficacy of oxolinic acid caused by foodborne pathogens of The use of feed medications in ods. J.Am.Vet.Med.Assoc.226, and other bactericides in suppres- animal origin. Clin. Infect. Dis. 34, swine and poultry facilities in 589–594. sion of Erwinia amylovora in pear S111–S122. the Weser-Ems region [Article Schwartz, T., Volkmann, H., Kirchen, orchards in Israel. Phytoparasitica Szczepanowski, R. H., Carpenter, in German]. Dtsch. Tierarztl. S., Kohnen, W., Schön-Hölz, K., 29, 143–154. M. A., Czapinska, H., Zaremba, Wochenschr. 103, 244–249. Jansen, B., et al. (2006). Real- Smith, P. R., Le Breton, A., Horsberg, M., Tamulaitis, G., Siksnys, V., Reinthaler, F. F., Posch, J., Feierl, G., time PCR detection of Pseudomonas T. E., and Corsin, F. (2009). et al. (2008). Central base pair Wüst, G., Haas, D., Ruckenbauer, aeruginosa in clinical and munic- “Guidelines for antimicrobial flipping and discrimination by G., et al. (2003). Antibiotic resis- ipal wastewater and genotyping use in aquaculture,” in Guide to PspGI. Nucleic Acids Res. 36, tance of E. coli in sewage and sludge. of the ciprofloxacin-resistant iso- Antimicrobial Use in Animals, 6109–6117. Water Res. 37, 1685–1690. lates. FEMS Microbiol. Ecol. 57, eds L. Guardabassi, L. B. Jensen, Szczepanowski, R., Linke, B., Krahn, Rhodes, G., Huys, G., Swings, J., 158–167. and H. Kruse (Oxford: Blackwell I., Gartemann, K.-H., Gutzkow, McGann, P., Hiney, M., Smith, Segura,P.A.,Francois,M.,Gagnon, Publishing Ltd.), 207–218. T., Eichler, W., et al. (2009). P., et al. (2000). Distribution of C., and Sauve, S. (2009). Review Soonthornchaikul, N., Garelick, H., Detection of 140 clinically relevant oxytetracycline resistance plasmids of the occurrence of anti-infectives Jones,H.,Jacobs,J.,Ball,D.,and antibiotic- resistance genes in the between Aeromonads in hospital in contaminated wastewaters Choudhury, M. (2006). Resistance plasmid metagenome of wastewater and aquaculture environments: and natural and drinking waters. to three antimicrobial agents of treatment plant bacteria showing implication of Tn1721 in dissemi- Environ. Health Perspect. 117, Campylobacter isolated from inten- reduced susceptibility to selected nation of the tetracycline resistance 675–684. sively and organically reared chick- antibiotics. Microbiology 155, determinant Tet, A. Appl. Environ. Seiler, C., and Berendonk, T. U. ens purchased from retail out- 2306–2319. Microbiol. 66, 3883–3890. (2012). Heavy metal driven lets. Int. J. Antimicrob. Agents 27, Tablan, O. C., Anderson, L. J., Besser, Riesenfeld, C. S., Goodman, R. M., and co-selection of antibiotic resis- 125–130. R., Bridges, C., and Hajjeh, R. Handelsman, J. (2004a). Uncultured tance in soil and water bodies Soonthornchaikul, N., and Garelick, (2004). Guidelines for preventing soil bacteria are a reservoir of new impacted by agriculture and aqua- H. (2009). Antimicrobial resistance health-care-associated pneumonia, antibiotic resistance genes. Environ. culture. Front. Microbiol. doi: of Campylobacter species isolated 2003: recommendations of CDC Microbiol. 6, 981–989. 10.3389/fmicb.2012.00399 from edible bivalve molluscs pur- and the Healthcare Infection Riesenfeld, C. S., Schloss, P. D., Sen, D., Van Der Auwera, G., Rogers, chased from Bangkok markets, Control Practices Advisory and Handelsman, J. (2004b). L.,Thomas,C.M.,Brown,C.J., Thailand. Foodborne Pathog. Dis. 6, Committee. MMWR Recomm. Metagenomics: genomic analysis of and Top, E. M. (2011). Broad- 947–951. Rep. 53, 1–36. microbial communities. Annu. Rev. host-range plasmids from agricul- Sørum, H. (2006). “Antimicrobial drug Tennstedt, T., Szczepanowski, R., Genet. 38, 525–552. tural soils have IncP-1 backbones resistance in fish pathogens,” in Braun, S., Puhler, A., and Schluter, Rizzo, L., Manaia, C. M., Merlin, C., with diverse accessory genes. Appl. Antimicrobial Resistance in Bacteria A. (2003). Occurrence of integron- Schwartz, T., Dagot, D., Ploy, M. Environ. Microbiol. 77, 7975–7983. of Animal Origin,edF.M.Aarestrup associated resistance gene cassettes C., et al. (2013). Urban wastew- Serrano, P. H. (2005). “Responsible (Washington, DC: ASM Press), located on antibiotic resistance ater treatment plants as hotspots use of antibiotics in aquaculture,” 213–238. plasmids isolated from a wastewater

www.frontiersin.org May 2013 | Volume 4 | Article 96 | 22 Cantas et al. The problem of antimicrobial resistance: a multi-disciplinary review

treatment plant. FEMS Microbiol. municipal wastewater using real- zoonotic pathogens in clinical Q. Q., et al. (2009). Phenotypic Ecol. 45, 239–252. time PCR (TaqMan). J. Microbiol. samples. Int. J. Med. Microbiol. 301, and genotypic characterisation of Thaker, M., Spanogiannopoulos, P., Methods 56, 277–286. 635–641. antimicrobial resistance in faecal and Wright, G. D. (2010). The tetra- Walsh, T. R., Weeks, J., Livermore, D. Wilke, M. H. (2010). Multiresistant bacteria from 30 Giant pandas. cycline resistome. Cell. Mol. Life Sci. M., and Toleman, M. A. (2011). bacteria and current therapy—the Int. J. Antimicrob. Agents 33, 67, 419–431. Dissemination of NDM-1 positive economical side of the story. Eur. J. 456–460. Thiele-Bruhn, S. (2003). Pharma- bacteria in the New Delhi envi- Med. Res. 15, 571–576. ceutical antibiotic compounds in ronment and its implications for Yang,C.,Zhang,W.,Liu,R.,Li,Q., Conflict of Interest Statement: The soils—a review. J. Plant Nutr. Soil human health: an environmental Li, B., Wang, S., et al. (2011). authors declare that the research Sci. 166, 145–167. point prevalence study. Lancet Phylogenetic diversity and was conducted in the absence of any Torres-Cortés, G., Millan, V., Ramirez- Infect. Dis. 11, 355–362. metabolic potential of activated commercial or financial relationships Saad, H.C., Nisa-Martinez, R., Toro, Watanabe, T. (1963). Infective hered- sludge microbial communities in that could be construed as a potential N., and Martinez-Abarca, F. (2011). ity of multiple drug resistance in full-scale wastewater treatment conflict of interest. Characterization of novel antibi- bacteria. Bacteriol. Rev. 27, 87–115. plants. Environ. Sci. Technol. 45, otic resistance genes identified by Watkinson, A. J., Micalizzi, G. R., 7408–7415. Received: 13 December 2012; paper functional metagenomics on soil Bates, J. R., and Costanzo, S. D. Ye, L., and Zhang, T. (2011). Pathogenic pending published: 04 January 2013; samples. Environ. Microbiol. 13, (2007). Novel method for rapid bacteria in sewage treatment plants accepted: 04 April 2013; published 1101–1114. assessment of antibiotic resistance as revealed by 454 pyrosequenc- online: 14 May 2013. Vaz-Moreira, I., Nunes, O. C., and in Escherichia coli isolates from ing. Environ. Sci. Technol. 45, Citation: Cantas L, Shah SQA, Cavaco Manaia, C. M. (2011). Diversity environmental waters by use of a 7173–7179. LM, Manaia CM, Walsh F, Popowska and antibiotic resistance pat- modified chromogenic agar. Appl. Ye, L., and Zhang, T. (2013). Bacterial M, Garelick H, Bürgmann H and Sørum terns of Sphingomonadaceae Environ. Microbiol. 73, 2224–2229. communities in different sections of H (2013) A brief multi-disciplinary isolated from drinking water. Weese, J. S. (2010). Methicillin-resistant a municipal wastewater treatment review on antimicrobial resistance in Appl. Environ. Microbiol. 77, Staphylococcus aureus in animals. plant revealed by 16S rDNA 454 medicine and its linkage to the global 5697–5706. ILAR J. 51, 233–244. pyrosequencing. Appl. Microbiol. environmental microbiota. Front. Vazquez-Moreno, L., Bermudez, A., Welch, T. J., Fricke, W. F., McDermott, Biotechnol. 97, 2681–2690. Microbiol. 4:96. doi: 10.3389/fmicb. Langure, A., Higuera-Ciapara, I., P. F., White, D. G., Rosso, M. Yoon, J. W., Lee, K. J., Lee, S. Y., Chae, 2013.00096 Diaz De Aguayo, M., and lores, L., Rasko, D. A., et al. (2007). M.J.,Park,J.K.,Yoo,J.H.,etal. This article was submitted to Frontiers E. (1990). Antibiotic residues and Multiple antimicrobial resistance (2010). Antibiotic resistance profiles in Antimicrobials, Resistance and drug resistant bacteria in beef, and in plague: an emerging public of Staphylococcus pseudintermedius Chemotherapy, a specialty of Frontiers in chicken tissues. J. Food Sci. 55, health risk. PLoS ONE 2:e309. doi: isolates from Canine patients in Microbiology. 632–634. 10.1371/journal.pone.0000309 Korea. J. Microbiol. Biotechnol. 20, Copyright © 2013 Cantas, Shah, Verschuere, L., Rombaut, G., Sorgeloos, Wieler, L. H., Ewers, C., Guenther, 1764–1768. Cavaco, Manaia, Walsh, Popowska, P., and Verstraete, W. (2000). S., Walther, B., and Lübke-Becker, Zhang,T.,Zhang,X.-X.,andYe, Garelick, Bürgmann and Sørum. This is Probiotic bacteria as biological A. (2011). Methicillin-resistant L. (2011). Plasmid metagenome an open-access article distributed under control agents in aquaculture. staphylococci (MRS) and reveals high levels of antibiotic the terms of the Creative Commons Microbiol. Mol. Biol. Rev. 64, extended-spectrum beta- resistance genes and mobile genetic Attribution License,whichpermitsuse, 655–671. lactamases (ESBL)-producing elements in activated sludge. PLoS distribution and reproduction in other Volkmann, H., Schwartz, T., Bischoff, Enterobacteriaceae in companion ONE 6:e26041. doi: 10.1371/ forums, provided the original authors P., Kirchen, S., and Obst, U. (2004). animals: nosocomial infections journal.pone.0026041 and source are credited and subject to Detection of clinically relevant as one reason for the rising Zhang, A. Y., Wang, H. N., Tian, any copyright notices concerning any antibiotic-resistance genes in prevalence of these potential G. B., Zhang, Y., Yang, X., Xia, third-party graphics etc.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy May2013|Volume4|Article96| 23 REVIEW ARTICLE published: 06 March 2013 doi: 10.3389/fmicb.2013.00015 Antibiotic resistance shaping multi-level population biology of bacteria

Fernando Baquero1,2,3*, Ana P.Tedim1,2 andTeresa M. Coque1,2,3 1 Department of Microbiology, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain 2 Centros de Investigación Biomédica en Red de Epidemiología y Salud Pública, Madrid, Spain 3 Unidad de Resistencia a Antibióticos y Virulencia Bacteriana asociada al Consejo Superior de Investigaciones Cientíﬁcas, Madrid, Spain

Edited by: Antibiotics have natural functions, mostly involving cell-to-cell signaling networks. The Fiona Walsh, Agroscope Changins anthropogenic production of antibiotics, and its release in the microbiosphere results in Wädenswil, Switzerland a disturbance of these networks, antibiotic resistance tending to preserve its integrity.The Reviewed by: cost of such adaptation is the emergence and dissemination of antibiotic resistance genes, Jose L. Martinez, Centro Nacional de Biotecnología, Spain and of all genetic and cellular vehicles in which these genes are located. Selection of the Andres Moya, University of Valencia, combinations of the different evolutionary units (genes, integrons, transposons, plasmids, Spain cells, communities and microbiomes, hosts) is highly asymmetrical. Each unit of selection *Correspondence: is a self-interested entity, exploiting the higher hierarchical unit for its own benefit, but in Fernando Baquero, Department of doing so the higher hierarchical unit might acquire critical traits for its spread because of the Microbiology, Hospital Universitario Ramón y Cajal, Instituto Ramón y exploitation of the lower hierarchical unit. This interactive trade-off shapes the population Cajal de Investigación Sanitaria, biology of antibiotic resistance, a composed-complex array of the independent “population Carretera de Colmenar Viejo, km. biologies.” Antibiotics modify the abundance and the interactive field of each of these 9.100, 28034 Madrid, Spain. e-mail: [email protected] units. Antibiotics increase the number and evolvability of “clinical” antibiotic resistance genes, but probably also many other genes with different primary functions but with a resistance phenotype present in the environmental resistome. Antibiotics influence the abundance, modularity, and spread of integrons, transposons, and plasmids, mostly acting on structures present before the antibiotic era. Antibiotics enrich particular bacterial lineages and clones and contribute to local clonalization processes. Antibiotics amplify particular genetic exchange communities sharing antibiotic resistance genes and platforms within microbiomes. In particular human or animal hosts, the microbiomic composition might facilitate the interactions between evolutionary units involved in antibiotic resistance. The understanding of antibiotic resistance implies expanding our knowledge on multi-level population biology of bacteria.

Keywords: antibiotics, resistance, population biology, multi-level selection, evolution, evolvability, resistome, microbiome

Antibiotics produced by natural organisms play a role in their 2012). It is of note that natural antibiotic production, decreasing interactions shaping the lifestyle and homeostasis of bacterial pop- the growth rate of the competing population, not only restricts ulations and communities (Waksman, 1961; Davies, 2006; Fajardo its local predominance, but also assures that this population is and Martínez, 2008; Aminov, 2009). Such interactions might be preserved, as antibiotic-promoted cessation of growth is a highly of antagonistic nature as the production of antibiotics serves to effective system to avoid antibiotic killing. inhibit other bacterial populations. Inhibition does not necessarily However, the production of natural antibiotics might only have intend to kill competitive bacterial organisms, but rather prevent functions unrelated with inter-bacterial antagonisms. Antagonism undesirable local overgrowth of partners in shared ecosystems. might arise in particular contexts as a side-effect of cell-to-cell The diffusion of antibiotics in the environment assures an “exclu- signaling effects resulting in self-regulation of growth, viru- sive zone” at a certain distance from the producer population. At lence, sporulation, motility, mutagenesis, SOS stress response, the borders of such a zone, the potentially competing organisms phage induction, transformation, lateral gene transfer, intra- are confronted with very low antibiotic concentrations, proba- chromosomal recombination, or bioﬁlm formation (Goh et al., bly sufﬁcient to decrease their growth rate, but not to kill the 2002; Ubeda et al., 2005; Linares et al., 2006; Yim et al., 2007; competing neighbor. In this sense, it is highly possible that the Martínez, 2008; Couce and Blázquez, 2009; Kohanski et al., 2010; production of antagonistic (allelopathic) substances by microor- Allen et al., 2011; Baharoglu and Mazel, 2011; Pedró et al., 2011; ganisms has more a defensive than offensive nature (Chao and Looft and Allen, 2012; Looft et al., 2012). Levin, 1981). In addition, mutual inhibition is frequently desir- Natural antibiotic resistance modulates the effect of the natural able for the maintenance of healthy species diversity in a particular production of antibiotics, so antibiotic production and antibiotic ecosystem (Czárán et al., 2002; Becker et al., 2012; Cordero et al., resistance act as two complementary sides of the same process

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 24

“fmicb-04-00015” — 2013/3/5 — 11:06 — page1—#1 Baquero et al. Multi-level population biology of resistance

assuring the homeostasis of microbial populations and commu- anthropogenic action, greatly exceeding the amount of natu- nities. In fact, communities with a cohesive habitat association ral antibiotics signaling and controlling the homeostasis of the might act as units in terms of antibiotic production and resistance. bacterial world. Moreover, the amount of antimicrobials of In these clusters, antibiotics are frequently produced by few bac- anthropogenic origin entering into the environment assures the terial organisms, whereas other members of the club are resistant presence of every possible antibiotic concentration in contact (Cordero et al., 2012). with bacteria. The consequences of such an extensive release of As in the case of natural antibiotic production, natural antibi- inhibitory and regulatory molecules have an important impact on otic resistance might not only focus on“defense against antibiotics” the population biology and evolutionary biology of bacteria. or“self-protection”in antibiotic producers. In fact, this“defense”is frequently a side-effect of other functions of the“natural resistance POPULATION BIOLOGY OF THE UNITS OF SELECTION mechanisms,” including nutrition, metabolism, detoxification of The units of selection define the evolutionary individuals (Lewon- noxious substances, and catabolic processes (Dantas et al., 2008; tin, 1970; Brandon, 1987; Mayr, 1997; Okasha, 2004; Dupré and Martínez, 2008; Alvarez-Ortega et al., 2011; Martínez and Rojo, O’Malley, 2007; Lloyd, 2008; Doolittle and Zhaxybayeva, 2010; 2011; Qu and Spain, 2011). Baquero, 2011; Rodriguez-Valera and Ussery, 2012). But what The so-called “intrinsic resistome,” the ensemble of non- is selected in the case of antibiotic resistance? Possible units of acquired genes and functions normally present in bacterial cells selection in antibiotic resistance are discrete genetic sequences, which influence the susceptibility to antibiotics, might account for genes, operons, functional genetic modules, mobile genetic ele- 3% of the bacterial genome (Fajardo et al., 2008). Obviously, such ments (MGEs) as integrons, transposons, integrative–conjugative a huge number of“defensive”genes reflects the unspecific nature of elements (ICEs), plasmids, or at the cellular and supra-cellular their functions in which antibiotic resistance is concerned (Fajardo levels, genomes and cells (organisms), clones, clonal complexes, et al., 2008; Alvarez-Ortega et al., 2011). That does not mean that species, communities, and ecosystems. Note that all these pos- these genes were not submitted to horizontal gene transfer before sible units belong to different hierarchical levels, ranging from the crisis provoked by the industrial antibiotics pollution, illus- the relatively simple to the complex, as resistance genes are part trating that besides direct selection by clinical antibiotics other of integrons, integrons part of transposons, transposons part of factors contribute to dissemination and maintenance of antibiotic plasmids, plasmids part of cells, cells part of clones, clones part resistance genes in bacterial populations (Biel and Hartl, 1983; of species, and so on. Each unit is a “vessel” for the other(s), Aminov and Mackie, 2007; Mindlin et al., 2008; Allen et al., 2009; affecting not only its potential dissemination but also its rate of D’Costa et al.,2011). In any case, it has been recently suggested that introgressive descent and evolution (Doolittle and Zhaxybayeva, the close identity of resistance genes (and resistance platforms) 2010; Baquero, 2011; Bapteste et al., 2012; Cordero et al., 2012). from clinical strains and environmental strains might indicate The investigation of such a trans-hierarchical landscape clearly recent exchange events (Forsberg et al., 2012). From this perspec- requires a multi-level population genetic approach (Baquero and tive, natural antibiotics and antibiotic “resistance” mechanisms Coque, 2011; Day et al., 2011; Cordero et al., 2012). have a natural regulatory role in shaping both population biology What we propose in this work is essentially a mental heuristic and evolutionary biology of bacterial organisms. However, the exercise. Let us imagine that we are aware of a kind of replicators amount of active antibiotic determining this physiological natural called genes but we still ignore the existence of cells. We could “antibiotic environment”is extremely low (Halling-Sørensen et al., observe changes in the frequency and variety of genes, and we 1998). Of course antibiotic-producer organisms are endowed with might consider populations of genes, submitted to evolutionary “mechanisms for self-protection,” which have been considered dynamics and natural selection. If we were only conscious of the as the origin of modern functions involved in clinical antibiotic existence of transposons, we would establish population biology resistance (Benveniste and Davies, 1973). However, phylogenetic of transposons. If we considered plasmids, we would refer to the studies suggest that current clinical resistance genes are not found pan-plasmidome, the plasmid population harbored by a particular in antibiotic producers, and its emergence in clinical strains can- microbiome or a particular bacterial group (Fondi and Fani, 2010; not be explained by recent horizontal gene transfer from these Mizrahi, 2012). This would apply for every unit of selection. We organisms. Nevertheless, they might have been historically sub- would of course be able to observe changes in the abundance and mitted to duplications and frequent horizontal gene transfer, so variety of each unit involving a resistance trait as a consequence of that “modern” resistance genes might have evolved along com- the presence of antibiotics in the environment. Each unit of selec- plex evolutionary processes pre-existing the industrial release of tion is a self-interested entity (Rankin et al., 2011b) exploiting the antibiotics. In fact many identical “resistance genes” are found in higher hierarchical unit for its own benefit (resistance plasmids environmental and clinical organisms (Cantón et al., 1999; Fors- exploit successful bacterial clones), but the higher unit might berg et al., 2012). In any case, what we denominate “antibiotic acquire critical traits for its spread because of the exploitation resistance” for clinical microorganisms is extremely rare in nature; of the lower hierarchy unit (bacterial clones, bacterial communi- other kinds of “resistance”genes, those of the“intrinsic resistome,” ties, or microbiomes might be successful because of resistance able to protect cells from tiny concentrations of natural antibiotics, plasmids, ICEs, or elements within those; Castillo et al., 2005; dominate in the wild environments. Mizrahi, 2012; Novais et al., 2012b). This trade-off of interac- The main problem that we are examining in this manuscript tions shapes the global population biology of antibiotic resistance. derives from the huge escalation of the amount of antibi- In a certain sense, this multi-level perspective represents a “sec- otics released into the microbial environments as an effect of ond line of complexity” in the classic view of antibiotics-driven

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 15 | 25

“fmicb-04-00015” — 2013/3/5 — 11:06 — page2—#2 Baquero et al. Multi-level population biology of resistance

natural selection processes, from the selection of a resistant cell to If antibiotics have polluted the entire globe, including wild multi-level selections. Such processes should increase the absolute environments, specialized antibiotic resistance genes, identical to density (number) of all pieces involved in antibiotic resistance, those found in hospitals, can be found everywhere else, including and consequently might favor their interactions and emergence the most remote and wild regions (Gilliver et al., 1999; Osterblad of novel combinatorial patterns (Baquero, 2004). Prediction of et al., 2001; Sjölund et al., 2008; Allen et al., 2009; Quinteira et al., which pieces and patterns will evolve is a crucial issue for the 2011; Stalder et al., 2012). However, the variety of natural bacterial management of multiantibiotic resistance, and might be possible genes that can provide antibiotic resistance in a heterologous host if we have the right data (Martínez et al., 2007; Baquero, 2011; is much larger than that actually found in human pathogens (Dan- Partridge, 2011). tas et al.,2008). Why only a very small fraction of“resistance genes” present in the“global resistome”have entered in human pathogens ANTIBIOTICS AND POPULATIONS OF RESISTANCE GENES is a poorly addressed question. Of course genes from phyloge- Have antibiotics increased the abundance of highly effective resis- netically remote organisms should have functional connectivity tance genes in the bacterial world? Confronted with antibiotics, and concert with the host systems, and that certainly constitutes bacterial populations might adapt by selecting “more effective” an important bottleneck for their acceptance (Halary et al., 2010; mutants of wild genes endowed with other functions, but pro- Martínez, 2011). However, relatively “independent” functionally viding low level of resistance. Such process is favored by gene connected gene clusters (Zheng et al.,2005) determining resistance duplication, so that wild genes having a “small effect” on resis- might be better tolerated and eventually fixed (Popa et al., 2011). tance could increase in number to increase protection. It is of In any case, as stated recently (Skippington and Ragan, 2011), the note that this process might be much more frequent than muta- network and evolutionary dynamics that allow the stoichiometric tion (Näsvall et al., 2012). A high number of gene-copies might participation of horizontally transferred genes in cellular net- in fact transitorily accumulate during selection, producing a full works remains poorly addressed, even though new bioinformatic resistance phenotype. The question is if such gene duplications advances have recently been made available (Cohen et al., 2012). might contribute to the emergence of novel resistance genes. Once Considering potential sets of “acceptable” resistance genes able the permanence of a functional copy of a given gene is guar- to evolve in bacterial populations, eventually only the “fittest anteed, the second (or n-) copy has the evolutionary freedom genes” resulting from competition among genes might finally (liberation from purifying selection) to be modified, eventually reach high densities. Competition is expected to occur mostly leading to a variant or novel gene (Kondrashov et al., 2002; Näs- among orthologs or paralogs (for instance resulting from recent vall et al., 2012). It is of note that not all genes have equal chances duplications) occupying the same functional niches (Kondrashov of duplication, and certainly there are adaptive genes with a et al., 2002; Francino, 2012). Antibiotics could have enriched higher potential variability, containing highly variable regions the more efficient adaptive genes among competing genes (for interspersed among well-conserved, “segmentally variable genes,” instance the more detoxifying ones; Novais et al.,2010b). However, as ABC transporters involved in multidrug resistance (Zheng et al., the “fittest genes” are not necessarily those with the best intrinsic 2004). Mutational changes in genes, leading to novel resistance activity in terms of providing antibiotic resistance. Different resis- genes, are facilitated under circumstances of enhanced mutagen- tance genes impose different biological costs for the host strain. As esis in the host strain. Hyper-mutable bacteria (“mutators”) are it was stated above, successful novel resistance genes should be fit enriched in allelic variants of resistance genes, eventually provid- in a particular genetic context, that is, the epigenetic compatibility ing wider resistance spectrum, as in the case of beta-lactamases of a new gene with the host genome is critical in the acquisition (Baquero et al., 2005). Indeed organisms with enhanced muta- of resistance (Sánchez and Martínez, 2012). Such fitness bottle- tion rates (frequently involving failures in the mismatch repair neck will select, in combination with the detoxifying efficiency, system) see their possibility of survival increased, and inside the novel successful genes. these strains, other genes could be modified to provide antibi- Alternatively, the quantitative success of a particular gene (as otic resistance. Note that hyper-mutation and gene variation at blaTEM−1 in Escherichia coli,orblaZ in Staphylococcus aureus) large, might result from the effects of the antibiotic themselves might result only from a “founder effect” (Livermore, 2000; Mar- (Blázquez et al., 2012). tinez et al., 2009), that is, the first gene that by chance conferred Indeed, intra- and inter-bacterial gene movement and recom- a selective advantage in particular conditions was fixed and that binational events between genetic platforms contribute to the total resulted in a successful wide spread. This founder effect in human amount of resistance genes. In fact, the “biological success” of a and animal pathogens might have occurred because of multiple resistance gene is dependent on its wider genetic context (Walsh, selective events in environmental organisms exposed to dynamic 2006; Wozniak and Waldor, 2010; Bertels and Rainey, 2011). landscapes. In fact, founder effects are expected to occur in a con- Moreover, hybrid resistance genes resulting from recombinational tinuous and cumulative way (Aguilée et al., 2009). In turn, such events are not infrequent in nature (Goffin and Ghuysen, 1998; emergent events might have been facilitated by changes in envi- Maiden, 1998; Novais et al., 2012b). Under antibiotic exposure, ronmental conditions (as animal crowding in farms) resulting in bacterial pathogens in humans and animals (and commensals) local fluctuations in the size of particular bacterial populations, might fix and further refine acquired resistance genes originated thus favoring acquisition by lateral genetic transfer of adaptive in areas less exposed to antimicrobials, as in the soil (including the traits from environmental bacteria (Balloux, 2010). Similarly, the rhizosphere!), or water bodies (including sewage!) (Aminov, 2009; changes in the environment, the landscape dynamics, influence Lupo et al., 2012). the probability of founders fixation, as well as the possibilities

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 26

“fmicb-04-00015” — 2013/3/5 — 11:06 — page3—#3 Baquero et al. Multi-level population biology of resistance

for extinction and re-emergence (Aguilée et al., 2011). It is not the total amount of resistance genes should increase under selec- impossible that many resistance genes, even those rarely found tion. Interestingly, some “vehicles” (as MGEs are more frequently or never found in the clinical environment, could have also been associated with resistance genes than others. As complementary enriched by environmental antibiotic exposure (Martinez et al., explanations, we can recall here the founder effect (advantages for 2009; Sommer et al., 2009). the first gene-capturing MGEs), the influence of genes and plat- How can we explain that the same resistance gene of plausi- forms on the overall fitness of the recipient cells, or the higher ble environmental origin (as blaCTX−Ms or blaCMYs)appearsto prevalence of particular MGEs in the organisms more exposed to have been captured in separate events by different gene-capturing antibiotics, heavy metals, biocides, or other ecological stressors elements as ISEcp,ISCR1,orIS26?(Barlow and Hall, 2002; Tole- (Stokes and Gillings, 2011). man et al., 2006; Valverde et al., 2009; Partridge, 2011).Thereisa contemporary enrichment in organisms as Enterobacteriaceae of ANTIBIOTICS AND POPULATIONS OF RESISTANCE captured gene(s) with apparently the same function, and a bloom INTEGRONS of a diversity of new genes coding for resistance to beta-lactams Have antibiotics increased the abundance, in the microbial world, (Cantón and Coque, 2006; Coque et al., 2008; Poirel et al., 2012). of integrons able to capture resistance genes? Integrons possess This might be due to a dense interactive field resulting from an in a site-specific recombination system able to integrate, rearrange, the number of environmental species (donors) where it originated and express adaptive genes, including antibiotic resistance genes from, as well as an environmental increase in population density (Mazel, 2006; Partridge, 2011; Stokes and Gillings, 2011; Moura of a variety of “good recipients” as E. coli or Klebsiella pneumo- et al.,2012). These genetic platforms are ancient structures (several niae. Recipients increase could result from both the augmentation hundred million years old) that were already involved in the initial of the total number of Enterobacteriaceae in the gut microbiota outbreaks of antibiotic resistance in the 1950s (Liebert et al., 1999; of mammals, probably due to antibiotic exposure, and the mas- Mazel, 2006; Revilla et al., 2008). The same type of integrons, now sive release of human-and animal sewage to the environment (see carrying a diversity of antibiotic resistance genes, are preserved in later). the current bacterial world, and have installed themselves in nat- Other less-successful resistance or pre-resistance genes might ural environments along extended periods of time (Petrova et al., not be relevant in the clinical setting, but constitute an increasing 2011; Stokes and Gillings, 2011; Stalder et al., 2012). Integrons reservoir of unpredictable consequences, and undoubtedly might evolution often results in the local array of resistance genes, and influence the population ecology of bacteria. On the other hand, other genes of adaptive nature (Labbate et al., 2009; Moura et al., the selection of variant genes might occur at very low antibiotic 2012; Stalder et al., 2012), which increases the possibilities of their concentrations (Henderson-Begg et al., 2006; Gullberg et al., 2011) selective multiplication. particularly among natural concentration gradients (Baquero and In other words, exposure to antibiotics, biocides, or heavy met- Negri, 1997; Negri et al., 2000; Hermsen et al., 2012). It can be sug- als and a high multiplicity of other different environmental factors gested that the overall increase in the amount of resistance genes on results in an increase of cells containing integrons (Gaze et al., Earth also has a positive effect in maintaining the desirable home- 2005, 2011; Wright et al., 2008; Stalder et al., 2012). Moreover, ostasis of bacterial populations in a heavily antibiotic-polluted exposure to different antibiotics (aminoglycosides, beta-lactams, environment. fluoroquinolones, trimethoprim, metronidazole) facilitates exten- An interesting point in gene population biology is the question sive gene cassette recombination; occasionally involving the of why a number of resistance genes maintain their full sequence SOS-triggered IntI1 integrase over-expression (Guerin et al., 2009; integrity through myriads of replications in spite of an appar- Hocquet et al., 2012). Other recombinational events (often medi- ently insufficient level of antibiotic selection. Even considering that ated by ISs) influence shuffling of resistance genes contained in they are co-selected with genes under active selection (for instance different integrons, giving rise to multi-resistance regions (Par- being part of the collection of gene cassettes of an integron), their tridge, 2011) and further facilitating the evolution and selection functionality seems to be better preserved than could be expected. of the upper-level host vehicles (Domingues et al., 2012). In fact, This might suggest that the current function of a number of clas- integrons are not mobile, but are frequently associated with sic “resistance genes” is something other than antibiotic resistance transposons and/or plasmids and therefore should increase in (exaptation; Alonso and Gready,2006; Petrova et al.,2011; Sánchez abundance as a result of conjugation or transposition events medi- and Martínez, 2012). ated by Tn21-like and IS26-like elements. For instance class 1 Resistance genes tend to be collected in particular clones and integrons located in Tn402, which are often part of Tn501-like clustered in common genetic platforms (Partridge and Hall, 2004; transposons on conjugative plasmids, have greatly contributed to Ktari et al., 2006; Partridge, 2011; Potron et al., 2013), probably the spread of integrons among γ and β-proteobacteria (Tato et al., following the “genetic capitalism principle,”that is, the more resis- 2010; Partridge, 2011). tant clones and the most fit resistance-providing platforms are The frequent association of integrons with a variety of special- selected, and then their ability to acquire novel adaptive traits is ized DNA recombination systems enhances both transferability favored (Lawrence, 1997; Baquero, 2004). As we will see along this and genetic diversification. For instance, insertion sequences (IS) review, the extensive antibiotic-promoted selection of resistant of the IS110/IS492 family as IS4321 and IS5075 (members of bacterial organisms is a selection of the “vehicles” where antibi- IS1111 subgroup) target the terminal inverted repeats of Tn21 otic resistance genes are located, and the selection of “vehicles” family transposons (Partridge and Hall, 2003; Novais et al., 2010a). assures the selection of the genes that they contain. Consequently, The outcome is the initiation of a non-standard transposition

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 15 | 27

“fmicb-04-00015” — 2013/3/5 — 11:06 — page4—#4 Baquero et al. Multi-level population biology of resistance

resulting in only a single copy appearing in the transposed prod- different ecological conditions and stressors (including antibi- uct (Cain et al., 2010; Martinez et al., 2012). Such mobilization otics) multiplies the chances for expansion, recombination, and of integrons by specialized transposition is a powerful mecha- diversification (Partridge and Iredell, 2012; Seputiene et al., 2012). nism for integrons spread in both environmental competent and The main group within dsDNA transposases corresponds to non-competent bacteria (Domingues et al., 2012; Stokes et al., DDE transposases (designation given due to the presence of a 2012). Also, IS-mediated mobilization of relevant antibiotic resis- highly conserved catalytic triad of two aspartate (D) and one glu- tance genes contained in the integron contributes to enhance gene tamate (E) residue), originally identified in the retrovirus integrase expression and mosaic genetic diversity, which should be reflected and having a role during the transfer of the DNA strand. Most in higher possibilities of dissemination. That is the case for inser- IS families use this catalytic reaction for transposition with the tion sequences targeting the pseudo-palindromes of integron attC exception of IS1/IS110 and the RCR IS91-like elements. IS-DDE sites, as the IS1111-attC group elements of the IS110/IS492 family transposases have been detected in more than 70 bacterial gen- (Tetu and Holmes, 2008), or IS4-like elements as IS1999 (Aubert era, more than one third being iso-elements (>95% of identity at et al., 2006; Post and Hall, 2009; Poirel et al., 2010). Note that the protein level, >90% at DNA level). They are frequently located antibiotic-mediated selection of strains, plasmids, or transposons on plasmids associated with composite transposons containing containing integrons necessarily implies selection of IS sequences, antibiotic resistance genes (Merlin et al., 2000). and therefore the capture of resistance genes and the combina- Tn7 poses an unique fine-tuned regulated transposition array torial evolution of resistance platforms. In fact, ISs mobilizing (TnsABCDE) involved in the regulation of transposition (the core adjacent sequences by rolling-circle (RC) transposition as, ISEcp1 machinery coding for TnsA, TnsB, TnsC) and the mobilization and the insertion sequence common regions (ISCRs), favor the of the element (TnsE, TnsD; Parks and Peters, 2009, Parks et al., capture and mobilization of a full series of antibiotic resistance 2009). Such regulation allows Tn7 to use two target-site selec- genes leading to complex multi-resistance class 1 integrons (del tion pathways and move to different hosts. Tn7 belongs to a Pilar Garcillán-Barcia et al., 2001; Garcillán-Barcia and de la Cruz, family of MGE that encodes a transposase and an ATP-utilizing 2002; Aubert et al., 2006; Toleman et al., 2006; Poirel et al., 2010). protein (TnsC) that controls the activity of such transposase and often its target site selection. Members of the ATP-subunit super- ANTIBIOTICS AND POPULATIONS OF RESISTANCE family comprise widespread AbR transposons that differ in the TRANSPOSABLE ELEMENTS transposase (also a DDE enzyme) and the number of proteins Have antibiotics increased the abundance of resistance trans- in the transposition module (for revision see Craig, 2002). Some posable elements in the microbiosphere? Transposable elements examples are Tn1825/Tn1826, Tn552-IS21, and Tn402–Tn5053 encode an enzyme, transposase, which is required for excis- (Craig, 2002). All are widely distributed and they are related with ing and inserting the mobile element. Transposases (revised in trimethoprim and heavy metals resistance (Kholodii et al., 2003; Hickman et al., 2010 and references herein) seem to be the most Partridge, 2011). Please note that in this case trimethoprim resis- abundant genes in known sequenced genomes and environmental tance is the currently recognizable phenotype, but the genes might metagenomes (Aziz et al., 2010). have been selected before trimethoprim exposure for other reasons Among transposases, class II dsDNA transposases constitute (Alonso and Gready, 2006). the most common group, followed by serine and tyrosine recom- Members of the Tn3 family are mainly derivatives of trans- binases and RC transposases which are linked to different MGEs poson subfamilies Tn3 (Tn3,Tn5393,Tn5403)orTn501 (Tn21, (IS, composite transposons, class II transposons, bacteriophages, Tn501,Tn1721) and all of them were already detectable in and genetic islands). The wide spread and maintenance of dif- ancient bacteria from permafrost (Tolmasky, 1990; Graidy, 2002; ferent classes of transposable elements in bacterial populations Kholodii et al., 2003; Mindlin et al., 2008). Members of the Tn501 has been obviously favored by antibiotic selection because of their subfamily of Tn3 transposons could have been enriched by mer- association with antibiotic resistance genes. However, most of the cury exposure, as they carry mercury-detoxifying genes. These contemporary antibiotic resistance transposable elements belong genes probably originated in hydrothermal environments, where to a few families that have been detected in ancient isolates, often geochemically derived mercury is at high concentrations (Boyd linked to alternative functional roles, thus suggesting antibiotic and Barkay, 2012). Mercury-transposons provide target sites for resistance might have overshadowed previous selection forces. Tn501-type transposons, and include a diversity of Tn21,Tn1696, For instance, some transposable elements, as IS or compos- and Tn501 related elements carrying class 1 integrons. Enrich- ite transposons, as Tn5 or Tn10, confer growth rate advantages ment of Tn3-type transposons by environmental pollutants, as under different conditions of nutrient availability, enabling pop- heavy metals, might have contributed to increase the connectivity ulations to rapidly adapt to different physiological conditions with organisms and genetic platforms harboring resistance genes, (Biel and Hartl, 1983; Hartl et al., 1983; Blot et al., 1994). Also, eventually included in integrons (most frequently of class 1), and the highly specialized targeting system of Tn7 able to selec- have converted this family in the “flagship” of floating resistance tively direct transposition into both mobile and stationary DNA genes (Liebert et al., 1999; Partridge and Hall, 2004; Partridge and pools (see later), avoids the occurrence of deleterious insertions Iredell, 2012). and allows the host population or community to recruit genes Beside classic antibiotic resistance gene cassettes, emerging through a variety of mobile DNAs, thus favoring the adaptation beta-lactamase genes as blaVIM, blaIMP blaVEB,orblaGES are of diverse groups of bacteria to survive or adapt to different con- located in integrons on different Tn501 derivatives (Partridge, ditions (Parks and Peters, 2009; Parks et al., 2009). Selection by 2011). Other widespread “new” beta-lactamase genes have been

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 28

“fmicb-04-00015” — 2013/3/5 — 11:06 — page5—#5 Baquero et al. Multi-level population biology of resistance

directly recruited by host-specific Tn3-like transposons as Tn1440 communities? Conjugative plasmids and ICEs are quite similar carrying blaKPC (a Tn5403-like transposon from Klebsiella which genomic objects, in fact they appear to be short-term variants of belongs to the Tn3 subfamily; (Naas et al., 2008). identical backbone elements (Guglielmini et al., 2011); the main Such process of gene capture contributes to further increase difference is that the replication of ICEs occurs only by integra- the number of these transposons, which in turn will facilitate its tion in the host’s chromosome (Wozniak and Waldor, 2010). For diversification and eventually novel resistance gene recruitments. instance a close relationship resulting from a common phylogeny Indeed the capture of blaTEM1a by Tn2 led to, in a landscape of high can be found between IncA/C plasmids and SXT/R391 ICEs (Woz- consumption of aminopenicillins in the 60s and 70s of the past niak and Waldor, 2010; Toleman and Walsh, 2011). Note that century, a huge amplification of this transposon, now widespread the traditional association of highly transmissible elements with in all environments. Other transposons were less-successful, as plasmids is not necessarily true. Tn1 (with blaTEM−2), and Tn3 (carrying blaTEM−1a; Novais et al., Plasmids are abundant in nature and consistently isolated from 2010a; Bailey et al., 2011), probably because of the reduced num- microbial communities of different habitats with and without bers and connectivities (influencing success) of the vehicles in anthropogenic exposure (Coombs, 2009; Sobecky and Hazen, which they were located (plasmids, clones; Cain et al.,2010; Novais 2009). In fact, contemporary resistance plasmids are based on et al., 2010a; Partridge, 2011). plasmid backgrounds existing in the pre-antibiotic era (Datta and Transposable elements also increase in number by inserting Hughes, 1983; Hughes and Datta, 1983). Maintenance of plasmids extra copies in the host genomes. Of course this might cause an and ICEs in bacterial populations results from both the selfish “intergenomic conflict,”as such insertions might affect chromoso- features that promote acquisition and persistence within bacte- mal balance and produce mutations, being so, transposons might rial populations (“the parasitic hypothesis”) and the beneficial be a “bitter–sweet” pill for host bacteria (Toleman and Walsh, effects they confer to individual bacterial hosts and communities 2011). This is why genomes have evolved suppressors limiting (“the evolvability hypothesis”; Werren, 2011). MGEs are increas- transposon spread (Pomiankowski, 1999). Eventually, equilibrium ingly being considered within this multi-hierarchical model, as is reached by diminishing the transposition frequency. Successful clonal-, species-, or community-specific mobile elements (Carat- transposons as Tn3 have this kind of “transposition immunity” to toli, 2009; Garcillán-Barcia et al., 2009; Lim et al., 2010; Shearer ensure a maximum of two copies per replicon. et al., 2011; Heuer et al., 2012; Williams et al., 2012; Clewell On the other hand, transposition might compete with the et al., 2013; Guglielmini et al., 2013). Between and also within host genome replication. Some transposases as that of Tn7 (and these hierarchical levels, plasmids may eventually evolve toward possibly Tn917) can bind to “processivity factors” involved DNA mutualism (Rankin et al., 2011b). Plasmids might assure their replication; competition for this interaction could limit their pro- permanent linkage with a particular host, bacterial lineage, or liferation. However, a benefit for the transposons appear to be multi-specific community by post-segregation killer strategies that derived from the fact that the interaction of transposases with cause the death of non-carrying bacterial offspring. This is caused processivity factors favors “target site selection,”so that activation by toxin–antitoxin (TA), restriction-modification (R-M), or clus- of transposition with Tn7 (transposon excision and insertion) tered regularly interspaced short palindromic repeats (CRISPR) does not occur until an appropriate target has been identified, systems, and also probably by “plasmid domestication,” which is most frequently mobile plasmids, providing Tn7 with a means of produced by changes either in the plasmid and host genome that spreading to a new host (Parks et al., 2009). lead to a more stable coexistence (Bouma and Lenski, 1988; Jones, As in the case of integrons, transposable elements are very 2010; Marraffini and Sontheimer, 2010; García-Quintanilla and ancient on Earth, but the very same molecular structures are found Casadesús, 2011). in modern resistance-bearing transposons (Bisercić and Ochman, The diversity of bacterial communities, the relative popula- 1993; Mindlin et al., 2005; Vishnivetskaya and Kathariou, 2005; tion densities of their components, the spatial separation, and Petrova et al., 2009; Aziz et al., 2010). The acquisition of resis- nutrient availability greatly influence plasmid host-range, content, tance genes seems to have occurred preferentially by particular and transferability (Coombs, 2009). It is interesting to suggest transposable elements that were afterward amplified by antibi- that the selective processes exerted by antibiotics will modify otics. Interestingly, a number of transposons carrying resistance bacterial diversity and population densities, forcing the coexis- genes have been recovered from ancient permafrost and seem to tence of plasmids and particular hosts, favoring recombination have been selected before the antibiotic era (Mindlin et al., 2008). and other processes that lead to plasmid domestication (Boyd Widespread transposons in our days, as those belonging to Tn7 et al., 1996). The robustness of interactions between particular or Tn3 superfamilies, certainly have a very ancient origin. Most plasmids and particular clones is shaped by epistatic processes probably they were selected by pre-antibiotic forces, increasing (Silva et al., 2011; San Millán et al., 2012) mediated by nucleoid- their absolute amount, followed by their spread and diversifi- associated transcriptional regulators (Doyle et al., 2007; Yun et al., cation in different plasmids and organisms. Exposure to early 2010; Fernández-Alarcón et al., 2011; Humphrey et al., 2012) and chemotherapeutic agents has reinforced these evolutionary events. the clonal interferences that might result from these interactions (Hughes et al.,2012). A robust interaction is reflected in a non-cost ANTIBIOTICS AND POPULATIONS OF RESISTANCE MOBILE or even negative-cost coexistence, and will tie the fate of plasmid GENETIC ELEMENTS (PLASMIDS, ICEs) density to the abundance of their specific bacterial hosts, resulting Have antibiotics increased the abundance of plasmids and in a necessary“in-host”plasmid evolution linked to“with plasmid” ICEs carrying resistance genes in bacterial populations and host evolution (Dionisio et al., 2005; Halary et al., 2010).

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 15 | 29

“fmicb-04-00015” — 2013/3/5 — 11:06 — page6—#6 Baquero et al. Multi-level population biology of resistance

A major topic in plasmid population biology is the consid- antibiotics might modulate phage–bacteria population dynamics eration of advantages and inconveniences of plasmid or ICE by processes as “phage-antibiotic synergy,”a non-SOS mechanism broad-host-range. It is of note that host-range does not nec- of virulent phage induction caused by exposure to sub-inhibitory essarily mean transfer ability to a particular host or long-term concentrations of beta-lactams (Comeau et al., 2007; Allen et al., maintenance in bacterial populations, but stable replication in a 2011; Looft et al., 2012). Antibiotics promote the number of new host (De Gelder et al., 2007; Suzuki et al., 2010). Certainly, phages and pro-phages linked to antibiotic resistance platforms, broad-host-range conjugative plasmids favor the penetration of favoring dissemination of these platforms, and consequently adaptive traits as “new” antibiotic resistance genes (Fernández- amplifying the dissemination of resistance and virulence genes Alarcón et al., 2011; Sen et al., 2011; Hamprecht et al., 2012; Heuer (Allen et al., 2011). and Smalla,2012) and, in turn, antibiotics can favor the abundance of these plasmids promoting transfer and by selection of plasmid ANTIBIOTICS AND POPULATIONS OF BACTERIAL CLONES containing bacteria (Lang et al.,2012). The frequent observation of AND SPECIES different systems of replication (recognizing host primases) in the Bacterial clones are constituted by isolates that have a close com- same plasmid suggests adaptive ways of increasing host-range (Del mon phylogenetic origin. High-risk clones are defined as clones Solar et al., 1996; Clewell et al., 2013). Globally spread plasmids with an enhanced ability to colonize, spread, and persist in a variety identified in hospitals, soils, agriculture, and marine habitats have of niches, which acquire adaptive traits that increase pathogenicity a complex mosaic structure that reflects inter-genomic historical or antibiotic resistance (Baquero and Coque, 2011). They consti- adaptations to phylogenetically distant bacterial hosts (Schlüter tute the main vehicles dispersing antibiotic resistance at a global et al., 2007; Norberg et al., 2011; Heuer et al., 2012; Partridge and scale (Willems et al., 2011; Woodford et al., 2011). Examples of Iredell, 2012). In addition, broad-host-range plasmids lacking these high-risk clones are E. coli ST131 (phylogroup B2), ST155 transfer systems can be transferred to phylogenetically close or and ST393 (phylogroup B1), or ST69, ST405, and ST648 (phy- distantly related bacteria by helper conjugation systems located logroup D); K. pneumoniae ST258, ST14 or ST37; Enterococcus in narrow-host-range plasmids containing a conjugation system faecium, ST18, ST17, ST78; Enterococcus faecalis ST6; S. aureus, (Smorawinska et al., 2012). ST45, ST5, ST8, ST30, or ST22. A number of these clones were Long-term maintenance and dispersion of broad-host-range ancient lineages, well-adapted to colonization and transmission plasmids in bacterial populations and communities seems to between particular hosts, that acquired antibiotic resistance and be related with the local availability of hosts (De Gelder et al., consequently enhanced capabilities of dispersal (McBride et al., 2007), but also with the “social interactions between plasmids” 2007; Brisse et al., 2009; Chambers and Deleo, 2009; Willems eventually leading to unbearable costs for their hosts (Smith, et al., 2012). Multiplication and spread of highly successful clones 2012). Eventually, exclusion mechanisms between plasmids (one implies multiplication and spread of all the antibiotic resistance plasmid excludes the incoming one) might be softened by inter- genes and platforms they contain, increasing their absolute num- plasmid recombination that might result in hybrids able to bers. In fact, it is not unusual that a single successful clone might evade exclusion. CRISPR is a genetic interference system by contribute to the spread of different plasmids, genetic platforms, which bacteria with CRISPR regions carrying DNA copies of and resistance genes, both in Gram-positives (Chambers and previously encountered plasmids can abort the replication of Deleo, 2009) and in Gram-negatives (Carattoli, 2009; Andrade plasmids with these sequences. Hypothetically that might favor et al., 2011; Woodford et al., 2011; Novais et al., 2012a; Partridge plasmid dispersal among different strains, providing a weak and Iredell, 2012). Such multi-lateral collaboration probably con- selective advantage for the host cell (Levin, 2010), although an tributes to the local ecological success of variants arising in these increased benefit could be predicted for host coalitions, as genetic clones, leading to a clonal diversification (clonalization) which exchange communities (GECs; see later). This system also controls assures a long-term permanence in complex adaptive landscapes, phages, but the possible populational interactions (competition– following the “never put all the eggs in the same basket” principle cooperation) between phages and plasmids have scarcely been (Wiedenbeck and Cohan, 2011; de Regt et al., 2012; Freitas et al., investigated. 2013). Focusing only on mutational evolution, it has been sug- Antibiotic exposure might have increased the absolute number gested that there is an acceleration of emergence of bacterial antibi- of plasmids with resistance determinants in bacterial populations otic resistance in connected microenvironments (Zhang et al., due to the selection of clones harboring them. The possibility that 2011; Hallatschek, 2012) but this might also occur in the case of broad-host conjugative plasmids have been submitted to a more gene flow. effective enrichment than narrow-host plasmids (because of selec- Local clonalization might result in a restricted gene flow tion in multiple hosts and environmental compartments) poses an among resulting subpopulations (Willems et al., 2012). However, interesting question. Eventually, the biological cost of resistance recombinational events might spare those regions required for plasmids for the host could be compensated by higher transmis- adaptation to local microniches, assuring divergence, and be main- sion (Garcillán-Barcia et al., 2011). As Stokes and Gillings pointed tained for other regions (ecological speciation with-gene-flow;Via, out, an increase in the general tempo of resistance genes dissemi- 2012). The increased recovery of isolates belonging to high-risk nation is highly probable, due to selection of cells with inherently clonal complexes of important human pathogens as E. coli, S. higher rates of lateral transfer (Stokes and Gillings, 2011). aureus, E. faecalis,orE. faecium, that cause both human infec- Finally, we can consider bacteriophages as mobile mediators tions and mucosal colonization, and even the expansion of these of inter-bacterial transfer of resistance genes. Also in this case clones to novel hosts is most probably related with the acquisition

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 30

“fmicb-04-00015” — 2013/3/5 — 11:06 — page7—#7 Baquero et al. Multi-level population biology of resistance

of antibiotic resistance genes (Hidron et al., 2008; Baquero, 2012; good” are frequently encoded by conjugative elements (Norman Novais et al., 2012a, 2013) et al., 2009; Rankin et al., 2011b). The distribution among GECs An interesting question is if the selection of particular clones members of such plasmid-mediated “public goods” is favored because of antibiotic resistance might decrease the overall clonal by common characteristics in the consortium, as nearly iden- diversity as might be inferred from recent studies (Ghosh et al., tical immune phenotypes mediated by CRISPR, or common 2011). Actually, that might be compensated by clonal diversifi- DNA uptake mechanisms or quorum sensing responses. Plasmid- cation, in a “ex pluribus unum/ex unibus plurum” evolutionary mediated common benefits will probably lead to GEC-plasmid dynamics (Baquero, 2011). It is easy to conclude that any outbreak coevolution (Skippington and Ragan, 2012). Along the same line, produced by antibiotic resistant bacteria will locally enrich the addiction-type TA complexes can spread on plasmids, favoring involved evolutionary units, facilitating further events of antibi- coexistence and/or competition in spatially structured environ- otic resistance development and possibly transmission (Chambers ments (Rankin et al., 2012) highlighting the role of kin effects and Deleo, 2009; Freitas et al., 2013). in GECs selection (taking “kin” in a wider sense than just intra- specific ties). It is of note, however, that possibly most organisms ANTIBIOTICS AND POPULATIONS OF BACTERIAL and environments might act as conduits for resistance gene flow COMMUNITIES (Stokes and Gillings, 2011), acting as “sources” from where resis- Imbedded into the high complexity of the microbiomes, of tance genes are directed to GECs, acting as “sinks,” according humans and animals, in the soil or in the water sediments, it to the Perron et al. (2007) metaphor. The possibility of “go- is possible to recognize “clubs” of bacterial clones and species between” organisms traveling from GECs of different microbiotic where genes and genetic platforms circulate via lateral trans- systems (humans, animals, rhizosphere, and water sediments) fer, the GECs. In a very restrictive manner, Skippington and should be taken into consideration, as they might contribute Ragan (2011) have recently defined a GEC as a group of organ- to the inter-environmental globalization of antibiotic resistance isms (entities) in which each entity has over time both donated genes. The existence of “ubiquitous” organisms or species able genetic material to, and received genetic material from every to transit in different environments has been suggested (Fondi other entity in that GEC, via a path of lateral gene transfer. and Fani, 2010; Freilich et al., 2010; Tamames et al., 2010). Can- What do the members of such clubs have in common? Network didates for efficient “go-between” organisms are groups of the modeling and co-occurrence statistical approaches indicate that same bacterial species but specialized in particular environments, lifestyle and shared environments, functional complementarities, as the case of human or animal-adapted versus environmental and most probably, continued physical clustering (granularity) E. coli clades, where probably only ecological barriers prevent gene determine the size and connectivity of GECs (Freilich et al., flow (Freilich et al., 2010; Luo et al., 2011). Mixing of human 2010; Smillie et al., 2011; Faust and Raes, 2012; Faust et al., or animal derived water effluents into the environment, a prac- 2012). In many cases, this also means a closed shared phy- tice that is surprisingly perpetuated even in modern societies logeny (Skippington and Ragan, 2012). In fact, GECs members will facilitate conduits for resistance gene flow (Baquero et al., are linked not only by genetic exchanges, but also by metabolic 2008; Czekalski et al., 2012; Lupo et al., 2012). Such flow occurs and functional cooperation, providing a certain ecological com- because of the confluence of human microbiota from different partmentalization inside particular microbial megasystems (as human hosts, different animals and the indigenous environmental intestinal microbiota; Faust and Raes, 2012; Faust et al., 2012). We microbiota. can consider here some kind of cooperative “niche construction” Among these GECs, the most relevant for the transmis- (Laland et al., 1999; Kylafis and Loreau, 2011). Genetic trans- sion of antibiotic resistance are those including species from fer, particularly considering mixed-granular “surface-associated Gammaproteobacteria (as E. coli) and Firmicutes (as Enterococ- populations” with a kind of “lattice reciprocity” (Zhong et al., cus; Antonopoulos et al., 2009; Freilich et al., 2010; Skippington 2010, 2012) might assure a “collective” adaptation of such func- and Ragan, 2011; Faust et al., 2012). These groups of organ- tional GEC units, increasing relatedness among members and isms are enriched in the microbiota during antibiotic exposure fixing common evolutionary boundaries (Nogueira et al., 2009; (Antonopoulos et al., 2009; Sommer et al., 2009). Antibiotic- Rankin et al., 2011a,b). The development of more studies on the mediated reduction in number or loss of some species, favors “physics” of genetic transfer, is certainly advisable, for instance, bacterial species able to explore and temporarily exploit empty to investigate to what extent genetic transfer can be influenced niches due to short generation times (Allen et al., 2011; Looft and by mixing movements or the viscosity or fluidity of the sur- Allen, 2012). Antibiotic exposure will increase the absolute num- rounding medium, as in the intestinal content (particularly during ber (overgrowth) of GECs-associated organisms, for instance, by enteric diseases) or in water bodies, influencing cell-to-cell con- antibiotic exposure of the infant gut (Fouhy et al., 2012; Looft and tacts and therefore GECs integrities (Zhong et al., 2010; Jeffery Allen, 2012). Probably the same might occur in environmental et al., 2012). GECs submitted either to antibiotic pollution or sanitation proce- An important topic is the possibility of “multiclonal GECs” dures (Baquero et al., 2008). For instance, metagenomic analysis as a form of organization of the lifestyle of a particular species indicates that drinking water chlorination could concentrate pop- or closely phylogenetically related coalitions in changing envi- ulations containing insertion sequences, integrons, and antibiotic ronments (Skippington and Ragan, 2012), where different sub- resistance plasmids (Shi et al., 2013). These effects will contribute specific ecotypes exploiting neighbor nano-niches (Wiedenbeck to the dissemination of resistance genes and the genetic platforms and Cohan, 2011) and taking advantage of a common “public in which they are located.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 15 | 31

“fmicb-04-00015” — 2013/3/5 — 11:06 — page8—#8 Baquero et al. Multi-level population biology of resistance

The identification of GECs among members of the “microbial in promoting antibiotic resistance will be significantly increased. guilds” in the three identified major microbiome “enterotypes” Finally, it could be considered, under certain circumstances, as (Arumugam et al., 2011) needs to be investigated. These major during the colonization of the neonatal intestinal tract, that enterotypes (clusters of species) should probably be tightly main- rapidly growing populations might be more prone to the dis- tained into host populations, and therefore the local spread of semination of antibiotic resistance. Also the unexpected possi- antibiotic resistance genes could serve to maintain their integrity bility of resistance gene exchange between Actinobacteria (Bifi- in an antibiotic-polluted environment. At the same time, low- dobacterium belongs to this group!) and Gammaproteobacteria abundance enterotype-species, but providing critical functions, has been recently shown under the same conditions (Tamminen could have low possibilities of getting resistance genes in the et al., 2012). absence of GECs. Consequently, a deep and permanent contamination by antibiotic resistance genes of the normal microbiota ANTIBIOTICS IN THE ANTHROPOCENE: EFFECTS ON GLOBAL of humans and animals is a reasonable possibility. Complex ECOLOGY AND EVOLUTION microbiota of humans and animals are reproducible systems, Evolution is a natural trend of complex systems, and might be not only along time in the same individual, but also across accelerated by changing and stressful conditions. The Anthro- individuals. These systems are frequently based in a microbi- pocene is the current human-dominated geological epoch where otic “core” composed by organisms belonging to a relatively nature is changed and stressed by the action of humans small number of phylogroups, and probably metabolically linked (Zalasiewicz et al., 2011; Biermann et al., 2012). Industrial antibi- with the host (Dethlefsen et al., 2006; Ley et al., 2006; Marchesi, otics are a paradigmatic example of substances exerting a powerful 2011). Newborns have a sterile gut, but the human micro- effect of anthropogenic origin on the bacterial communities of the biota is “reproduced” with a relatively low number of variations microbiosphere. Not only most of these substances are unspecif- on each of them (Baquero and Nombela, 2012; Vallès et al., ically killing bacterial organisms, and selecting for resistance, but 2012). Possibly there is also an “epidemiology of bacterial con- directly influence the mechanisms of genetic variation (muta- sortia” even in hospitals, which remains to be investigated. tion, recombination, transposition, modularization, gene transfer; Exposure to antimicrobial agents might affect the frequency Baquero, 2009; Gillings and Stokes, 2012). and absolute number of GECs; in a number of cases, antibi- Such effects on microorganisms will be further enhanced by a otic resistance might contribute to the temporal stability and diversity of other anthropogenic effects as the release of biophar- resilience of microbiomes in an antibiotic-polluted environ- maceuticals, biocides, heavy metals, industrial and agricultural ment (Allison and Martiny, 2008; Antonopoulos et al., 2009). residues, and plastic materials or changes in the environmental Of course that occurs at the expenses of maintenance and conditions. The mixing of bacterial populations (human organ- spread of the full range of antibiotic resistance evolutionary isms with other human, animal, or environmental organisms), units. that makes the emergence and spread of resistance possible is Finally, we cannot discard individual variations in the microbi- also favored by poor sanitation, facilitating contact of human otic communities caused by diet, host genetics, particular illnesses, or animal sewage with the soil. Some of these effects might inflammation, or infectious agents including viruses and para- escalate with other anthropogenic effects as destruction of diver- sites which might lead to microbial communities more prone sity in food animals (Baquero, 2012) or even global warming to capture and propagate antibiotic resistance (Marchesi, 2010; (Baquero et al., 2008; Baquero, 2009; Balbus et al., 2013). The Claesson et al., 2012; Looft and Allen, 2012). For instance, a fight against antibiotic resistance should focus not only on act- high-fat diet determines the composition of the murine gut micro- ing on its appropriate usage in human and veterinary medicine, biome independently of obesity, with an increase of Proteobacteria but by considering possible initiatives at ecological and evolu- and Firmicutes, heavily involved in resistance gene mobilization tionary levels, as eco-evo drugs and strategies (Baquero et al., (Hildebrandt et al., 2009; Tagliabue and Elli, 2012). In several 2011) in accordance with the environmental distribution of bac- microbiota communities studied in the elderly, the proportion of terial organisms (Tamames et al., 2010), in the scope of progre- phylum Proteobacteria, very active in the mobilization of antibi- ssing toward a protective and restorative planet medicine otic resistance genes and vehicles, was ten times higher than (Baquero, 2009). average (20 versus 2%; Claesson et al., 2011). E. coli numbers are higher in the microbiota of women with excessive weight gain than ACKNOWLEDGMENTS in women with normal weight gain during pregnancy (Santacruz The authors’ work was sponsored by grants from the European et al., 2010). Union (PAR-241476 and EvoTAR-282004), the Instituto de Salud A number of surgical interventions (as surgery for morbid obe- Carlos III – Ministry of Economy and Competitiveness of Spain sity) increases Proteobacteria even in a higher proportion (50 (FIS-PS09-02381; FIS-PI10-02588, PI12-01581), and the Regional times increase; Li et al., 2011; Graessler et al., 2012). Unfortu- Government of Madrid in Spain (PROMPT- S2010/BMD2414). nately, these populational microbiotic shifts favoring the active The authors are also grateful to the Spanish Network for the populations and communities contributing to the emergence, dis- Study of Plasmids and Extrachromosomal Elements (REDEEX) persal and maintenance of antibiotic resistance might also occur for encouraging and funding cooperation among Spanish micro- as a consequence of undernutrition (10 times increase in Pro- biologists working on the biology of mobile genetic elements teobacteria, 46 versus 5% in healthy children in Bangladesh; (grant BFU 2012-0079-E/BMC; Spanish Ministry of Science and Monira et al., 2011). Possibly the deleterious effect of antibiotics Innovation).

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 32

“fmicb-04-00015” — 2013/3/5 — 11:06 — page9—#9 Baquero et al. Multi-level population biology of resistance

REFERENCES Aubert, D., Naas, T., Héritier, C., Poirel, Baquero, F., Coque, T. M., and de of genetic variation. Curr. Opin. Aguilée, R., Claessen, D., and Lambert, L., and Nordmann, P. (2006). Func- la Cruz, F. (2011). Ecology and Microbiol. 15, 561–569. A. (2009). Allele fixation in a dynamic tional characterization of IS1999, evolution as targets: the need for Blot, M., Hauer, B., and Monnet, G. metapopulation: founder effects vs an IS4 family element involved in novel eco-evo drugs and strategies (1994). The Tn5 bleomycin resistance refuge effects. Theor. Popul. Biol. 76, mobilization and expression of beta- to fight antibiotic resistance. Antimi- gene confers improved survival and 105–117. lactam resistance genes. J. Bacteriol. crob. Agents Chemother. 55, 3649– growth advantage on Escherichia coli. Aguilée, R., Lambert, A., and Claessen, 188, 6506–6514. 3660. Mol. Gen. Genet. 242, 595–601. D. (2011). Ecological speciation in Aziz, R. K., Breitbart, M., and Edwards, Baquero, F.,Martínez, J.-L., and Cantón, Bouma, J. E., and Lenski, R. E. (1988). dynamic landscapes. J. Evol. Biol. 24, R. A. (2010). Transposases are the R. (2008). Antibiotics and antibi- Evolution of a bacteria/plasmid asso- 2663–2677. most abundant, most ubiquitous otic resistance in water environments. ciation. Nature 335, 351–352. Allen, H. K., Looft, T., Bayles, D. genes in nature. Nucleic Acids Res. 38, Curr. Opin. Biotechnol. 19, 260–265. Boyd, E. F., Hill, C. W., Rich, S. M., and O., Humphrey, S., Levine, U. Y., 4207–4217. Baquero, F., and Negri, M. C. (1997). Hartl, D. L. (1996). Mosaic structure Alt, D., et al. (2011). Antibiotics Baharoglu, Z., and Mazel, D. (2011). Selective compartments for resistant of plasmids from natural popula- in feed induce prophages in swine Vibrio cholerae triggers SOS and microorganisms in antibiotic gradi- tions of Escherichia coli. Genetics 143, fecal microbiomes. MBio 2, e00260– mutagenesis in response to a ents. Bioessays 19, 731–736. 1091–1100. e00211. wide range of antibiotics: a route Baquero, F., and Nombela, C. (2012). Boyd, E. S., and Barkay, T. (2012). Allen, H. K., Moe, L. A., Rodbum- towards multiresistance. Antimi- The microbiome as a human organ. The mercury resistance operon: from rer, J., Gaarder, A., and Handelsman, crob. Agents Chemother. 55, 2438– Clin. Microbiol. Infect. 18, 2–4. an origin in a geothermal environ- J. (2009). Functional metagenomics 2441. Baquero, M.-R., Galán, J. C., Del ment to an efficient detoxification reveals diverse beta-lactamases in a Bailey, J. K., Pinyon, J. L., Anantham, S., Carmen Turrientes, M., Cantón, machine. Front. Microbiol. 3:349. doi: remote Alaskan soil. ISME J. 3, and Hall, R. M. (2011). Distribution R., Coque, T. M., Martínez, J. L., 10.3389/fmicb.2012.00349 243–251. of the blaTEM gene and blaTEM- et al. (2005). Increased mutation Brandon, R. (1987). The levels of selec- Allison, S. D., and Martiny, J. B. H. containing transposons in commen- frequencies in Escherichia coli iso- tion: a hierarchy of interactors,” in (2008). Colloquium paper: resis- sal Escherichia coli. J. Antimicrob. lates harboring extended-spectrum The Role of Behavior in Evolution,ed. tance, resilience, and redundancy Chemother. 66, 745–751. beta-lactamases. Antimicrob. Agents H. Plotkin (Cambridge: MIT Press), in microbial communities. Proc. Balbus, J. M., Boxall, A. B. A., Fenske, Chemother. 49, 4754–4756. 124–146. Natl. Acad. Sci. U.S.A. 105, 11512– R. A., McKone, T. E., and Zeise, L. Barlow, M., and Hall, B. G. (2002). Brisse, S., Fevre, C., Passet, V., 11519. (2013). Implications of global cli- Origin and evolution of the AmpC Issenhuth-Jeanjean, S., Tournebize, Alonso, H., and Gready, J. E. mate change for the assessment and beta-lactamases of Citrobacter fre- R., Diancourt, L., et al. (2009). Vir- (2006). Integron-sequestered dihy- management of human health risks undii. Antimicrob. Agents Chemother. ulent clones of Klebsiella pneumo- drofolate reductase: a recently rede- of chemicals in the natural environ- 46, 1190–1198. . niae: identification and evolution- ployed enzyme. Trends Microbiol. 14, ment. Environ. Toxicol. Chem. 32, Becker, J., Eisenhauer, N., Scheu, S., ary scenario based on genomic and 236–242. 62–78. and Jousset, A. (2012). Increasing phenotypic characterization. PloS Alvarez-Ortega, C., Wiegand, I., Oli- Balloux, F. (2010). “Demographic influ- antagonistic interactions cause bacte- ONE 4:e4982. doi: 10.1371/jour- vares, J., Hancock, R. E. W., and ences on bacterial population struc- rial communities to collapse at high nal.pone.0004982 Martínez, J. L. (2011). The intrinsic ture,” in Bacterial Population Genet- diversity. Ecol. Lett. 15, 468–474. Cain, A. K., Liu, X., Djordjevic, resistome of Pseudomonas aeruginosa ics in Infectious Disease, eds D. A. Benveniste, R., and Davies, J. S. P., and Hall, R. M. (2010). to β-lactams. Virulence 2, 144–146. Robinson, D. Falush, and E. J. Feil (1973). Aminoglycoside antibiotic- Transposons related to Tn1696 in Aminov, R. I. (2009). The role of (Hoboken: John Wiley & Sons, Inc.), inactivating enzymes in actino- IncHI2 plasmids in multiply antibi- antibiotics and antibiotic resistance 103–120. mycetes similar to those present in otic resistant Salmonella enterica in nature. Environ. Microbiol. 11, Bapteste, E., Lopez, P., Bouchard, F., clinical isolates of antibiotic-resistant serovar Typhimurium from Aus- 2970–2988. Baquero, F., McInerney, J. O., and bacteria. Proc. Natl. Acad. Sci. U.S.A. tralian animals. Microb. Drug Resist. Aminov, R. I., and Mackie, R. I. (2007). Burian, R. M. (2012). Evolutionary 70, 2276–2280. 16, 197–202. Evolution and ecology of antibiotic analyses of non-genealogical bonds Bertels, F., and Rainey, P. B. Cantón, R., and Coque, T. M. (2006). resistance genes. FEMS Microbiol. produced by introgressive descent. (2011). Within-genome evolution The CTX-M beta-lactamase pan- Lett. 271, 147–161. Proc. Natl. Acad. Sci. U.S.A. 109, of REPINs: a new family of demic. Curr. Opin. Microbiol. 9, Andrade, L. N., Curiao, T., Ferreira, 18266–18272. miniature mobile DNA in bacte- 466–475. J. C., Longo, J. M., Clímaco, E. C., Baquero, F. (2009). Environmental ria. PLoS Genet. 7:e1002132. doi: Cantón, R., Mir, N., Sánchez, M., and Martinez, R., et al. (2011). Dissem- stress and evolvability in microbial 10.1371/journal.pgen.1002132 Baquero, F. (1999). MIC distribution ination of blaKPC-2 by the spread systems. Clin. Microbiol. Infect. 15, Biel, S. W., and Hartl, D. L. (1983). Evo- and inoculum effect of LY333328: of Klebsiella pneumoniae clonal com- 5–10. lution of transposons: natural selec- a study of vancomycin-susceptible plex 258 clones (ST258, ST11, ST437) Baquero, F. (2004). From pieces to pat- tion for Tn5 in Escherichia coli K12. and VanA-type and VanC-type ente- and plasmids (IncFII, IncN, IncL/M) terns: evolutionary engineering in Genetics 103, 581–592. rococci obtained from intensive care among Enterobacteriaceae species in bacterial pathogens. Nat. Rev. Micro- Biermann, F., Abbott, K., Andresen, unit patient surveillance cultures. Brazil. Antimicrob. Agents Chemother. biol. 2, 510–518. S., Bäckstrand, K., Bernstein, S., Clin. Microbiol. Infect. 5, 554–559. 55, 3579–3583. Baquero, F. (2012). On the shifting Betsill, M. M., et al. (2012). Sci- Carattoli, A. (2009). Resistance plas- Antonopoulos, D. A., Huse, S. M., Mor- balance: the case of Staphylococcus ence and government. Navigating the mid families in Enterobacteriaceae. rison, H. G., Schmidt, T. M., Sogin, aureus CC398. MBio 3, e00078–12. anthropocene: improving Earth sys- Antimicrob. Agents Chemother. 53, M. L., andYoung,V.B. (2009). Repro- Baquero, F. (2011). The 2010 Garrod tem governance. Science 335, 1306– 2227–2238. . ducible community dynamics of the Lecture: the dimensions of evolution 1307. Castillo, A., Eguiarte, L. E., and Souza,V. gastrointestinal microbiota follow- in antibiotic resistance: ex unibus Bisercić, M., and Ochman, H. (1993). (2005). A genomic population genet- ing antibiotic perturbation. Infect. plurum et ex pluribus unum. J. Natural populations of Escherichia ics analysis of the pathogenic entero- Immun. 77, 2367–2375. Antimicrob. Chemother. 66, 1659– coli and Salmonella typhimurium har- cyte effacement island in Escherichia Arumugam, M., Raes, J., Pelletier, E., 1672. bor the same classes of insertion coli: the search for the unit of selec- Le Paslier, D., Yamada, T., Mende, D. Baquero, F., and Coque, T. M. (2011). sequences. Genetics 133, 449–454. tion. Proc. Natl. Acad. Sci. U.S.A. 102, R., et al. (2011). Enterotypes of the Multilevel population genetics in Blázquez, J., Couce, A., Rodríguez- 1542–1547. human gut microbiome. Nature 473, antibiotic resistance. FEMS Micro- Beltrán, J., and Rodríguez-Rojas, A. Chambers, H. F.,and Deleo, F.R. (2009). 174–180. biol. Rev. 35, 705–706. (2012). Antimicrobials as promoters Waves of resistance: Staphylococcus

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 15 | 33

“fmicb-04-00015” — 2013/3/5 — 11:06 — page 10 — #10 Baquero et al. Multi-level population biology of resistance

aureus in the antibiotic era. Nat. Rev. Switzerland. Front. Microbiol. 3:106. transfer of transposons, integrons Francino, M. P. (2012). The ecology of Microbiol. 7, 629–641. doi: 10.3389/fmicb.2012.00106 and gene cassettes between bacte- bacterial genes and the survival of the Chao, L., and Levin, B. R. (1981). Czárán, T. L., Hoekstra, R. F., and Pagie, rial species. PLoS Pathog. 8:e1002837. new. Int. J. Evol. Biol. 2012, 394026. Structured habitats and the evolution L. (2002). Chemical warfare between doi: 10.1371/journal.ppat.1002837 Freilich, S., Kreimer, A., Meilijson, of anticompetitor toxins in bacte- microbes promotes biodiversity. Doolittle, W. F., and Zhaxybayeva, O. I., Gophna, U., Sharan, R., and ria. Proc. Natl. Acad. Sci. U.S.A. 78, Proc. Natl. Acad. Sci. U.S.A. 99, (2010). Metagenomics and the units Ruppin, E. (2010). The large-scale 6324–6328. 786–790. of biological organization. BioScience organization of the bacterial network Claesson, M. J., Cusack, S., O’Sullivan, Dantas, G., Sommer, M. O. A., Oluwase- 60, 102–112. of ecological co-occurrence interac- O., Greene-Diniz, R., De Weerd, H., gun, R. D., and Church, G. M. Doyle, M., Fookes, M., Ivens, A., Man- tions. Nucleic Acids Res. 38, 3857– Flannery, E., et al. (2011). Composi- (2008). Bacteria subsisting on antibi- gan, M. W., Wain, J., and Dor- 3868. tion, variability, and temporal stabil- otics. Science 320, 100–103. man, C. J. (2007). An H-NS-like Freitas, A. R., Novais, C., Tedim, A. ity of the intestinal microbiota of the Datta, N., and Hughes, V. M. (1983). stealth protein aids horizontal DNA P., Francia, M. V., Baquero, F., and elderly. Proc. Natl. Acad. Sci. U.S.A. Plasmids of the same Inc groups in transmission in bacteria. Science 315, Coque, T. M. (2013). Microevolu- 108, 4586–4591. Enterobacteria before and after the 251–252. tionary events involving narrow host Claesson, M. J., Jeffery, I. B., Conde, medical use of antibiotics. Nature Dupré, J., and O’Malley, M. A. (2007). plasmid influences local fixation of S., Power, S. E., O’Connor, E. M., 306, 616–617. Metagenomics and biological ontol- vancomycin-resistance in Enterococ- Cusack, S., et al. (2012). Gut micro- Davies, J. (2006). Are antibiotics nat- ogy. Stud. Hist. Philos. Biol. Biomed. cus. PloS ONE (in press). biota composition correlates with urally antibiotics? J. Ind. Microbiol. Sci. 38, 834–846. Garcillán-Barcia, M. P., Alvarado, A., diet and health in the elderly. Nature Biotechnol. 33, 496–499. Fajardo, A., and Martínez, J. L. (2008). and De la Cruz, F. (2011). Identi- 488, 178–184. Day, M. D., Beck, D., and Foster, J. Antibiotics as signals that trigger spe- fication of bacterial plasmids based Clewell, D. B., Weaver, K. E., Dunny, G. A. (2011). Microbial communities cific bacterial responses. Curr. Opin. on mobility and plasmid population M., Coque, T. M., Francia, M. V., and as experimental units. Bioscience 61, Microbiol. 11, 161–167. biology. FEMS Microbiol. Rev. 35, Hayes, F. (2013). “Extrachromosomal 398–406. Fajardo, A., Martínez-Martín, N., Mer- 936–956. and mobile elements,” in Enterococci. D’Costa, V. M., King, C. E., Kalan, L., cadillo, M., Galán, J. C., Ghysels, Garcillán-Barcia, M. P., Francia, M. V., Transmission, Maintenance, and Epi- Morar, M., Sung, W. W. L., Schwarz, B., Matthijs, S., et al. (2008). The and De la Cruz, F. (2009). The diver- demiology, ed. M. S. Gilmore (in C., et al. (2011). Antibiotic resistance neglected intrinsic resistome of bac- sity of conjugative relaxases and its press). is ancient. Nature 477, 457–461. terial pathogens. PloS ONE 3:e1619. application in plasmid classification. Cohen, O., Ashkenazy, H., Burstein, D., De Gelder, L., Ponciano, J. M., Joyce, doi: 10.1371/journal.pone.0001619 FEMS Microbiol. Rev. 33, 657–687. and Pupko, T. (2012). Uncovering P., and Top, E. M. (2007). Stability Faust, K., and Raes, J. (2012). Micro- Garcillán-Barcia, M. P., and de la the co-evolutionary network among of a promiscuous plasmid in differ- bial interactions: from networks to Cruz, F. (2002). Distribution of IS91 prokaryotic genes. Bioinformatics 28, ent hosts: no guarantee for a long- models. Nat. Rev. Microbiol. 10, family insertion sequences in bac- i389–i394. term relationship. Microbiology 153, 538–550. terial genomes: evolutionary impli- Comeau, A. M., Tétart, F., Trojet, S. 452–463. Faust, K., Sathirapongsasuti, J. F., cations. FEMS Microbiol. Ecol. 42, N., Prère, M.-F., and Krisch, H. del Pilar Garcillán-Barcia, M., Bernales, Izard, J., Segata, N., Gevers, D., 303–313. M. (2007). Phage-Antibiotic Synergy I., Mendiola, M. V., and De la Raes, J., et al. (2012). Microbial García-Quintanilla, M., and Casadesús, (PAS): beta-lactam and quinolone Cruz, F.(2001). Single-stranded DNA co-occurrence relationships in the J. (2011). Virulence plasmid inter- antibiotics stimulate virulent phage intermediates in IS91 rolling-circle human microbiome. PLoS Comput. change between strains ATCC 14028, growth. PloS ONE 2:e799. doi: transposition. Mol. Microbiol. 39, Biol. 8:e1002606. doi: 10.1371/jour- LT2, and SL1344 of Salmonella enter- 10.1371/journal.pone.0000799 494–501. nal.pcbi.1002606 ica serovar Typhimurium. Plasmid Coombs, J. M. (2009). Potential for Del Solar, G., Alonso, J. C., Espinosa, M., Fernández-Alarcón, C., Singer, R. 65, 169–175. horizontal gene transfer in micro- and Díaz-Orejas, R. (1996). Broad- S., and Johnson, T. J. (2011). Gaze, W. H., Abdouslam, N., Hawkey, P. bial communities of the terrestrial host-range plasmid replication: an Comparative genomics of multidrug M., and Wellington, E. M. H. (2005). subsurface. Methods Mol. Biol. 532, open question. Mol. Microbiol. 21, resistance-encoding IncA/C plasmids Incidence of class 1 integrons in a 413–433. 661–666. from commensal and pathogenic quaternary ammonium compound- Coque, T. M., Baquero, F., and Canton, de Regt, M. J. A., Van Schaik, W., Escherichia coli from multiple ani- polluted environment. Antimicrob. R. (2008). Increasing prevalence of Van Luit-Asbroek, M., Dekker, H. mal sources. PloS One 6:e23415. doi: Agents Chemother. 49, 1802–1807. ESBL-producing Enterobacteriaceae A. T., Van Duijkeren, E., Koning, 10.1371/journal.pone.0023415 Gaze, W. H., Zhang, L., Abdouslam, in Europe. Euro Surveill. 13, 19044. C. J. M., et al. (2012). Hospital Fondi, M., and Fani, R. (2010). The N. A., Hawkey, P. M., Calvo-Bado, Cordero, O. X., Wildschutte, H., Kirkup, and community ampicillin-resistant horizontal flow of the plasmid resis- L., Royle, J., et al. (2011). Impacts of B., Proehl, S., Ngo, L., Hussain, F., Enterococcus faecium are evolution- tome: clues from inter-generic sim- anthropogenic activity on the ecology et al. (2012). Ecological populations arily closely linked but have diversi- ilarity networks. Environ. Microbiol. of class 1 integrons and integron- of bacteria act as socially cohesive fied through niche adaptation. PloS 12: 3228–3242. associated genes in the environment. units of antibiotic production and ONE 7:e30319. doi: 10.1371/jour- Forsberg, K. J., Reyes, A., Wang, B., ISME J. 5, 1253–1261. resistance. Science 337, 1228–1231. nal.pone.0030319 Selleck, E. M., Sommer, M. O. A., Ghosh, A., Dowd, S. E., and Zurek, L. Couce, A., and Blázquez, J. (2009). Dethlefsen, L., Eckburg, P.B., Bik, E. M., and Dantas, G. (2012). The shared (2011). Dogs leaving the ICU carry a Side effects of antibiotics on genetic and Relman, D. A. (2006). Assembly antibiotic resistome of soil bacteria very large multi-drug resistant ente- variability. FEMS Microbiol. Rev. 33, of the human intestinal microbiota. and human pathogens. Science 337, rococcal population with capacity 531–538. Trends Ecol. Evol. 21, 517–523. 1107–1111. for biofilm formation and horizon- Craig, N. L. (2002). “Tn7,” in Mobile Dionisio, F., Conceição, I. C., Mar- Fouhy, F., Guinane, C. M., Hussey, tal gene transfer. PloS ONE 6:e22451. DNA II, eds. N. Craig, R. Craigie, ques, A. C. R., Fernandes, L., and S., Wall, R., Ryan, C. A., doi: 10.1371/journal.pone.0022451 M. Gellet, and A. M. Lambowitz Gordo, I. (2005). The evolution of a Dempsey, E. M., et al. (2012). Gillings, M. R., and Stokes, H. W. (Washington: ASM Press), 432–456. conjugative plasmid and its ability to High-throughput sequencing reveals (2012). Are humans increasing bac- Czekalski, N., Berthold, T., Caucci, increase bacterial fitness. Biol. Lett. 1, the incomplete, short-term recovery terial evolvability? Trends Ecol. Evol. S., Egli, A., and Bürgmann, H. 250–252. of infant gut microbiota following 27, 346–352. (2012). Increased levels of mul- Domingues, S., Harms, K., Fricke, parenteral antibiotic treatment with Gilliver, M. A., Bennett, M., Begon, M., tiresistant bacteria and resistance W. F., Johnsen, P. J., Da Silva, ampicillin and gentamicin. Antimi- Hazel, S. M., and Hart, C. A. (1999). genes after wastewater treatment and G. J., and Nielsen, K. M. (2012). crob. Agents Chemother. 56, 5811– Antibiotic resistance found in wild their dissemination into lake geneva, Natural transformation facilitates 5820. rodents. Nature 401, 233–234.

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 34

“fmicb-04-00015” — 2013/3/5 — 11:06 — page 11 — #11 Baquero et al. Multi-level population biology of resistance

Goffin, C., and Ghuysen, J. M. carbapenemase GIM-1 in Enterobac- SOS response in a clinical set- Proc. Natl. Acad. Sci. U.S.A. 96, (1998). Multimodular penicillin- ter cloacae in Germany. J. Antimicrob. ting. PLoS Pathog. 8:e1002778. doi: 10242–10247. binding proteins: an enigmatic family Chemother. doi: 10.1093/jac/dks447 10.1371/journal.ppat.1002778 Lang, K. S., Danzeisen, J. L., Xu, W., and of orthologs and paralogs. Microbiol. [Epub ahead of print]. Hughes, J. M., Lohman, B. K., Deck- Johnson, T. J. (2012). Transcriptome Mol. Biol. Rev. 62, 1079–1093. Hartl, D. L., Dykhuizen, D. E., Miller, ert, G. E., Nichols, E. P., Settles, mapping of pAR060302, a blaCMY- Goh, E.-B., Yim, G., Tsui, W., McClure, R. D., Green, L., and De Framond, M., Abdo, Z., et al. (2012). The 2-positive broad-host-range IncA/C J., Surette, M. G., and Davies, J. J. (1983). Transposable element IS50 role of clonal interference in the plasmid. Appl. Environ. Microbiol. 78, (2002). Transcriptional modulation improvesgrowthrateofE. coli evolutionary dynamics of plasmid- 3379–3386. of bacterial gene expression by subin- cells without transposition. Cell 35, host adaptation. MBio 3, e00077–12. Lawrence, J. G. (1997). Selfish oper- hibitory concentrations of antibi- 503–510. Hughes, V. M., and Datta, N. (1983). ons and speciation by gene transfer. otics. Proc. Natl. Acad. Sci. U.S.A. 99, Henderson-Begg, S. K., Livermore, D. Conjugative plasmids in bacteria of Trends Microbiol. 5, 355–359. 17025–17030. M., and Hall, L. M. C. (2006). the “pre-antibiotic” era. Nature 302, Levin, B. R. (2010). Nasty viruses, costly Graessler, J., Qin, Y., Zhong, H., Effect of subinhibitory concentra- 725–726. plasmids, population dynamics, Zhang, J., Licinio, J., Wong, M.-L., tions of antibiotics on mutation Humphrey, B., Thomson, N. R., and the conditions for establishing et al. (2012). Metagenomic sequenc- frequency in Streptococcus pneumo- Thomas, C. M., Brooks, K., Sanders, and maintaining CRISPR-mediated ing of the human gut microbiome niae. J. Antimicrob. Chemother. 57, M., Delsol, A. A., et al. (2012). Fitness adaptive immunity in bacteria. before and after bariatric surgery in 849–854. of Escherichia coli strains carrying PLoS Genet. 6:e1001171. doi: obese patients with type 2 diabetes: Hermsen, R., Deris, J. B., and Hwa, expressed and partially silent IncN 10.1371/journal.pgen.1001171 correlation with inflammatory and T. (2012). On the rapidity of antibi- and IncP1 plasmids. BMC Microbiol. Lewontin, R. C. (1970). The units of metabolic parameters. Pharmacoge- otic resistance evolution facilitated 12:53. doi: 10.1186/1471-2180-12-53 selection. Annu. Rev. Ecol. Syst. 1, nomics J. doi: 10.1038/tpj.2012.43 by a concentration gradient. Proc. Jeffery, I. B., Claesson, M. J., O’Toole, 1–18. [Epub ahead of print]. Natl. Acad. Sci. U.S.A. 109, 10775– P. W., and Shanahan, F. (2012). Cat- Ley, R. E., Turnbaugh, P.J., Klein, S., and Graidy, N. G. (2002). “The movement 10780. egorization of the gut microbiota: Gordon, J. I. (2006). Microbial ecol- of Tn3-like elements. Transposase Heuer, H., Binh, C. T. T., Jechalke, enterotypes or gradients? Nat. Rev. ogy: human gut microbes associated and cointegrate resolution,”in Mobile S., Kopmann, C., Zimmerling, U., Microbiol. 10, 591–592. with obesity. Nature 444, 1022–1023. DNA II, eds. N. L. Craig, R. Craigie, Krögerrecklenfort, E., et al. (2012). Jones, B. V. (2010). The human Li, J. V., Ashrafian, H., Bueter, M., M. Gellet, and A. M. Lambowitz IncP-1ε plasmids are important vec- gut mobile metagenome: a meta- Kinross, J., Sands, C., Le Roux, C. (Washington: ASM Press), 272–304. tors of antibiotic resistance genes in zoan perspective. Gut Microbes 1, W., et al. (2011). Metabolic surgery Guerin, E., Cambray, G., Sanchez- agricultural systems: diversification 415–431. profoundly influences gut microbial- Alberola, N., Campoy, S., Erill, I., driven by class 1 integron gene cas- Kholodii, G., Mindlin, S., Petrova, M., host metabolic cross-talk. Gut 60, Da Re, S., et al. (2009). The SOS settes. Front. Microbiol. 3:2. doi: and Minakhina, S. (2003). Tn5060 1214–1223. response controls integron recombi- 10.3389/fmicb.2012.00002 from the Siberian permafrost is most Liebert, C. A., Hall, R. M., and nation. Science 324, 1034. Heuer, H., and Smalla, K. (2012). closely related to the ancestor of Tn21 Summers, A. O. (1999). Transpo- Guglielmini, J., De la Cruz, F., and Plasmids foster diversification and prior to integron acquisition. FEMS son Tn21, flagship of the floating Rocha, E. P. C. (2013). Evolution adaptation of bacterial populations in Microbiol. Lett. 226, 251–255. genome. Microbiol. Mol. Biol. Rev. 63, of conjugation and type IV secretion soil. FEMS Microbiol. Rev. 36, 1083– Kohanski, M. A., DePristo, M. A., 507–522. systems. Mol. Biol. Evol. 30, 315–331. 1104. and Collins, J. J. (2010). Sublethal Lim, J. Y., Yoon, J., and Hovde, Guglielmini, J., Quintais, L., Garcillán- Hickman, A. B., Chandler, M., and antibiotic treatment leads to mul- C. J. (2010). A brief overview of Barcia, M. P., De la Cruz, F., and Dyda, F. (2010). Integrating prokary- tidrug resistance via radical-induced Escherichia coli O157:H7 and its plas- Rocha, E. P. C. (2011). The repertoire otes and eukaryotes: DNA trans- mutagenesis. Mol. Cell 37, 311–320. mid O157. J. Microbiol. Biotechnol. of ICE in prokaryotes underscores the posases in light of structure. Crit. Kondrashov, F. A., Rogozin, I. B., 20, 5–14. unity, diversity, and ubiquity of con- Rev. Biochem. Mol. Biol. 45, Wolf, Y. I., and Koonin, E. V. Linares, J. F., Gustafsson, I., Baquero, F., jugation. PLoS Genet. 7:e1002222. 50–69. (2002). Selection in the evolution of and Martinez, J. L. (2006). Antibiotics doi: 10.1371/journal.pgen.1002222 Hidron, A. I., Edwards, J. R., Patel, J., gene duplications. Genome Biol. 3, as intermicrobial signaling agents Gullberg, E., Cao, S., Berg, O. G., Ilbäck, Horan, T. C., Sievert, D. M., Pol- research0008.1–research0008.9. instead of weapons. Proc. Natl. Acad. C., Sandegren, L., Hughes, D., et al. lock, D. A., et al. (2008). NHSN Ktari, S., Arlet, G., Mnif, B., Gau- Sci. U.S.A. 103, 19484–19489. (2011). Selection of resistant bac- annual update: antimicrobial- tier, V., Mahjoubi, F., Ben Jmeaa, Livermore, D. M. (2000). Antibiotic teria at very low antibiotic concen- resistant pathogens associated with M., et al. (2006). Emergence of resistance in staphylococci. Int. J. trations. PLoS Pathog. 7:e1002158. healthcare-associated infections: multidrug-resistant Klebsiella pneu- Antimicrob. Agents 16, S3–S10. doi:10.1371/journal.ppat.1002158 annual summary of data reported moniae isolates producing VIM-4 Lloyd, E. (2008). “Units and levels of Halary, S., Leigh, J. W., Cheaib, to the National Healthcare Safety metallo-beta-lactamase, CTX-M-15 selection,” in The Stanford Encyclope- B., Lopez, P., and Bapteste, E. Network at the Centers for Disease extended-spectrum beta-lactamase, dia of Philosophy, ed. E. N. Zalta. (2010). Network analyses struc- Control and Prevention, 2006-2007. and CMY-4 AmpC beta-lactamase Looft, T., and Allen, H. K. (2012). ture genetic diversity in independent Infect. Control Hosp. Epidemiol. 29, in a Tunisian university hospital. Collateral effects of antibiotics on genetic worlds. Proc. Natl. Acad. Sci. 996–1011. Antimicrob. Agents Chemother. 50, mammalian gut microbiomes. Gut U.S.A. 107, 127–132. Hildebrandt, M. A., Hoffmann, C., 4198–4201. Microbes 3, 463–467. Hallatschek, O. (2012). Bacteria evolve Sherrill-Mix, S. A., Keilbaugh, S. Kylafis, G., and Loreau, M. (2011). Looft, T., Johnson, T. A., Allen, H. K., to go against the grain. Physics 5, 93. A., Hamady, M., Chen, Y.-Y., et al. Niche construction in the light of Bayles, D. O., Alt, D. P., Stedtfeld, Halling-Sørensen, B., Nors Nielsen, S., (2009). High-fat diet determines niche theory. Ecol. Lett. 14, 82–90. R. D., et al. (2012). In-feed antibiotic Lanzky, P. F., Ingerslev, F., Holten the composition of the murine gut Labbate, M., Case, R. J., and Stokes, H. effects on the swine intestinal micro- Lützhøft, H. C., and Jørgensen, S. E. microbiome independently of obe- W.(2009). The integron/gene cassette biome. Proc. Natl. Acad. Sci. U.S.A. (1998). Occurrence, fate and effects sity. Gastroenterology 137, 1716– system: an active player in bacterial 109, 1691–1696. of pharmaceutical substances in the 1724. adaptation. Methods Mol. Biol. 532, Luo, C., Walk, S. T., Gordon, environment–a review. Chemosphere Hocquet, D., Llanes, C., Thouverez, 103–125. D. M., Feldgarden, M., Tiedje, 36, 357–393. M., Kulasekara, H. D., Bertrand, Laland, K. N., Odling-Smee, F. J., and J. M., and Konstantinidis, K. T. Hamprecht, A., Poirel, L., Göttig, S., X., Plésiat, P., et al. (2012). Evi- Feldman, M. W. (1999). Evolutionary (2011). Genome sequencing of envi- Seifert, H., Kaase, M., and Nord- dence for induction of integron- consequences of niche construction ronmental Escherichia coli expands mann, P. (2012). Detection of the based antibiotic resistance by the and their implications for ecology. understanding of the ecology and

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 15 | 35

“fmicb-04-00015” — 2013/3/5 — 11:06 — page 12 — #12 Baquero et al. Multi-level population biology of resistance

speciation of the model bacterial PloS ONE 2:e582. doi: 10.1371/jour- Norman, A., Hansen, L. H., and Partridge, S. R. (2011). Analysis of species. Proc. Natl. Acad. Sci. U.S.A. nal.pone.0000582 Sørensen, S. J. (2009). Conjuga- antibiotic resistance regions in Gram- 108, 7200–7205. Merlin, C., Mahillon, J., Nesvera, J., and tive plasmids: vessels of the com- negative bacteria. FEMS Microbiol. Lupo, A., Coyne, S., and Beren- Touissant,A. (2000). “Gene recruiters munal gene pool. Philos. Trans. R. Rev. 35, 820–855. donk, T. U. (2012). Origin and and transporters: the modular struc- Soc. Lond. B Biol. Sci. 364, 2275– Partridge, S. R., and Hall, R. M. (2004). evolution of antibiotic resistance: ture of bacterial mobile elements,” in 2289. Complex multiple antibiotic and the common mechanisms of The Horizontal Gene Pool. Bacterial Novais, A., Baquero, F., Machado, mercury resistance region derived emergence and spread in water Plasmids and Gene Spread, ed. C. M. E., Cantón, R., Peixe, L., and from the r-det of NR1 (R100). bodies. Front. Microbiol. 3:18. doi: Thomas (Harwood Academic Press), Coque, T. M. (2010a). Interna- Antimicrob. Agents Chemother. 48, 10.3389/fmicb.2012.00018 363–409. tional spread and persistence of 4250–4255. Maiden, M. C. (1998). Horizontal Mindlin, S., Minakhin, L., Petrova, TEM-24 is caused by the confluence Partridge, S. R., and Hall, R. M. genetic exchange, evolution, and M., Kholodii, G., Minakhina, S., of highly penetrating enterobacteri- (2003). The IS1111 family members spread of antibiotic resistance in bac- Gorlenko, Z., et al. (2005). Present- aceae clones and an IncA/C2 plasmid IS4321 and IS5075 have subtermi- teria. Clin. Infect. Dis. 27, S12–S20. day mercury resistance transposons containing Tn1696::Tn1 and IS5075- nal inverted repeats and target the Marchesi, J. R. (2010). Prokaryotic and are common in bacteria preserved in Tn21. Antimicrob. Agents Chemother. terminal inverted repeats of Tn21 eukaryotic diversity of the human permafrost grounds since the Upper 54, 825–834. family transposons. J. Bacteriol. 185, gut. Adv. Appl. Microbiol. 72, 43–62. Pleistocene. Res. Microbiol. 156, 994– Novais, A., Comas, I., Baquero, F., 6371–6384. Marchesi, J. R. (2011). Shifting 1004. Cantón, R., Coque, T. M., Moya, Partridge, S. R., and Iredell, J. R. (2012). from a gene-centric to metabolite- Mindlin, S. Z., Soina, V. S., Petrova, A., et al. (2010b). Evolutionary tra- Genetic Contexts of blaNDM-1. centric strategy to determine the core M. A., and Gorlenko, Z. M. (2008). jectories of beta-lactamase CTX-M-1 Antimicrob. Agents Chemother. 56, gut microbiome. Bioeng. Bugs 2, Isolation of antibiotic resistance bac- cluster enzymes: predicting antibiotic 6065–6067. 309–314. terial strains from Eastern Siberia resistance. PLoS Pathog. 6:e1000735. Pedró, L., Baños, R. C., Aznar, S., Marraffini, L. A., and Sontheimer, E. J. permafrost sediments. Russ. J. Genet. doi: 10.1371/journal.ppat.1000735 Madrid, C., Balsalobre, C., and (2010). CRISPR interference: RNA- 44, 27–34. Novais, A., Pires, J., Ferreira, H., Costa, Juárez, A. (2011). Antibiotics shaping directed adaptive immunity in bacte- Mizrahi, I. (2012). The rumen plas- L., Montenegro, C., Vuotto, C., et al. bacterial genome: deletion of an IS91 ria and archaea. Nat. Rev. Genet. 11, midome: a genetic communication (2012a). Characterization of glob- flanked virulence determinant upon 181–190. hub for the rumen microbiome. Mob. ally spread Escherichia coli ST131 exposure to subinhibitory antibiotic Martinez, E., Marquez, C., Ingold, A., Genet. Elements 2, 152–153. isolates (1991 to 2010). Antimi- concentrations. PloS ONE 6:e27606. Merlino, J., Djordjevic, S. P., Stokes, Monira, S., Nakamura, S., Gotoh, K., crob. Agents Chemother. 56, 3973– doi: 10.1371/journal.pone.0027606 H. W., et al. (2012). Diverse mobi- Izutsu, K., Watanabe, H., Alam, N. 3976. Perron, G. G., Gonzalez, A., and Buck- lized class 1 integrons are common H., et al. (2011). Gut microbiota of Novais, C., Freitas, A. R., Silveira, E., ling, A. (2007). Source-sink dynam- in the chromosomes of pathogenic healthy and malnourished children in Baquero, F., Peixe, L., Roberts, A. ics shape the evolution of antibiotic Pseudomonas aeruginosa clinical iso- bangladesh. Front. Microbiol. 2:228. P., et al. (2012b). A tet(S/M) hybrid resistance and its pleiotropic fitness lates. Antimicrob. Agents Chemother. doi: 10.3389/fmicb.2011.00228 from CTn6000 and CTn916 recom- cost. Proc. Biol. Sci. 274, 2351– 56, 2169–2172. Moura, A., Pereira, C., Henriques, I., bination. Microbiology 158, 2710– 2356. Martinez, J. L., Fajardo, A., Garmen- and Correia, A. (2012). Novel gene 2711. Petrova, M., Gorlenko, Z., and Mindlin, dia, L., Hernandez, A., Linares, J. cassettes and integrons in antibiotic- Novais, A., Vuotto, C., Pires, J., Mon- S. (2009). Molecular structure and F., Martínez-Solano, L., et al. (2009). resistant bacteria isolated from urban tenegro, C., Donelli, G., Coque, T. M., translocation of a multiple antibi- A global view of antibiotic resis- wastewaters. Res. Microbiol. 163, et al. (2013). Diversity and biofilm- otic resistance region of a Psy- tance. FEMS Microbiol. Rev. 33, 92–100. production ability among isolates chrobacter psychrophilus permafrost 44–65. Naas, T., Cuzon, G., Villegas, M.-V.,Lar- of epidemic clonal groups (ST69, strain. FEMS Microbiol. Lett. 296, Martínez, J. L. (2008). Antibiotics and tigue, M.-F., Quinn, J. P., and Nord- ST393 and ST405) of Escherichia coli 190–197. antibiotic resistance genes in natural mann, P.(2008). Genetic structures at phylogroup D. Antimicrob. Agents Petrova, M., Gorlenko, Z., and Mindlin, environments. Science 321, 365–367. the origin of acquisition of the beta- Chemother. (in press). S. (2011). Tn5045, a novel integron- Martínez, J. L. (2011). Bottlenecks in lactamase bla KPC gene. Antimi- Näsvall, J., Sun, L., Roth, J. R., and containing antibiotic and chromate the transferability of antibiotic resis- crob. Agents Chemother. 52, 1257– Andersson, D. I. (2012). Real-time resistance transposon isolated from a tance from natural ecosystems to 1263. evolution of new genes by innova- permafrost bacterium. Res. Microbiol. human bacterial pathogens. Front. Negri, M.-C., Lipsitch, M., Blazquez, J., tion, amplification, and divergence. 162, 337–345. Microbiol. 2:265. doi: 10.3389/fmicb. Levin, B. R., and Baquero, F. (2000). Science 338, 384–387. Poirel, L., Bonnin, R. A., and Nord- 2011.00265 Concentration-dependent selection Okasha, S. (2004). Multilevel selection mann, P. (2012). Genetic support Martínez, J. L., Baquero, F., and Ander- of small phenotypic differences and the partitioning of covariance: and diversity of acquired extended- sson, D. I. (2007). Predicting antibi- in TEM beta-lactamase-mediated a comparison of three approaches. spectrum β-lactamases in Gram- otic resistance. Nat. Rev. Microbiol. 5, antibiotic resistance. Antimicrob. Evolution 58, 486–494. negative rods. Infect.Genet.Evol.12, 958–965. Agents Chemother. 44, 2485–2491. Osterblad, M., Norrdahl, K., Kor- 883–893. Martínez, J. L., and Rojo, F. (2011). Nogueira, T., Rankin, D. J., Touchon, pimäki, E., and Huovinen, P. Poirel, L., Rodríguez-Martínez, J.- Metabolic regulation of antibiotic M., Taddei, F., Brown, S. P., and (2001). Antibiotic resistance. How M., Al Naiemi, N., Debets- resistance. FEMS Microbiol. Rev. 35, Rocha, E. P. C. (2009). Horizontal wild are wild mammals? Nature 409, Ossenkopp, Y. J., and Nordmann, 768–789. gene transfer of the secretome drives 37–38. P. (2010). Characterization of DIM- Mayr, E. (1997). The objects of selec- the evolution of bacterial coopera- Parks, A. R., Li, Z., Shi, Q., Owens, 1, an integron-encoded metallo- tion. Proc. Natl. Acad. Sci. U.S.A. 94, tion and virulence. Curr. Biol. 19, R. M., Jin, M. M., and Peters, J. E. beta-lactamase from a Pseudomonas 2091–2094. 1683–1691. (2009). Transposition into replicat- stutzeri clinical isolate in the Nether- Mazel, D. (2006). Integrons: agents of Norberg, P., Bergström, M., Jethava, ing DNA occurs through interaction lands. Antimicrob. Agents Chemother. bacterial evolution. Nat. Rev. Micro- V., Dubhashi, D., and Hermansson, with the processivity factor. Cell 138, 54, 2420–2424. biol. 4, 608–620. M. (2011). The IncP-1 plasmid back- 685–695. Pomiankowski, A. (1999). “Intrage- McBride, S. M., Fischetti, V. A., bone adapts to different host bacterial Parks, A. R., and Peters, J. E. (2009). Tn7 nomic conflict,” in Levels of Leblanc, D. J., Moellering, R. C., species and evolves through homolo- elements: engendering diversity from Selection in Evolution, ed. L. and Gilmore, M. S. (2007). Genetic gous recombination. Nat. Commun. chromosomes to episomes. Plasmid Keller (Princeton University Press), diversity among Enterococcus faecalis. 2, 268. 61, 1–14. 121–152.

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 36

“fmicb-04-00015” — 2013/3/5 — 11:06 — page 13 — #13 Baquero et al. Multi-level population biology of resistance

Popa, O., Hazkani-Covo, E., Lan- Gut microbiota composition is asso- E. J. (2011). Ecology drives a global Tamminen, M., Virta, M., Fani, R., and dan, G., Martin, W., and Dagan, ciated with body weight, weight network of gene exchange connect- Fondi, M. (2012). Large-scale analy- T. (2011). Directed networks reveal gain and biochemical parameters in ing the human microbiome. Nature sis of plasmid relationships through genomic barriers and DNA repair pregnant women. Br. J. Nutr. 104, 480, 241–244. gene-sharing networks. Mol. Biol. bypasses to lateral gene transfer 83–92. Smith, J. (2012). Tragedy of the com- Evol. 29, 1225–1240. among prokaryotes. Genome Res. 21, Schlüter, A., Szczepanowski, R., Pühler, mons among antibiotic resistance Tato, M., Coque, T. M., Baquero, 599–609. A., and Top, E. M. (2007). Genomics plasmids. Evolution 66, 1269–1274. F., and Cantón, R. (2010). Disper- Post, V., and Hall, R. M. (2009). Inser- of IncP-1 antibiotic resistance plas- Smorawinska, M., Szuplewska, M., sal of carbapenemase blaVIM−1 gene tion sequences in the IS1111 family mids isolated from wastewater treat- Zaleski, P., Wawrzyniak, P., Maj, associated with different Tn402 vari- that target the attC recombination ment plants provides evidence for A., Plucienniczak, A., et al. (2012). ants, mercury transposons, and con- sites of integron-associated gene cas- a widely accessible drug resistance Mobilizable narrow host range plas- jugative plasmids in Enterobacteri- settes. FEMS Microbiol. Lett. 290, gene pool. FEMS Microbiol. Rev. 31, mids as natural suicide vectors aceae and Pseudomonas aeruginosa. 182–187. 449–477. enabling horizontal gene transfer Antimicrob. Agents Chemother. 54, Potron, A., Nordmann, P., Rondinaud, Sen, D., Van der Auwera, G. A., Rogers, among distantly related bacterial 320–327. E., Jaureguy, F., and Poirel, L. (2013). L. M., Thomas, C. M., Brown, species. FEMS Microbiol. Lett. 326, Tetu, S. G., and Holmes, A. J. (2008). A mosaic transposon encoding OXA- C. J., and Top, E. M. (2011). 76–82. A family of insertion sequences that 48 and CTX-M-15: towards pan- Broad-host-range plasmids from Sobecky, P. A., and Hazen, T. H. impacts integrons by specific target- resistance. J. Antimicrob. Chemother. agricultural soils have IncP-1 (2009). Horizontal gene transfer and ing of gene cassette recombination 68, 476–477. backbones with diverse accessory mobile genetic elements in marine sites, the IS1111-attC Group. J. Bac- Qu,Y.,and Spain, J. C. (2011). Catabolic genes. Appl. Environ. Microbiol. 77, systems. Methods Mol. Biol. 532, teriol. 190, 4959–4970. pathway for 2-nitroimidazole 7975–7983. 435–453. Toleman, M. A., Bennett, P. M., and involves a novel nitrohydrolase Seputiene, V., Povilonis, J., and Sommer, M. O. A., Dantas, G., and Walsh, T. R. (2006). ISCR elements: that also confers drug resistance. Suziedeliene, E. (2012). Novel vari- Church, G. M. (2009). Functional novel gene-capturing systems of the Environ. Microbiol. 13, 1010–1017. ants of AbaR resistance islands with characterization of the antibiotic 21st century? Microbiol. Mol. Biol. Quinteira, S., Novais, C., Antunes, acommonbackboneinAcinetobacter resistance reservoir in the human Rev. 70, 296–316. P., Campos, J., Freitas, A. R., baumannii isolates of European clone microflora. Science 325, 1128– Toleman,M. A., and Walsh,T. R. (2011). Abdukadir, A., et al. (2011). Track- II. Antimicrob. Agents Chemother. 56, 1131. Combinatorial events of insertion ing antibiotic resistance along the Silk 1969–1973. Stalder, T., Barraud, O., Casellas, M., sequences and ICE in Gram-negative Road. Clin. Microbiol. Infect. 17, Shearer, J. E. S., Wireman, J., Dagot, C., and Ploy, M.-C. (2012). bacteria. FEMS Microbiol. Rev. 35, s58–s59. Hostetler, J., Forberger, H., Bor- Integron involvement in environ- 912–935. Rankin, D. J., Ginty, S. E. M., Nogueira, man, J., Gill, J., et al. (2011). Major mental spread of antibiotic resis- Tolmasky, M. E. (1990). Sequencing and T., Touchon, M., Taddei, F., Rocha, E. families of multiresistant plasmids tance. Front. Microbiol. 3:119. doi: expression of aadA, bla, and tnpR P. C., et al. (2011a). Bacterial cooper- from geographically and epidemio- 10.3389/fmicb.2012.00119 from the multiresistance transposon ation controlled by mobile elements: logically diverse staphylococci. G3 1, Stokes, H. W., and Gillings, M. R. Tn1331. Plasmid 24, 218–226. kin selection and infectivity are part 581–591. (2011). Gene flow, mobile genetic ele- Ubeda, C., Maiques, E., Knecht, E., of the same process. Heredity 107, Shi, P., Jia, S., Zhang, X.-X., Zhang, ments and the recruitment of antibi- Lasa, I., Novick, R. P., and Penadés, 279–281. T., Cheng, S., and Li, A. (2013). otic resistance genes into Gram- J. R. (2005). Antibiotic-induced Rankin, D. J., Rocha, E. P.C., and Brown, Metagenomic insights into chlorina- negative pathogens. FEMS Microbiol. SOS response promotes horizontal S. P. (2011b). What traits are carried tion effects on microbial antibiotic Rev. 35, 790–819. dissemination of pathogenicity on mobile genetic elements, and why? resistance in drinking water. Water Stokes, H. W.,Martinez, E., Roy Chowd- island-encoded virulence factors in Heredity 106, 1–10. Res. 47, 111–120. hury, P., and Djordjevic, S. (2012). staphylococci. Mol. Microbiol. 56, Rankin, D. J., Turner, L. A., Heinemann, Silva, R. F., Mendonça, S. C. M., Class 1 integron-associated spread 836–844. J. A., and Brown, S. P. (2012). The Carvalho, L. M., Reis, A. M., of resistance regions in Pseudomonas Vallès, Y., Gosalbes, M. J., De Vries, L. coevolution of toxin and antitoxin Gordo, I., Trindade, S., et al. aeruginosa: plasmid or chromosomal E., Abellán, J. J., and Francino, M. P. genes drives the dynamics of bacte- (2011). Pervasive sign epistasis platforms? J. Antimicrob. Chemother. (2012). Metagenomics and develop- rial addiction complexes and intrage- between conjugative plasmids and 67, 1799–1800. ment of the gut microbiota in infants. nomic conflict. Proc. Biol. Sci. 279, drug-resistance chromosomal muta- Suzuki, H., Yano, H., Brown, C. J., and Clin. Microbiol. Infect. 18, 21–26. 3706–3715. tions. PLoS Genet. 7:e1002181. doi: Top, E. M. (2010). Predicting plas- Valverde, A., Cantón, R., Garcillán- Revilla, C., Garcillán-Barcia, M. P., 10.1371/journal.pgen.1002181 mid promiscuity based on genomic Barcia, M. P., Novais, A., Galán, J. Fernández-López, R., Thomson, N. Sjölund, M., Bonnedahl, J., Hernan- signature. J. Bacteriol. 192, 6045– C., Alvarado, A., et al. (2009). Spread R., Sanders, M., Cheung, M., et al. dez, J., Bengtsson, S., Cederbrant, G., 6055. of bla(CTX-M-14) is driven mainly (2008). Different pathways to acquir- Pinhassi, J., et al. (2008). Dissemina- Sánchez, M. B., and Martínez, J. L. by IncK plasmids disseminated ing resistance genes illustrated by the tion of multidrug-resistant bacteria (2012). Differential epigenetic com- among Escherichia coli phylogroups recent evolution of IncW plasmids. into the Arctic. Emerg. Infect. Dis. 14, patibility of qnr antibiotic resis- A, B1, and D in Spain. Antimi- Antimicrob. Agents Chemother. 52, 70–72. tance determinants with the chro- crob. Agents Chemother. 53, 5204– 1472–1480. Skippington, E., and Ragan, M. A. mosome of Escherichia coli. PloS 5212. Rodriguez-Valera, F., and Ussery, D. (2011). Lateral genetic transfer and ONE 7:e35149. doi: 10.1371/jour- Via, S. (2012). Divergence hitchhik- W. (2012). Is the pan-genome also a the construction of genetic exchange nal.pone.0035149 ing and the spread of genomic iso- pan-selectome? F1000 Res. 1:16. communities. FEMS Microbiol. Rev. Tagliabue, A., and Elli, M. (2012). The lation during ecological speciation- San Millán, A., Heilbron, K., and 35, 707–735. role of gut microbiota in human obe- with-gene-flow. Philos. Trans. R. Soc. MacLean, R. C. (2012). Positive epis- Skippington, E., and Ragan, M. A. sity: Recent findings and future per- Lond. B Biol. Sci. 367, 451–460. tasis between co-infecting plasmids (2012). Phylogeny rather than ecol- spectives. Nutr. Metab. Cardiovasc. Vishnivetskaya, T. A., and Kathariou, promotes plasmid stability and main- ogy or lifestyle biases the con- Dis. (in press). S. (2005). Putative transposases con- tenance of multidrug resistance. in struction of Escherichia coli-Shigella Tamames, J., Abellán, J. J., Pignatelli, served in Exiguobacterium isolates IPBC (Santander). genetic exchange communities. Open M., Camacho, A., and Moya, A. from ancient Siberian permafrost and Santacruz, A., Collado, M. C., García- Biol. 2, 120112. (2010). Environmental distribution from contemporary surface habitats. Valdés, L., Segura, M. T., Martín- Smillie, C. S., Smith, M. B., Friedman, J., of prokaryotic taxa. BMC Microbiol. Appl. Environ. Microbiol. 71, 6954– Lagos, J. A., Anjos, T., et al. (2010). Cordero, O. X., David, L. A., and Alm, 10:85. doi: 10.1186/1471-2180-10-85 6962.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 15 | 37

“fmicb-04-00015” — 2013/3/5 — 11:06 — page 14 — #14 Baquero et al. Multi-level population biology of resistance

Waksman, S. A. (1961). “The role of arrays of accessory genes on com- regulator that alters host function. J. for mating pair formation in plasmid antibiotics in nature,” in Prespec- mon replicon families. Plasmid 69, Bacteriol. 192, 4720–4731. population dynamics. J. Theor. Biol. tives in Biology and Medicine, Vol. 4 36–48. Zalasiewicz, J., Williams, M., Haywood, 262, 711–719. (Chicago: University of Chicago Woodford, N., Turton, J. F., and Liv- A., and Ellis, M. (2011). The Anthro- Press), 271–287. ermore, D. M. (2011). Multiresistant pocene: a new epoch of geological Conflict of Interest Statement: The Walsh, T. R. (2006). Combinatorial Gram-negative bacteria: the role of time? Philos. Transact. A Math. Phys. authors declare that the research was genetic evolution of multiresistance. high-risk clones in the dissemina- Eng. Sci. 369, 835–841. conducted in the absence of any com- Curr. Opin. Microbiol. 9, 476–482. tion of antibiotic resistance. FEMS Zhang, Q., Lambert, G., Liao, D., mercial or financial relationships that Werren, J. H. (2011). Selfish genetic Microbiol. Rev. 35, 736–755. Kim, H., Robin, K., Tung, C., et al. could be construed as a potential con- elements, genetic conflict, and evolu- Wozniak, R. A. F., and Waldor, M. K. (2011). Acceleration of emergence of flict of interest. tionary innovation. Proc. Natl. Acad. (2010). Integrative and conjugative bacterial antibiotic resistance in con- Sci. U.S.A. 108, 10863–10870. elements: mosaic mobile genetic nected microenvironments. Science Received: 14 December 2012; paper pend- Wiedenbeck, J., and Cohan, F. M. elements enabling dynamic lateral 333, 1764–1767. ing published: 08 January 2013; accepted: (2011). Origins of bacterial diver- gene flow. Nat. Rev. Microbiol. 8, Zheng, Y., Anton, B. P., Roberts, R. 22 January 2013; published online: 06 sity through horizontal genetic trans- 552–563. J., and Kasif, S. (2005). Phylogenetic March 2013. fer and adaptation to new ecological Wright, M. S., Baker-Austin, C., Lin- detection of conserved gene clusters Citation: Baquero F, Tedim AP and niches. FEMS Microbiol. Rev. 35, dell, A. H., Stepanauskas, R., Stokes, in microbial genomes. BMC Bioin- Coque TM (2013) Antibiotic resistance 957–976. H. W., and McArthur, J. V. (2008). formatics 6:243. doi:10.1186/1471- shaping multi-level population biology Willems, R. J. L., Hanage, W. P., Influence of industrial contamina- 2105-6-243 of bacteria. Front. Microbiol. 4:15. doi: Bessen, D. E., and Feil, E. J. (2011). tion on mobile genetic elements: Zheng, Y., Roberts, R. J., and Kasif, S. 10.3389/fmicb.2013.00015 Population biology of Gram-positive class 1 integron abundance and (2004). Segmentally variable genes: This article was submitted to Fron- pathogens: high-risk clones for dis- gene cassette structure in aquatic a new perspective on adaptation. tiers in Antimicrobials, Resistance and semination of antibiotic resistance. bacterial communities. ISME J. 2, PLoS Biol. 2:E81. doi: 10.1371/jour- Chemotherapy, a specialty of Frontiers in FEMS Microbiol. Rev. 35, 872–900. 417–428. nal.pbio.0020081 Microbiology. Willems, R. J. L., Top, J., Van Schaik, W., Yim, G., Wang, H. H., and Davies, Zhong, X., Droesch, J., Fox, R., Top, Copyright © 2013 Baquero, Tedim and Leavis, H., Bonten, M., Sirén, J., et al. J. (2007). Antibiotics as signalling E. M., and Krone, S. M. (2012). Coque. This is an open-access article dis- (2012). Restricted gene flow among molecules. Philos. Trans. R. Soc. Lond. On the meaning and estimation of tributed under the terms of the Creative hospital subpopulations of Enterococ- B. Biol. Sci. 362, 1195–1200. plasmid transfer rates for surface- Commons Attribution License, which cus faecium. MBio 3, e00151–12. Yun, C.-S., Suzuki, C., Naito, K., Takeda, associated and well-mixed bacterial permits use, distribution and reproduc- Williams, L. E., Wireman, J., Hilliard, T., Takahashi, Y., Sai, F., et al. (2010). populations. J. Theor. Biol. 294, tion in other forums, provided the origi- V. C., and Summers, A. O. (2012). Pmr, a histone-like protein H1 (H- 144–152. nal authors and source are credited and Large plasmids of Escherichia coli NS) family protein encoded by the Zhong, X., Krol, J. E., Top, E. M., subject to any copyright notices concern- and Salmonella encode highly diverse IncP-7 plasmid pCAR1, is a key global and Krone, S. M. (2010). Accounting ing any third-party graphics etc.

www.frontiersin.org March 2013 | Volume 4 | Article 15 | 38

“fmicb-04-00015” — 2013/3/5 — 11:06 — page 15 — #15 REVIEW ARTICLE published: 08 February 2013 doi: 10.3389/fmicb.2013.00009 Antibiotics as selectors and accelerators of diversity in the mechanisms of resistance: from the resistome to genetic plasticity in the β-lactamases world

Juan-Carlos Galán1,2,3*, Fernando González-Candelas4,5, Jean-Marc Rolain6,7 and Rafael Cantón1,3 1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain 2 Centros de Investigación Biomédica en Red en Epidemiología y Salud Pública, Instituto Ramón y Cajal de Investigación Sanitaria, Madrid, Spain 3 Unidad de Resistencia a Antibióticos y Virulencia Bacteriana Asociada al Consejo Superior de Investigaciones Cientíﬁcas, Madrid, Spain 4 Joint Research Unit “Genomics and Health”,Centro Superior de Investigación en Salud Pública, Universitat de València, Barcelona, Spain 5 Centros de Investigación Biomédica en Red en Epidemiología y Salud Pública, Barcelona, Spain 6 Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergents, Centre National de la Recherche Scientiﬁque – Institut de Recherche pour le Développement, UMR 6236, Institut Hospitalo-Universitaire Méditerranée Infection, Aix-Marseille Université Faculté de Médecine et de Pharmacie, Marseille, France 7 Fédération de Microbiologie Clinique, Hôpital de la Timone, Marseille, France

Edited by: Antibiotics and antibiotic resistance determinants, natural molecules closely related to Fiona Walsh, Agroscope Changins bacterial physiology and consistent with an ancient origin, are not only present in antibiotic- Wädenswil, Switzerland producing bacteria. Throughput sequencing technologies have revealed an unexpected Reviewed by: reservoir of antibiotic resistance in the environment. These data suggest that co-evolution Dmitri Debabov, NovaBay Pharmaceuticals, USA between antibiotic and antibiotic resistance genes has occurred since the beginning of Yoshimi Matsumoto, Osaka time.This evolutionary race has probably been slow because of highly regulated processes University, Japan and low antibiotic concentrations. Therefore to understand this global problem, a new *Correspondence: variable must be introduced, that the antibiotic resistance is a natural event, inherent to Juan-Carlos Galán, Servicio de life. However, the industrial production of natural and synthetic antibiotics has dramatically Microbiología, Hospital Universitario Ramón y Cajal, Carretera de accelerated this race, selecting some of the many resistance genes present in nature and Colmenar, km. 9.1, Madrid contributing to their diversification. One of the best models available to understand the 28034, Spain. biological impact of selection and diversification are β-lactamases.They constitute the most e-mail: [email protected] widespread mechanism of resistance, at least among pathogenic bacteria, with more than 1000 enzymes identified in the literature. In the last years, there has been growing concern about the description, spread, and diversification of β-lactamases with carbapenemase activity and AmpC-type in plasmids. Phylogenies of these enzymes help the understanding of the evolutionary forces driving their selection. Moreover, understanding the adaptive potential of β-lactamases contribute to exploration the evolutionary antagonists trajectories through the design of more efficient synthetic molecules. In this review, we attempt to analyze the antibiotic resistance problem from intrinsic and environmental resistomes to the adaptive potential of resistance genes and the driving forces involved in their diversification, in order to provide a global perspective of the resistance problem.

Keywords: environmental resistome, intrinsic resistome, plasticity, β-lactamase

INTRODUCTION constant arms-race over a long time (Fajardo and Martínez, 2008). Nowadays and thanks to the high-throughput sequencing tools So today the intrinsic resistome is a wider concept and prob- and bioinformatics software, knowledge on high bacterial diver- ably universal to the bacterial world. The description of the sity in bacterial communities (metagenome) is increasing. A huge intrinsic resistome has expanded our knowledge about poten- diversity of resistance mechanisms to practically all antibiotic tial new resistance mechanisms (Dantas et al., 2008; Toleman families has been found in both antibiotic- and non-antibiotic- et al., 2012). They could become true antibiotic resistance genes if producing bacteria (D’Costa et al., 2006; Bhullar et al., 2012). appropriate driving forces were exerted. Moreover, the potential Three types of resistome can be defined: intrinsic, environmen- adaptiveness of these pre-resistome genes can be accelerated if, by tal, and unknown (Fajardo et al., 2008; Martínez, 2008; Dantas chance, they are transferred to new genetic contexts (exaptation; and Sommer, 2012). In the intrinsic resistome or pre-resistome, Figure 1), where these genes may evolve toward more efficient the antibiotic resistant elements belong to bacterial metabolic net- enzymes without having a physiological role (Baquero et al., 2009; works, reflecting their role in microbial physiology. They might Gullberg et al., 2011). As a consequence, this silent and non- be coupled to signaling molecules (antibiotics) facilitating the predictive resistome (unknown resistome) is ready to be selected co-selection of antibiotics and antibiotic resistance genes in a (Baquero, 2012).

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 39

“fmicb-04-00009” — 2013/2/7 — 12:09 — page1—#1 Galán et al. Exploring the diversity of β-lactamases

FIGURE 1 | Overview evolutionary processes of the antibiotic true and more efficient antibiotic resistance genes. However, the resistance. During millions of years antibiotics and antibiotic resistance great evolutionary transition was the discovery, mass production and genes have co-evolved slowly. In this long period the first transition consumption of antibiotics. Antibiotics accelerated dramatically the was the acquisition of pre-resistance genes by different bacteria diversification of resistance genes and selection for reaching extraordinary (exaptation). This genetic transference allowed the evolution toward efficient variants.

Efforts to understand the spread of antibiotic resistance and high concentration of antibiotics (Martínez et al., 2007). There- find previously undescribed antibiotic resistance mechanisms in fore, antibiotics are not only selector agents of mechanisms of non-human environments have yielded a more complex view of resistance; they are also accelerator agents of the evolution of the antibiotic resistance problem (D’Costa et al., 2006; Dantas resistance (Figure 1). This is a consequence of the huge plas- et al., 2008; Bhullar et al., 2012). Usually, the bacterial population- ticity observed in antibiotic resistance genes for acquiring a new nutrient concentration ratio is low in the environment and a model spectrum of action or more efficient capacities with respect to the of competitive interactions among microorganisms is established. original spectrum (Deng et al., 2012). This observation suggests Microbial competitors are able to produce high levels of natural that the diversification force of resistance elements can be as fast antibiotics that become toxic compounds killing non-producer and strong as the antibiotic diversification. For instance, the golden competitor strains in order to access the limited nutrients. How- age in the commercialization for clinical use of β-lactam antibiotics ever, antibiotic susceptible strains were able to develop strategies was between the late 1970s and mid 1980s. Based on dated phy- conferring resistance to these antibiotics, constituting the environ- logenetic reconstructions of the most important β-lactamases in mental resistome, starting adaptation, and co-adaptation cycles the clinical setting, the majority of diversification events occurred between antibiotics and antibiotic-resistant proteins (Martínez, recently, even though the enzymes have been present for millions 2009). New sequencing platforms have revealed that our old of years (Barlow and Hall, 2002a,b). In vitro models with CTX-M concept that only the antibiotic-producing bacteria were carri- β-lactamases suggest that the explosive molecular diversification of ers of antibiotic resistance mechanisms was a very simple view. these enzymes could only be explained by the simultaneous pres- The scientific community has gradually begun to understand that ence of different extended spectrum cephalosporins (Novais et al., antibiotic resistance has a global distribution in nature, even with- 2010). Similar results were found with TEM enzymes (Salverda out the presence of humans, and that antibiotics and antibiotic et al., 2011), suggesting that both the environmental contamina- resistance mechanisms have been evolving for millions of years tion with different β-lactams and the potential plasticity of these (Wright, 2010). enzymes could have been the main diversifying forces. Tradition- The capture of antibiotic resistance or pre-resistance genes from ally, the problem of antibiotic resistance has been focused as a intrinsic, environmental, or unknown resistomes is a stochastic, clinical problem. Obviously, human health is the main reason, unpredictable phenomenon. Once the determinants of resistance but we will never be able to cope with the antibiotic resistance reach the human environments or environments related to human problem if it is only seen as such. In this review, we attempt activity, the evolutionary race between antibiotics and antibiotic to analyze the antibiotic resistance problem from new perspec- resistance genes is drastically accelerated, as a consequence of the tives. From the intrinsic resistome to the potential adaptiveness

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 40

“fmicb-04-00009” — 2013/2/7 — 12:09 — page2—#2 Galán et al. Exploring the diversity of β-lactamases

of determinants of resistance, from the environmental resistome physiological role in the normal course of peptidoglycan synthe- to the driving forces involved in the diversity of variants related sis, remodeling and recycling the bacterial envelope (Jacobs et al., to specific determinants of resistance, as well as to provide a 1994; Henderson et al., 1997). global view of the antibiotic resistance problem (Davies, 2007; For years recognition of the intrinsic resistome has not been Martínez et al., 2009). an easy task. This will not be the case in the coming years as the implementation of whole genome sequencing strategies and INTRINSIC RESISTANCE OR INTRINSIC RESISTOME bioinformatic tools for genome comparisons and knockout pro- In the EUCAST expert rules on antimicrobial susceptibility testing, cedures will increase the recognition of determinants that might “intrinsic resistance”or“inherent resistance”is understood as a fea- be involved in resistance phenotypes, even if they are very lowly ture of all or almost all isolates of a bacterial species and in contrast expressed (Didelot et al., 2012; Huang et al., 2012; Schmieder to the acquired and/or mutational resistance concepts (Leclercq and Edwards, 2012). The increased expression of these systems et al., 2011). From a microbiological point of view, intrinsic resis- increases resistance levels (increased MIC values) whereas their tance can be a result of: (i) inherent difficulties for the antibiotic absence increases bacterial susceptibility (decreased MIC values; to reach its corresponding target due to impaired permeability of Alekshun and Levy, 2007). the bacterial envelope or efficient drug export systems, the so- Hence, in bacterial resistance the differentiation between called multi-drug resistance (MDR) efflux pumps; (ii) the absence intrinsic resistome and intrinsic resistance is a thin line, but low- of antimicrobial target(s) or presence of targets with low affin- level resistance can be associated at a certain point to intrinsic ity; or even (iii) the presence of a mechanism that inhibits or resistome and high-level resistance to intrinsic resistance (Leclercq destroys the antibiotics (enzymes that neutralize antibiotics in the et al., 2011). cytoplasm or periplasmic space). Some examples of these mechanisms are included in Table 1. Nevertheless, all microorganisms ANTIBIOTICS AND ANTIBIOTIC RESISTANCE GENES HAVE contain efflux pumps involved in bacterial physiology, which can ANCIENT ORIGINS participate in resistance to different extents, although the clini- During the 1970s, it was shown that resistance genes related to cal consequences might be of minor importance unless coupled aminoglycoside modifying enzymes (AMEs) in clinical bacteria with other resistance mechanisms (Li et al., 1994a,b; Piddock, had their origin in common soil bacteria belonging to Acti- 2006; Martínez, 2012; Nessar et al., 2012). Often, bacteria com- nomycetes, which also produce AMEs (Benveniste and Davies, bine different mechanisms affecting several antimicrobial drugs. 1973). However, metagenome studies from different sources have Conventionally, the intrinsic resistome has been defined as the set questioned the “hypothesis of the antibiotic producers” because of chromosomal genes that are involved in intrinsic resistance and antibiotic resistance genes have been found in the microbiome of whose presence in strains of a bacterial species is independent of ancient isolated environments dating back a million years (D’Costa previous antibiotic exposure and is not due to horizontal gene et al., 2006; Allen et al., 2010; Torres-Cortés et al., 2011; Bhullar transfer (Martínez, 2012). et al., 2012). The functional role of antibiotic resistance genes in Nowadays, the “intrinsic resistome” is a wider concept than antibiotic-producing bacteria seems clear: they need to protect intrinsic resistance and includes genetic elements normally themselves from the activity of their own antimicrobials; however, belonging to bacterial metabolic networks that can eventually par- in non-producers their role is less evident. Some explanations ticipate in resistance to antimicrobial agents. Pre-resistance genes have been suggested. First, antibiotic resistance genes have or have constituting the intrinsic resistome has two main characteristics: had a physiological role in bacteria (see below). Second, antimi- firstly, they confer low level of resistance which is also known as crobial agents are more prevalent than suspected. Analyses of “pre-resistome” (Fajardo et al., 2008); secondly, the different resis- bacterial genome sequences suggest that only 10% of the natural tance mechanisms are expressed at low levels, but their expression antibiotics have been discovered and probably only 1% of antimi- can increase due to the involvement of regulatory genes or as a crobial molecules from producers are known (Fischbach, 2009), consequence of mutational events, the so called “silent resistome” possibly because they are natural products of bacterial physiol- (Dantas and Sommer, 2012). Global transcriptome analyses of ogy (Osbourn, 2010). More probably both scenarios are true and microorganisms grown in the presence of β-lactams induce the non-exclusionary. The complex network of physiological interac- expression of ampC (Bagge et al., 2004). This chromosomal gene, tions that constitute the intrinsic resistome is coupled to small present in the genome of Pseudomonas aeruginosa and of many natural molecules, such as antibiotics, which might have a role Enterobacteriaceae for several hundred millions of years, plays a as signaling molecules. According to this concept, antibiotics and

Table 1 | Different examples of intrinsic resistance with clinical relevance.

Intrinsic resistance Resistance mechanism Antibiotics affected Microorganisms mechanisms class

Inadequate target PBP5 mutations Cephalosporins Enterococcus faecium Inactivating process L1 beta-lactamase Carbapenems Stenotrophomonas maltophilia Impaired permeability Impermeable cell barrier Vancomycin Enterobacteriaceae

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 41

“fmicb-04-00009” — 2013/2/7 — 12:09 — page3—#3 Galán et al. Exploring the diversity of β-lactamases

antibiotic resistance genes have evolved during millions of years by 2012). Most of these genes have physiological functions but forming interactions in coupled bacterial-specific networks, both express resistance in the presence of antibiotics, again suggesting by the same and different species (Arifuzzaman et al., 2006). For that antibiotic resistance genes are the result of specific adaptive instance, different Staphylococcus aureus strains synthesize pep- responses in genes with previous physiological roles. tides that are recognized as signals by strains belonging to the The idea that intrinsic resistance is a consequence of the global same group and are competitive inhibitors against other S. aureus bacterial physiology was later demonstrated with the study of strains belonging to different lineages (Ji et al., 1997). the intrinsic resistomes of P. aeruginosa (Fajardo et al., 2008) and An accepted hypothesis is that these low molecular weight nat- Escherichia coli (Tamae et al., 2008). P. aeruginosa is thought to be ural products (antibiotics) could originally be signaling molecules intrinsically resistant to several antimicrobial agents mainly due that help shaping the structure of microbial communities (Yim to a reduced cellular permeability and the activity of MDR efflux et al., 2007; Linares et al., 2006; Fajardo and Martínez, 2008). Even pumps. Fajardo et al. (2008) performed a transposon-tagging quinolone compound derivatives, which are produced by a vari- experiment to determine the elements involved in the intrinsic ety of plants and microorganisms, have been found to act as resistance of P. aeruginosa. They demonstrated that ∼2% of the P. quorum-sensing signal molecules, controlling the expression of aeruginosa genome is involved in the altered susceptibility against virulence genes as a function of cell population density. This has different categories of antibiotics, but surprisingly only one gene been particularly investigated in P.aeruginosa. In this species, these had been annotated as an antibiotic resistance gene (Fajardo et al., compounds play multiple roles as membrane-interacting com- 2008). Many of the genes detected as related to antibiotic resis- pounds, inhibitors of cytochrome complexes and iron chelators, as tance are involved in basic bacterial metabolism. However, their well as in the regulation of their biosynthesis and their integration phenotypic effect is weak; therefore, the development of a real into the intricate quorum-sensing regulatory networks governing intrinsic resistance phenotype requires a complex assemblage of virulence and secondary metabolite gene expression (Heeb et al., mutations, such as those previously commented in efflux pumps. 2011). This could also be the case of other antibiotics, includ- Those genes with a physiological function in the bacteria rep- ing small peptide molecules that might have a potential role in resenting genes that do not confer clinical resistance to their native complex bacterial communities (microbiomes; Belda-Ferre et al., host, but are capable of expressing resistance when up-regulated or 2012). Therefore, the production of antibiotics is under strict expressed in other hosts (exaptation) can eventually be involved genetic control. In resource-limited environments or when bacte- in resistance will be part of the so-called “unknown resistome.” rial cells reach the stationary phase, the production of microbial Elements from the intrinsic resistome and the unknown resis- secondary metabolites, such as microcins, is increased in order to tome have been stressed as potential targets for the design of new yield a survival advantage to the producing bacteria (Romero et al., interventions to prevent antimicrobial resistance (Martínez,2012). 2011). Microcins are DNA-damaging agents and, in consequence, the SOS regulon is induced and the chromosomal gene encod- THE ENVIRONMENTAL RESISTOME ing the physiological inhibitor DNA gyrase, gyrI, is over-expressed Although the origin of antibiotic resistance was mysterious in the (Baquero et al., 1995). This case is one of the examples showing past, it has become evident over the last decade that environmental that antibiotics and antibiotic resistance genes are natural products bacteria were highly resistant to antibiotics (Wright, 2010). In fact in constant and ancestral co-evolution (Chatterji and Nagaraja, this was discovered 40 years ago by Benveniste and Davies (1973) 2002). Another interesting example is that of β-lactamases, which who found that AMEs from various species of the genus Strepto- are structurally similar to PBPs (penicillin-binding proteins), myces were similar to those found in clinical bacteria. Similarly, the target of β-lactam antibiotics, the enzymes involved in the resistance to vancomycin, first reported in 1988 in Enterococcus metabolism of peptidoglycan (Massova and Mobashery, 1998). faecium clinical isolates in Europe (Leclercq et al., 1988), was later Some PBPs such as PBP5 have weak β-lactamase activity confer- found to be associated with glycopeptide antibiotic-producing ringalowβ-lactam resistance phenotype (Sarkar et al., 2011). In Actinomycetes from soil (Marshall et al., 1997, 1998)aswellas conclusion, antibiotics and also antibiotic resistance genes might in non-antibiotic-producing bacteria of the genera Paenibacil- have a dual functional and ecological role. At low concentrations lus and Rhodococcus from soil (Guardabassi et al., 2004). More antibiotics are signaling systems and at high concentrations they recently it has been demonstrated that several antibiotic resistance are weapons (Fajardo and Martínez, 2008). genes (genes conferring resistance to β-lactams, aminoglycosides, tetracyclines, sulfonamides, and phenicoles) have been exchanged PHYSIOLOGICAL ROLE OF THE INTRINSIC RESISTOME between environmental bacteria and clinical pathogens on the ELEMENTS AND THE UNKNOWN OR SILENT RESISTOME basis of perfect nucleotide sequence identities of these genes with It has been anticipated that the intrinsic resistome elements have those from diverse human pathogens (Forsberg et al., 2012). In a physiological role in bacteria other than conferring resistance to 2010, Knapp et al. (2010) quantitatively analyzed the abundance antibiotics currently used in the clinical practice (Wright, 2007, of 18 antibiotic resistance genes from five long-term-soil extracted 2010). A recent review addressing the importance of Mycobac- DNAs from different areas in The Netherlands covering the years terium abscessus, a rapidly growing bacteria involved in soft-tissue 1940 to 2008. They found that antibiotic resistance genes from infections and chronic pulmonary diseases, showed the presence all classes of antibiotics have increased since 1940, especially for of a high number of resistance mechanisms responsible for natural tetracycline and β-lactam resistance encoding genes that have a resistance in this species, including efflux pumps, antibiotic- higher abundance in the twenty-first century when compared modifying enzymes, and target-modifying enzymes (Nessar et al., with the 1970s, suggesting that levels of antibiotic resistance genes

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 42

“fmicb-04-00009” — 2013/2/7 — 12:09 — page4—#4 Galán et al. Exploring the diversity of β-lactamases

in the environment are now high and increasing (Knapp et al., (Poeta et al., 2009; Literak et al., 2010), pigs, and rodents (Literak 2010). Although resistant organisms in the environment may be et al., 2009). Wild birds may also be a source for antibiotic resis- the result of contamination by the recent use and misuse of antibi- tance genes and their dissemination by migratory birds, as recently otics by humans, this previous dogma is now seriously disputed exemplified using functional metagenomics of a huge diversity of thanks again to the advances in new high throughput sequencing antibiotic resistance genes in fecal samples from a breeding colony technologies (Rolain et al., 2012). of gulls in the United States (Martiny et al., 2011) or ESBL E. coli Aquatic environments are also a source for antibiotic resis- in great cormorans and mallards in Europe (Tausova et al., 2012). tance genes. The bacteria and/or antibiotic resistance genes could Finally, it has been recently shown that feces from urban pigeons be transmitted directly or indirectly from these environments to in Brazil contain MDR enterococci (da Silva et al., 2012). Similarly, humans (Zhang et al., 2009) as result of contamination by the MDR enterococci including Enterococcus faecalis and Enterococcus recent use of antibiotics in agriculture and aquaculture selecting gallinarum isolates resistant to vancomycin and MDR E. coli have these intrinsic resistance genes (Baquero et al., 2008; Zhang et al., been recovered from feces of feral pigeons in the Czech Republic 2009; Wright, 2010). Many different antibiotic resistance genes (Radimersky et al., 2010), suggesting that pigeons should be con- from different classes of antibiotics (tetracyclines, aminoglyco- sidered a risk species that may contribute to the spread of resistance sides, macrolides, chloramphenicol, vancomycin, sulfonamides, in the urban environment. and β-lactams) have been detected in different water environ- In recent years, new technological approaches, especially ments worldwide using different molecular techniques as reviewed functional metagenomics, have led to the characterization and dis- recently (Baquero et al., 2008; Zhang et al., 2009). The recent covery of many unknown antibiotic resistance genes, the so-called emergence and spread of carbapenemase encoding genes in Gram- resistome, i.e., all antibiotic resistance genes at a global scale in negative bacteria seems to have originated from bacterial species anybacteria(Wright, 2007). Functional metagenomic approaches in water environments (Lupo et al., 2012). For example, par- have been developed and used to decipher the resistome in soil tial OXA-23 like carbapenemase sequences have been detected in samples by Handelsman (2004) in 2004 that led to the discovery groundwater samples from the Katmandu Valley of Nepal (Tanaka of new antibiotic resistance genes such as AMEs and β-lactamases. et al., 2012) that were 100% similar to those of sequences found The first study of the extent of this resistome was reported by in human head lice from Senegal (Kempf et al., 2012), suggest- D’Costa et al. (2006) from bacteria isolated from soils showing ing that arthropods may also be a source of antibiotic resistance that many environmental bacteria were MDR and that half of genes in human pathogens. Recently, MDR E. coli and Klebsiella the antibiotic resistance genes discovered were unknown. More- spp. with extended spectrum β-lactamase (ESBLs) have been cul- over, novel mechanisms of resistance involving the inactivation tured from municipal wastewater treatment plant effluents in the of telithromycin and daptomycin, two antibiotics approved in the Czech Republic (Dolejska et al., 2011). Similarly, several bacterial last decade were discovered thanks to this approach (D’Costa et al., species including human pathogens carrying NDM-1, recently dis- 2006). Later, Dantas et al. (2008) confirmed the presence of bac- covered (Yong et al., 2009) and spreading worldwide (Rolain et al., teria from soils intrinsically resistant to many different classes of 2010), have been detected in tap and drinking water as well as antibiotics. drain and sewage in India (Walsh et al., 2011; Shahid et al., 2012) Nowadays, there is evidence that these antibiotic resistance and in seepage water (rivers, lakes, and water pools in streets) genes have an ancient origin as demonstrated by the recent from Vietnam (Isozumi et al., 2012). These data implicate con- discovery using metagenomic analysis of genes conferring resis- taminated aquatic environments, as a likely site for the exchange of tance to β-lactams, tetracyclines, and glycopeptides in permafrost antibiotic resistance genes between the clinic and the environment sediments dating from 30,000 years ago (D’Costa et al.,2011). Sim- (Baquero et al., 2008). ilarly, MDR bacteria have been recently cultured from soil samples Wild animals and pets, especially their gut microbiota, may also of an over 4 million year old cave in New Mexico (Bhullar et al., be a source for antibiotic resistance genes that come in contact with 2012), revealing an unexpected reservoir of antibiotic resistance humans (Allen et al., 2010). Pets, cats and dogs in particular, may genes in the environment. Although environmental reservoirs are be a source for antibiotic resistance genes that could be transmitted well known as a possible source of antibiotic resistance genes to humans and the environment and vice versa (Guardabassi et al., detected in human pathogens, reports of antibiotic resistance 2004). It has been shown that resistance to antibiotics in bacteria genes from environmental bacteria with a high level of sequence recovered from pets has increased markedly over the last decade, similarity to those from human pathogens previously reported are especially the emergence of MDR Acinetobacter baumannii, E. coli, limited. CTX-M-8 β-lactamase in environmental bacteria of the Salmonella enterica, Staphylococcus intermedius, or methicillin- genus Kluyvera (Poirel et al., 2002)orqnrA-like genes from marine resistant S. aureus that could be a risk for transmission to humans and freshwater bacteria of the genus Shewanella (Poirel et al.,2005) (Lloyd, 2007). Gilliver et al. (1999) reported more than 10 years are examples of antibiotic resistance transferred from environ- ago that antibiotic resistance was highly prevalent even in wild mental bacteria to human pathogens. But, the environmental rodents that were believed not to be exposed to antibiotics. Sim- resistome is not only a huge reservoir of antibiotic resistance genes; ilarly, vancomycin-resistant enterococci carrying the vanA gene it is also a generator of new mechanisms of resistance. The finding have been isolated from feces of wild mammals in the United of a new bifunctional β-lactamase in a remote Alaskan soil sam- Kingdom (Mallon et al., 2002), OXA-23 carbapenemase encoding ple (Allen et al., 2009) or the chimeric origin of the New Delhi gene in Acinetobacter species from the gut microbiota of cattle in metallo-β-lactamase NDM as the consequence of a recombina- France (Poirel et al., 2012), or MDR E. coli in European wild boars tion event between AME and β-lactamase (Toleman et al., 2012)

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 43

“fmicb-04-00009” — 2013/2/7 — 12:09 — page5—#5 Galán et al. Exploring the diversity of β-lactamases

are good examples of new antibiotic resistance arising in the β-lactamases. In some cases, such as class D (OXA type) enzymes, environment. they are present both in Gram-positive and Gram-negative bac- In summary, antibiotic resistance is a natural and ancient phe- teria whereas Class C enzymes are only present in Gram-negative nomenon and its recent increase of antibiotic resistance among organisms (unlike Mycobacterium smegmatis), reflecting different pathogenic bacteria has been the result of the ancient and recent evolutionary trajectories. Along these trajectories, β-lactamases mobilization of resistance genes from these environmental and have had to undergo structural alterations to become efficient as animal reservoirs to human pathogens. Nevertheless, in spite of β-lactam-hydrolyzing enzymes, avoiding the interaction with pep- the efforts to clearly identify transfer events from environmental tidoglycan (or peptidoglycan precursors), which are the substrates to pathogenic strains, only a few examples have been documented. of PBPs. It has been hypothesized that this gives an advantage to The paucity of genetic evidence to date this transference is more β-lactamase-producing bacteria, particularly in soil communities, likely the consequence of under sampling rather than that these where natural β-lactam-producing bacteria might be normally exchanges have not occurred. Our knowledge of the environmen- present (Bush, 1997). tal resistome remains low and future studies should be focused on One of the most studied β-lactamases is the Class A enzymes. discovery of these new antibiotic resistance genes for the better Within this group the most widely distributed enzyme is TEM- understanding of the magnitude of this phenomenon in environ- 1, which was first described in 1963 (Datta and Kontomichalou, mental reservoirs and its potential impact in human pathogens. 1965). The bla gene encoding this enzyme is carried in a Another reason for the few cases documented so far could be transposable element (Tn3) that is widely present in plasmids related to the complex interplay between resistome genotypes of different incompatibility groups (Hedges and Jacob, 1974). and resistance phenotypes, which could be context-depend. Only Despite its widespread presence all over the world and in many some specific genetic combinations of resistance genes, plasmids, different pathogenic bacteria, including among others all Enter- strains, and microbial community can be successful. Therefore, obacteriaceae, Haemophilus influenzae, Neisseria gonorrhoeae, in the absence of experimental evidence the spectrum of action and P. aeruginosa, its ancestor remains undetermined. This is will be difficult to assess (Dantas and Sommer, 2012). The high not the case of the CTX-M enzymes, which were recognized prevalence and diversity of both antibiotic resistance genes and as plasmid-mediated enzymes hydrolyzing extended-spectrum bacterial species in the environment make these ecosystems an cephalosporins (i.e., cefotaxime) in 1989 (Bauernfeind et al., opportunity to decipher the mechanisms of transfer and usage of 1990), from environmental bacteria of the genus Kluyvera (Poirel these genes in bacteria living in a sympatric lifestyle which is a key et al., 2002). component for bacterial evolution (Diene et al., 2012). We need Nowadays, the CTX-M enzymes are widely disseminated all to understand the magnitude of this phenomenon in the future over the world within Enterobacteriaceae (Cantón and Coque, in order to be able to define new strategies to face the problem of 2006). The corresponding genes, the blaCTX−M, have been found antibiotic resistance globally and not only from a human medical in chromosomal location in different species of the genus Kluyvera, viewpoint. which is generally accepted as the origin of these genes (Can- tón et al., 2012). In the Kluyvera species, cefotaxime resistance MOBILIZATION AND EXPRESSION OF INTRINSIC RESISTOME has low levels of expression. Mobilization by insertion sequences ELEMENTS (IS), such as ISEcp1 or ISCR1, has resulted in higher expression Comparisons of soil microbiota with clinical pathogens have proof resistance in E. coli, K. pneumoniae, and other clinically rele- vided evidence of an ancient and recent exchange of antibiotic vant Enterobacteriaceae (Poirel et al., 2003; Lartigue et al., 2006; resistance genes (β-lactams, aminoglycosides, amphenicols, sul- Rodriguez-Martinez et al., 2006). fonamides, and tetracyclines) as well as the multiple mobilizations Similar to Class A enzymes, the mobilization of blaOXA genes of sequences between these communities. They demonstrate not has been estimated to have occurred at different times, the first only evidence of lateral exchange but also a mechanism for the one a hundred million years ago, whereas the last mobilization dissemination of antibiotic resistance (Forsberg et al., 2012). If occurred very recently. This could have been the case of the the assumption that the intrinsic resistance has an ancient origin carbapenem-hydrolyzing β-lactamase OXA-48 and its derivative in environmental genes, then it can also be accepted that these OXA-181, which differs from the former by four amino acid genes are transmitted to human pathogenic bacteria. Admittedly substitutions, which have been related with Shewanella xiame- these genes could be mobilized from their ancestors. Among these nensis (Potron et al., 2011). This is an environmental species intrinsic resistance genes, β-lactamases genes (bla genes) are con- from marine and freshwaters that contains the blaOXA−181 gene. sidered one of the best examples and are also discussed in this OXA-48-producing Enterobacteriaceae are increasingly recog- review. nized worldwide whereas the detection of OXA-181-producing As previously commented, amino acid sequence analysis of isolates remains scarce (Poirel et al., 2012). Similarly to some of PBPs and β-lactamases point to a common origin of these proteins the blaCTX−M genes, the blaOXA−181 gene was also preceded by (Bush, 1997; Massova and Mobashery, 1998). Nevertheless, dif- its insertion in the element ISEcp1, which might also have been ferent ancestors have been recognized and different evolutionary responsible for its mobilization. trajectories might have occurred (Cantón, 2007). The analysis of Interestingly, other species from Shewanella might also play an β-lactamase phylogeny is not congruent with the phylogenetic his- important role as the origin and reservoirs of resistance deter- tory of its carriers, as horizontal gene transfer, frequently produced minants. They have been found to harbor not only different β- over time, might have interfered in the evolutionary process of lactamases genes but also resistance genes affecting other antibiotic

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 44

“fmicb-04-00009” — 2013/2/7 — 12:09 — page6—#6 Galán et al. Exploring the diversity of β-lactamases

classes such as fluoroquinolones (qnrA gene in Shewanella algae; the mid-twentieth century, the pace of this evolutionary race has Poirel et al., 2005; Rodriguez-Martinez et al., 2005). In the case accelerated tremendously. The most relevant player in this game of β-lactamases, Shewanella oneidensis and Shewanella algae har- is natural selection, which results from the advantage gained by a bor chromosomal class D β-lactamases genes (blaOXA−54 and mutant organism that can overcome more efficiently the deleteri- blaOXA−55, respectively) and Shewanella livingstonensis and She- ous effects of antibiotics, and also by antibiotic-producers, which wanella frigidimarina harbor chromosomal metallo-β-lactamase are able to find a new way of evading these defenses. In fact, genes (blaSLB−1 and blaSFB−1, respectively; Poirel et al., 2005). this important role of natural selection has been demonstrated None of these genes has been found yet in plasmids but they could at different scales, from the observed changes in the prevalence of eventually be mobilized to pathogenic bacteria. Unlike these cases, antibiotic resistance genes to the molecular level, by identification the blaOXA−23 gene, whose progenitor has been found in Acineto- of those amino acid residues that are the main targets of selection bacter radioresistens,isnowadayswidelyspreadinA. baumannii (Novais et al., 2010). (Walsh, 2008). Notwithstanding the relevance of natural selection, other All these examples illustrate how the intrinsic resistome can evolutionary mechanisms are also important in the evolution be mobilized from environmental bacteria and expressed at dif- of antibiotic resistance. Obviously, the most important chance ferent levels in new hosts. However, it is surprising that despite involved in evolutionary success is related to horizontal gene trans- the huge environmental and intrinsic resistome in environmental fer and the integration of antibiotic resistance gene in widespread and commensal bacteria we can identify transmission events in plasmids and/or clones, but this aspect is not considered in this only a few cases (Table 2; Davies, 1997; Davies and Davies, 2010), review. Other chance events such as mutations may play an impor- suggesting the existence of biochemical, metabolic, and genetic tant role in the evolutionary trajectories followed by antibiotic constrictions (Dantas and Sommer, 2012). When we are capable resistance strains. This has been experimentally shown to occur in of understanding the “global resistome,” i.e., the environmental the evolution of resistance to β-lactams on at least two occasions. and intrinsic (including silent) resistome, then all the bacterial Novais et al. (2010) showed that depending on the first mutation potential for fighting other bacteria will be known and only then to appear in a few critical positions, resistance to cefotaxime and will we be able to fight the antibiotic war on equal terms. Then, the ceftazidime might follow-up to three alternative routes in CTX- following steps in our understanding of the resistance process will M-type β-lactamases. A similar result was obtained after the in be to define and reveal the adaptive potential of those physiological vitro evolution of TEM-1 β-lactamase (Salverda et al., 2011). genes that evolved toward resistance genes. Apart from contingency, other stochastic processes may also be important for the evolution of antibiotic resistance. In their anal- PHYLOGENY AND EVOLUTION OF β-LACTAMASES ysis of CTX-M experimental evolution, Novais et al. (2010) found Antibiotic resistance is probably one of the best examples of “evo- that Darwinian selection alone could not explain the evolution lution in action.” The basic mechanisms of resistance appeared of the CTX-M variants with the highest resistance to cefotaxime early in the evolutionary time-scale and evolved to counter the and ceftazidime because the fitness gradient along the different new defenses put forward by other bacterial species to compete for alternative routes always involved at least some steps of reduced resources or survival against competitors and predators (Baquero fitness. The alternative possibility discussed by Novais et al. implies and Blázquez, 1997; Salmond and Welch, 2008; Davies and Davies, the action of genetic drift and selection in heterogeneous environ- 2010). It has been commented several times throughout this ments to cross these fitness valleys. In consequence, the action of review that antibiotic resistance genes were ancient elements with natural selection does not guarantee, on its own, the evolution a physiological role as components of complex networks. The of the genotype with the highest fitness. Therefore, it would be of decontextualization of these genes from their original background interest to extend these analyses to other antibiotic resistance genes (exaptation) allowed a slightly faster evolutionary process toward and to evaluate the prevalence and relevance of natural selection a real antibiotic resistance per se. However, it is certain that since and other evolutionary mechanisms in their recent and ancient the adoption of antibiotic therapy to combat infectious diseases in evolution.

Table 2 | Examples of resistance mechanisms in clinical isolates that evolved from natural functions in environmental bacteria.

Antimicrobial group Mechanisms Related natural protein Natural reservoirs

Aminoglycosides AcetylationPhosphorylation Histone-acetylasesProtein kinases Streptomyces Tetracyclines Efﬂux (mar) Major facilitator superfamily EF-Tu, EF-G Streptomyces Chloramphenicol AcetylationEfﬂux (mar) AcetylasesMajor facilitatorsuperfamily EF-Tu, EF-G Streptomyces Macrolides Target mutation 50S ribosomal subunit Streptomyces β-lactams (methicillin) PBP2a Homologous PBP2a Staphylococcus sciuri β-lactams (carbapenems) OXA-48 inactivating enzyme Proteins participating in peptidoglycan synthesis Shewanella xiamenensis OXA-23 inactivating enzyme Proteins participating in peptidoglycan synthesis Acinetobacter radioresistens Fluoroquinolones Topoisomerase protection Qnr-like protein Shewanella algae

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 45

“fmicb-04-00009” — 2013/2/7 — 12:09 — page7—#7 Galán et al. Exploring the diversity of β-lactamases

β-Lactams are the most widely used antibiotics in clinical prac- these results, it is likely that this group of sequences originated tice over the world. Strong selective pressures upon them have in Acinetobacter and has remained distributed mostly within this resulted in a continuous increase of resistant isolates (Pitout and genus. The only exception corresponds to a plasmid-borne rep- Laupland, 2008). In addition, they have also driven the diversi- resentative isolated in Klebsiella, where it probably arrived from fication of known β-lactamases genes (Bush and Jacoby, 2010), an Acinetobacter ancestor. The OXA-51 clade, the largest subin order to increase the spectrum of action, and facilitated the group, is a very homogeneous group of sequences, with an average acquisition of new mechanisms from the environmental resistome, nucleotide distance of 0.018 substitutions/site (s/s) and a maxi- such as CTX-M β-lactamases (Poirel et al., 2002). Since the 1990s, mum within-group distance of 0.036 s/s. These results indicate the identification of new β-lactamase variants has increased dra- that this group has diversified recently. Similarly, recent diversi- matically (Davies and Davies, 2010), especially as a consequence fication events were suggested in the OXA-10 (the second largest of new class A and D β-lactamase variants (Bush and Jacoby, group with 18 sequences) and OXA-2 clades. The average pairwise 2010). Moreover, the description of new β-lactamases with car- divergences were 0.022 s/s and 0.132 s/s and a maximum value of bapenemase activity such as NDM and KPC (Klebsiella producing 0.045 s/s and 0.288 s/s, respectively. The OXA-54 clade, with very carbapenemase; Bonomo, 2011; Cornaglia et al., 2011), outbreaks recently evolved sequences, was also found to be closely related to of known β-lactamases such as OXA-48 (Dimou et al., 2012) and the OXA-2 clade. the spread of ampC in plasmids (D’Andrea et al., 2011)havecon- These results are indicating that only some branches are evolv- tributed to the current state of alarm (Bush, 2010). We have ing quickly and they could be under positive selection. Hence, analyzed, for the better understanding of the factors and processes the selective processes might have left an imprint in the form underlying the recent spread of β-lactamases, the evolutionary of increased rates of non-synonymous substitutions (dN) com- dynamics of three β-lactamase families, i.e., ESBL-OXA, CYM, pared to those of synonymous substitutions (dS). In consequence, and IMP, representing enzymes belonging to classes D, C, and we have tested this hypothesis by comparing the rates of dS and B, respectively. We decided to exclude the most prevalent (Class dN substitutions at each codon. Based on the ML tree, estimates A), such as TEM and CTX-M, because they are well character- of the two substitution rates were obtained using the ancestral ized (Salverda et al., 2010; Cantón et al., 2012) and their extent states inferred by ML under a Muse–Gaut model (Muse and diversity is the consequence of recent evolution in response to the Gaut, 1994) of codon substitution and the Tamura–Nei model clinical use of β-lactam antibiotics (Weinreich et al., 2006; Novais of nucleotide substitution (Tamura and Nei, 1993). Computa- et al., 2010). We have also excluded other worrying cases for pub- tion of dN/dS was performed with the Hyphy software package lic health, such as NDM and KPC, because the diversity of their (Kosakovsky Pond et al., 2005). Positions with <95% sequence known variants is still very low for a phylogenetic analysis. coverage (i.e., with >5% of the sequences presenting a gap) were It is already well understood that the OXA family is very removed from the analysis. In contrast to the initial expectation, diverse (Sanschagrin et al.,1995) and old (Barlow and Hall,2002b). no codon presented a statistically significant higher rate of dN than Only one phylogenetic analysis has been published using 35 dS substitutions. The same general results were obtained when the OXA β-lactamases (Barlow and Hall, 2002b). The main conclu- different groups in the OXA phylogenetic tree were analyzed sepa- sions of Barlow’s work were: first, four branches were identified rately in search of positively selected codons as well as in an analysis (OXA-1/OXA-9; OXA-2; OXA-5; OXA-23); second, of the three with the seven ungrouped sequences of this gene (Figure 1). This mobilization events from a chromosomal site to plasmids detected result indicates that neither the ancient divergence nor the recent two were ancestral events (OXA-1); and third, three branches spread of some groups of OXA alleles have been driven primarily (OXA-1, OXA-2, and OXA-10) were under significant positive by selection acting at this (codon) level. selection at the beginning of the diversification process. A total Plasmid-encoded ampC genes have been known since 1989 of 192 ESBL-OXA sequences downloaded from GenBank were when CMY-1 was described in K. pneumoniae isolates (Bauern- used in our new approach. In order to facilitate the interpreta- feind et al., 1989), while the most common plasmid-mediated tion of the resulting maximum-likelihood (ML) phylogenetic tree, AmpC β-lactamase worldwide is CMY-2 (Jacoby, 2009). Generally, highly supported (BS > 95%) clades of closely related sequences CMY-2 enzymes are responsible for outbreaks across European have been grouped (Figure 2). Each group has been denoted countries (D’Andrea et al., 2011). CMY-1 and CMY-2 have differ- by a representative OXA variant present therein and the result ent phylogenetic origins (Figure 3A); whereas CMY-1 and related essentially matches the nomenclature of similar groups in previ- variants are close to chromosomally determined AmpC enzymes ous works (Couture et al., 1992; Poirel et al., 2010). Five major in Aeromonadaceae, CMY-2 and evolved variants are related to branches can be distinguished now. The new branch identified AmpC from Citrobacter freundii (Barlow and Hall, 2002a). A total corresponds to OXA-61/63. The OXA-1/OXA-9 branch shows two of 32 CMY alleles with known date and country of first isola- different dynamics. The OXA-9 clade differs from other previous tion were included in this analysis. These variants cluster into two groups in its ancient origin, with long internal branches indicating well-defined and distant groups in the reconstructed ML tree using long periods of evolution after divergence from the corresponding the Tamura-3P + gamma model (Figure 3), denoted CMY-1 and ancestors. On the contrary, the OXA-1 clade includes sequences CMY-2. The CMY-2 group is larger and more diverse. A large from several different species such as E. coli, P. aeruginosa, K. pneu- number of sequences (n = 22) from the five continents are very moniae, and others. They are very similar and suggest a very recent similar and have diversified very recently, while the five remaining spread from a common ancestor. The OXA-51/OXA-23 branch sequences in the CMY-2 group have a more ancient origin. This includes 120 sequences, all but one from Acinetobacter. From larger group is dominated by isolates from E. coli, but a recent

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 46

“fmicb-04-00009” — 2013/2/7 — 12:09 — page8—#8 Galán et al. Exploring the diversity of β-lactamases

FIGURE 2 | Summarized representation of the maximum-likelihood tree was investigated at the codon level by analyzing the difference between of OXA genes obtained with the K2P + GI model of evolution. Multiple dN–dS substitution rates at different positions. Based on the corresponding nucleotide sequence alignments were obtained using the corresponding maximum likelihood trees using a Muse–Gaut model (Muse and Gaut, 1994) amino acid sequences with Muscle (Edgar, 2004a,b). Maximum-likelihood of codon substitution and the Tamura–Nei model of nucleotide substitution trees were obtained using the most appropriate model of evolution resulting (Tamura and Nei, 1993). Computation of dN/dS was performed with the from the comparison of Bayesian Information Criterion (BIC) values for 24 Hyphy software package (Kosakovsky Pond et al., 2005). Additionally, two alternative models including gamma-distributed heterogeneous rates of tests of neutrality were applied to the three data sets: the Fisher’s exact test evolution among and invariant sites. Support of the inferred clusters was of neutrality and a test based on bootstrapping (1000 replicates) of dN–dS evaluated with 1000 bootstrap replicates; BS values >70% are indicated. All values from which a variance of the corresponding statistic is derived in these methods were used as implemented in MEGA 5 (Tamura et al., 2011). order to test the null hypothesis of neutrality (dN = dS). These tests were The scale bar corresponds to substitutions/site. The role of natural selection performed using MEGA 5 (Tamura et al., 2011). colonization and spread into Proteus mirabilis can be observed shown above, there are two main, anciently-diverged groups that in sequences isolated from European countries (D’Andrea et al., have been analyzed separately in search of positive selection. The 2011) with a possible origin in NorthAfrica (CMY-4 from Tunisia). phylogeny-based analysis of CMY-1 variants failed to identify any The CMY-1 group included only six sequences from Asian–Pacific codon with a significant positive difference between dN and dS, countries, which are distributed evenly in two subgroups of very with only seven codons yielding positive values of this parame- similar sequences. ter none of which reached statistical significance. Similar results The CMY alignment resulted in 376 codons available for selec- were obtained for the CMY-2 group, with average dN–dS values tion analysis. None of them presented significant deviations from of −0.362 (range: −10.484 to 1.000, minimum P = 0.444), and the expected dN−dS value under neutrality, although 123 codons the two pairwise comparison-based tests failing to reveal evidence had positive values for this difference. These analyses corre- for positive selection. Barlow and Hall (2002a) obtained similar sponded to the whole data set of CMY alleles considered but, as results using a lower number of variants. Although a diversification

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 47

“fmicb-04-00009” — 2013/2/7 — 12:09 — page9—#9 Galán et al. Exploring the diversity of β-lactamases

FIGURE 3 | Maximum-likelihood tree of CMY genes obtained with the models including gamma-distributed heterogeneous rates of evolution Tamura-3P + G model of evolution. (A) The upper tree corresponds to among and invariant sites. Support of the inferred clusters was CMY-1 group. (B) The lower one to the CMY-2 group of sequences. Multiple evaluated with 1000 bootstrap replicates, BS values >70% are indicated. sequence alignments were obtained using the corresponding amino acid All these methods were used as implemented in MEGA 5 (Tamura et al., sequences with Muscle (Edgar, 2004a,b). Maximum-likelihood trees were 2011). The scale bar corresponds to substitutions/site. Detection of obtained using the most appropriate model of evolution resulting from the positive selection and neutrality tests were performed as described comparison of Bayesian Information Criterion (BIC) values for 24 alternative in Figure 2. process has occurred in this group, especially in CMY-2, the newly probably because they are all resistant to many β-lactam antibi- arisen variants were not subject to positive selection, in contrast otics. Therefore, the plasmid-mediated AmpC enzymes are not to TEM and CTX-M enzymes. The hydrolytic activities con- evolving phenotypically because it is not necessary (Barlow and ferred by chromosomal ampC recovered from the pre-antibiotic Hall, 2002a). However, the impact of single-amino acid sub- era are essentially the same as plasmid-mediated ampC alleles, stitutions on the evolution of CMY proteins can be observed

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 48

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 10 — #10 Galán et al. Exploring the diversity of β-lactamases

in particular cases. For instance, the G214E change in CMY-2 In fact, our analyses in search of positive selection in these gene increases the catalytic efficiency against cefotaxime (Endimiani families failed to find evidence for it after using both phylogeny- et al., 2010), suggesting that although the selective pressure occurs and pairwise-based analysis of dN–dS differences in codons or it might be weak. the whole gene. In general, most isolates in recently spread OXA- β-Lactamases with capacity for hydrolyzing carbapenems are and IMP-groups are derived from A. baumannii and P. aerugi- very versatile in their origins and nucleotide sequence. Currently, nosa. Over 210 and 120 β-lactamases have been identified in A. almost 30 families of carbapenemases belonging to classes A, D, baumannii and P. aeruginosa, respectively, suggesting that these and B are known (Widmann et al., 2012), but new families are species are crucial reservoirs of β-lactam resistance determinants being described continuously (Pollini et al., 2012). The spread of (Zhao and Hu,2010,2012). Although they are ubiquitous in nature metalo-β-lactamases (MBL), classified as class B, presents a major also they are frequently isolated in nosocomial and chronic infec- challenge both for treatment of individual patients and for policies tions, exposed to a variety of antibiotic regimens, giving rise to of infection control (Cornaglia et al., 2011), because they confer selection and spread of mechanisms of resistance. In this scenario resistance to almost all β-lactams, except aztreonam. There are at these results suggest that these species are an excellent shut- least nine different types of MBL, but probably IMP and VIM are tle of resistance genes between the environment and pathogenic the most prevalent (Fukigai et al.,2007; Van der Bij et al., 2011) and strains. diversified1. The dendrogram of VIM enzymes suggests two recent events of diversification involving VIM-1 and VIM-2 (Cornaglia UNDERSTANDING THE ADAPTIVE POSSIBILITIES AND THEIR et al., 2011). CONSTRICTIONS TO PREDICT β-LACTAMASE EVOLUTION We have analyzed 21 IMP alleles with year and country of The ability of protein to evolve is dependent on tolerance to first isolation. The ML tree was constructed using the Tamura- change. TEM β-lactamases can be extremely tolerant to amino acid 3P model. According to this phylogeny (Figure 4), several closely substitutions both in the number of affected positions (>200 posi- related groups of likely recent origin and many other more tions can change; Salverda et al.,2010) as in number of amino acids anciently diverged sequences can be observed. The two recently in a particular position (one-third of the positions in TEM can tol- diverged groups include mostly sequences isolated from East Asian erate more than five different amino acids; Deng et al., 2012). The countries. A common feature of these recently spread groups is combination of potential mutations could increase the theoretical that they include most alleles isolated in species not belonging to diversity up to astronomically large numbers. However, only 82 P. aeruginosa and A. baumannii. The first IMP allele, IMP-1, was polymorphic positions have been described among the 204 TEM isolated in Japan and it is included in a group with three addi- mutants described so far and, strikingly, 85% of the substitutions tional alleles from the same country. Another group related to are located in only 17 positions.1 Moreover, the most polymorphic this one includes two sequences isolated in Hong Kong and Singa- positions show only 3–4 amino acid changes. So, why does not the pore, respectively. The second recently spread group includes two sequence plasticity observed in site-directed mutagenesis experi- sequences from Taiwan and one from Italy and France. In both ments translate easily in huge diversification of evolved proteins groups, the V67F change was selected on three occasions suggest- in nature? (Taverna and Goldstein, 2002). ing that this change could be under positive selection. However, Many researchers have studied the possible genetic constraints no mutation presented a statistically significant, positive deviation involved in the discrepancy between the potential and the actu- in the dN–dS parameters indicative of positive selection acting in ally observed biological plasticity of β-lactamases (Camps et al., the IMP phylogeny. Pairwise comparisons also failed to reveal 2007). Mutagenesis studies on TEM-1 did not yield TEM vari- positive selection as a driving force in the evolution of these alle- ants more efficient than TEM-1 in hydrolyzing natural β-lactams les. The V67F change is highly variable, non-essential for the such as ampicillin and cephalosporin C, suggesting that during protein function but it could have a role in antibiotic recogni- thousands of years of evolution TEM-1 has become the most tion (Widmann et al., 2012). Moreover, in the IMP-1 clade, the efficient enzyme in conferring the highest MIC values to natural mutation G235S contributes in increasing the hydrolytic activ- β-lactams (Deng et al., 2012). The kinetic effect of this evolved ity against meropenem (Liu et al., 2012), suggesting a process of enzyme is the result of a delicate network of hydrogen bonds selection. contributing to the enzymatic stabilization. Therefore, it is easy Despite their many differences at the genetic and ecological lev- to imagine that any mutation could alter this balance and con- els, the three families of β-lactamases present several remarkable sequently decrease its efficiency. Internal positions (close to the similarities. The three gene families have several anciently diverged active center) are less tolerant of substitutions than external ones variants along with others that have spread very recently. Most of because, presumably, they have more interactions and the equilib- these recently diverged groups include very similar sequences with rium is altered easily (Deng et al., 2012). Therefore, the majority of only a few nucleotide substitutions and usually only one or two mutations in internal positions are generally deleterious but they amino acid replacements. This distribution probably results from might also facilitate the acquisition of new functions (Tokuriki a biased screening of variants – only those with at least one amino et al., 2008). For instance, mutations such as R164H in TEM-1 acid difference are reported and deposited in the corresponding (TEM-29), D179N in SHV-1 (SHV-8), or P167T in CTX-M-1 databases, which may lead to the false impression that positive (CTX-M-58) increase the hydrolytic activity against third genera- selection is a major factor driving the evolution of these genes. tion β-lactam antibiotics (new function), but decrease the specific activity of the existing function as a consequence of a loss of sta- 1http://www.lahey.com bility (Poirel et al., 2001; Wang et al., 2002). The protein instability

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 49

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 11 — #11 Galán et al. Exploring the diversity of β-lactamases

FIGURE 4 | Maximum-likelihood tree of IMP genes obtained with the gamma-distributed heterogeneous rates of evolution among and invariant Tamura-3P model of evolution. Multiple sequence alignments were sites. Support of the inferred clusters was evaluated with 1000 bootstrap obtained using the corresponding amino acid sequences with Muscle replicates, BS values >70% are indicated. All these methods were used as (Edgar, 2004a,b). Maximum-likelihood trees were obtained using the most implemented in MEGA 5 (Tamura et al., 2011). The scale bar corresponds to appropriate model of evolution resulting from the comparison of Bayesian substitutions/site. Detection of positive selection and neutrality tests were Information Criterion (BIC) values for 24 alternative models including performed as described in Figure 2. observed with some mutations leads to lower levels of correctly- stability due to previous selection of mutations conferring new folded protein and, consequently, to a reduction in enzymatic functions (Kather et al., 2008). In other words, the evolution activity (Bloom et al., 2005). Therefore, the stability/instability toward new functions must be facilitated by mutations that act level of a mutant protein must be proportional to its fitness. as compensatory mutations of protein stability defects (Brown Almost 40% of TEM-1 artificial mutations cause a partial or et al., 2010). complete reduction in the level of folded protein (Tokuriki and The best studied example of compensatory mutation is M182T Tawfik, 2009). Hence as mutations accumulate, the likelihood of in TEM enzymes. This mutation has no effect on the catalytic declines in protein fitness increases exponentially (Soskine and activity of TEM-1 (Huang and Palzkill, 1997), however the ther- Tawfik, 2010). modynamic stability of TEM-1 is increased by 2.67 kcal/mol in According to the theoretical estimations, those proteins car- the presence of this mutation (Wang et al., 2002). Therefore, the rying, five mutations on average, with respect to the wild-type selection of M182T will be favored when previous mutations that variant, will reduce their fitness in > 80% (Tokuriki and Taw- increase the activity against new β-lactams have reduced protein fik, 2009) and the accumulation of >10 mutations per gene stability (Camps et al., 2007). For instance, TEM-15 has substi- would result in non-functionalization of 99% of the mutated tutions E104K:G238S which contribute synergistically to increase genes (Soskine and Tawfik, 2010). However, several TEM-1 the hydrolytic activity toward extended-spectrum cephalosporins. variants, such as TEM-121, TEM-162, or TEM-194, carrying These mutations rearrange the active site and, consequently, five or more mutations have been described in nature. The destabilize the enzyme (ΔΔG = 2.24 kcal/mol), whereas the increased tolerance to changes observed in these enzymes requires triple mutant, TEM-52, contains the additional substitution the presence of mutations capable of compensating the loss of M182T compensating the loss associated with the E104K:G238S

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 50

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 12 — #12 Galán et al. Exploring the diversity of β-lactamases

substitutions (ΔΔG =−1.76 kcal/mol), increasing the activity known to cause the greatest increase in CAZ and CTX resistance against extended-spectrum β-lactams (Wang et al.,2002). Progres- among all known single CTX-M and TEM mutations. However, sively, other compensatory mutations have been described in TEM there are more clinical isolates carrying the D240G and R164S enzymes (Brown et al., 2010). mutations in CTX-M and TEM, respectively. This observation is The presence of compensatory mutations is a global phe- contradictory to the Darwinian paradigm of evolution, as geno- nomenon, also described in other β-lactamases, such as mutations types with higher fitness must be favored by selection (“survival A77V in CTX-M (Novais et al., 2008), N70S in metallo-β- of the fitness”). lactamase BcII (Tomatis et al., 2008), or L169I in OXA and At high mutation rates or weak bottlenecks, selection favors ROB-β-lactamases (Poirel et al., 2002; Galán et al., 2003). From genotypes with larger networks of interactions, which will be more an evolutionary perspective, these point mutations are essential tolerant (robust) to the impact of deleterious mutations in spite because the extra stability promotes the evolvability of the protein of lower fitness. This phenomenon is known as “survival of the (Bloom et al., 2006), but potentially they can also have negative flattest” (Codoñer et al., 2006; Archetti, 2009). Depending on the effects if the excess stability hinders protein turnover (Brown et al., environmental conditions, selection may favor an organism that 2010). Therefore, an equilibrium between evolvability and robust- replicates faster (“survival of the fitness”) or is more robust (“sur- ness is essential for the plasticity of proteins, that is, between the vival of the flattest”), but not both at the same time (Codoñer necessity to adapt to new environments and the ability to maintain et al., 2006). The high connectedness of robust genotypes will be a phenotype in the presence of genotypic variations. a guarantee of innovation without drastically losing functional- The network model establishes strong associations between ity in the long time scale (Ferrada and Wagner, 2008), whereas a particular mutations. For instance, mutation M182T is more fre- high evolvability is a guarantee for survival to strong bottlenecks quently associated with mutations related to ESBL phenotypes in a short time but at the cost of losing many interactions and (Huang and Palzkill, 1997), whereas N276D is associated with IRT old functions. In general, β-lactamases carrying D240G or R164S phenotypes (Abriata et al., 2012), suggesting co-evolutionary pro- have accumulated more mutations and the old functions are less cesses (Guthrie et al., 2011). In other cases, mutation L210P is affected than in β-lactamases harboring P167S or G238S muta- co-selected with R244A (Marciano et al., 2008), whereas mutation tions in the first step. Therefore, D240G or R164S mutations in E240K is associated with R164H, suggesting different networks CTX-M and TEM enzymes will condition the “survival of the flat- of compensatory mutations depending on the initial mutation test” model; whereas P167S or G238S mutations will contribute to responsible for the ESBL phenotype. These epistatic constric- “survival of the fittest.” These results suggest that the first mutations required for maintaining stability while activity is increased tions not only determine the co-selection of specific compensatory drastically reduce the number of possible mutational pathways mutations but also condition the selection of certain evolution- (Weinreich et al.,2006; Novais et al.,2010). Therefore, initial muta- ary strategies. Nevertheless, the first mutations to be selected will tions drive evolutionary pathways of protein evolution (Salverda also depend on the frequency and intensity of changes in the et al., 2011). The genetic reconstruction based on a combina- environment. torial strategy of five mutations in TEM-1, which were chosen Antibiotic resistance is a public health problem and for many for their large joint phenotypic effect, revealed that only 18 of microbiologists and evolutionary researchers a goal to address the 120 evolutionary trajectories were accessible through Darwinian resistance problem has been to predict the selection of antibiotic selection (Weinreich et al., 2006). Our group, using the CTX-M resistance to new antibiotic (Martínez et al., 2007). In this situa- enzyme as model, obtained similar results but concluded that tion, which types of studies are necessary to analyze in more depth only the simultaneous presence of cefotaxime and ceftazidime the development of resistance in human bacterial pathogens? For in the environment permitted reaching the highest level of resis- many years, studies based on serial-passages experiments have tance (Novais et al.,2010). These studies of experimental evolution been used as the best model to predict the selection of muta- on β-lactamases show that only a small fraction of all possible tions involved in extending the spectrum of enzymatic activity mutational trajectories are accessible to evolution. (Martínez et al., 2011). However, this approach generally detected These lines of evidence suggest that many combinations of single mutants and, as a consequence of the strong bottlenecks mutations must be antagonistic. Pleiotropic antagonism has been involved in this experimental design, in most cases only those fit- used to describe the incompatibility between ESBL and inhibitor- ness variants corresponding to the“survival of the fitness”concept resistant β-lactamases phenotypes (Cantón et al., 2008; Ripoll were recovered (Novais et al., 2008; Ripoll et al., 2011). This sim- et al., 2011). However, in this case pleiotropic antagonism is ple vision does not predict the selection of complex mutants based defined as the incompatibility between mutations conferring the on the “survival of the flattest” concept. Then, the evolvability of same phenotype. For instance, mutations D240G and P167S known antibiotic resistance genes was explored by DNA shuffling increase the hydrolytic activity of CTX-M enzymes to ceftazidime. and error-prone PCR (Orencia et al., 2001). The combination of However, the double mutant confers lower MIC values than both approaches in fluctuating environments could be more real- both single mutants in every background (Novais et al., 2010). istic, because in these cases mutations representing the “survival Similar results were observed between mutations R164S and of the flattest,” such as R164S, are easily selected (Blázquez et al., G238S in TEM, which show a strong negative interaction (Salverda 2000; Barlow and Hall, 2002c). Two new approaches are contribut- et al., 2011). Laboratory-directed protein evolution experiments ing to improve our predictive capacity. One of them, defining revealed that P167S and G238S mutations were more frequently the driving forces the selection in a given combination of time selected (Barlow and Hall, 2002c; Novais et al., 2008)whichare and environment and understanding the possible evolutionary

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 51

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 13 — #13 Galán et al. Exploring the diversity of β-lactamases

trajectories in the diversiﬁcation process (Weinreich et al., 2006; FIS-PI-12/00567) and HEALTH-F3-2011-282004 (EVOTAR) and Novais et al., 2010). Secondly, the implementation of bioinfor- HEALTH-F3-2010-241476 (PAR) from European Commission. matics programs such as FoldX (Deng et al., 2012), determining Fernando González was supported by project BFU2011-24112 the ΔΔG of a new mutant and consequently its stability and ﬁt- from MINECO (Spanish Government). Rafael Cantón was sup- ness. The combinatorial approaches between bioinformatics and ported by national grant (PS09/00825) and LSHMCT-2008- in vitro procedures will give rise to a more complete vision on the 223031 and FP7-HEALTH-2010-28251from the European Union. complex process of antibiotic resistance evolution. Juan-Carlos Galán and Fernando González-Candelas belong to CIBERESP, Network for Biomedical Research in Epidemiology ACKNOWLEDGMENTS and Public Health (CB06/02/0053). Rafael Cantón also belongs Juan-Carlos Galán was supported by research grants from Min- to REIPI, Spanish Network for Research in Infectious Diseases isterio de Sanidad y Consumo (Instituto de Salud Carlos III, (RD12/0015).

REFERENCES Baquero, F., Alvarez-Ortega, C., and Blázquez, J., Morosini, M. I., Negri, A. Gutierrez-Fuentes (Washington: Abriata, L. A., Merijn, L., Salverda, Martinez, J. L. (2009). Ecology and M. C., and Baquero, F. (2000). ASM Press), 249–270. M., and Tomatis, P. E. (2012). evolution of antibiotic resistance. Selection of naturally occurring Cantón, R., Akóva, M., Carmeli, Y., Sequence-function-stability relation- Environ. Microbiol. Rep. 1, 469–476. extended-spectrum TEM beta- Giske, C. G., Glupczynski, Y., Gni- ships in proteins from datasets of Baquero, M. R., Bouzon, M., Varea, lactamase variants by fluctuating adkowski, M., et al. (2012). Rapid functionally annotated variants: the J., and Moreno, F. (1995). sbmC, beta-lactam pressure. Antimicrob. evolution and spread of carbapene- case of TEM β-lactamases. FEBS Lett. a stationary-phase induced SOS Agents Chemother. 44, 2182–2184. mases among Enterobacteriaceae in 586, 3330–3335. Escherichia coli gene, whose product Bloom, J. D., Labthavikul, S. T., Otey, Europe. Clin. Microbiol. Infect. 18, Alekshun, M. N., and Levy, S. B. (2007). protects cells from the DNA repli- C. R., and Arnold, F. H. (2006). Pro- 413–431. Molecular mechanisms of antibacte- cation inhibitor microcin B17. Mol. tein stability promotes evolvability. Cantón, R., and Coque, T. M. (2006). rial multidrug resistance. Cell 128, Microbiol. 18, 301–311. Proc. Natl. Acad. Sci. U.S.A. 103, The CTX-M beta-lactamase pan- 1037–1050. Barlow, M., and Hall, B. G. (2002a). 5869–5874. demic. Curr. Opin. Microbiol. 9, Allen, H. K., Donato, J., Wang, H. Origin and evolution of the AmpC Bloom, J. D., Silberg, J. J., Wilke, C. 466–475. H., Cloud-Hansen, K. A., Davies, J., β-lactamases of Citrobacter freundii. O., Drummond, D. A., Adami, C., Cantón, R., Morosini, M. I., de la and Handelsman, J. (2010). Call of Antimicrob. Agents Chemother. 46, and Arnold, F. H. (2005). Thermo- Maza, O. M., and de la Pedrosa, E. G. the wild: antibiotic resistance genes 1190–1198. dynamic prediction of protein neu- (2008). IRT and CMT b-lactamases in natural environments. Nat. Rev. Barlow, M., and Hall, B. G. (2002b). trality. Proc. Natl. Acad. Sci. U.S.A. and inhibitor resistance. Clin. Micro- Microbiol. 8, 251–259. Phylogenetic analysis shows that the 102, 606–611. biol. Infect. Suppl. 1, 53–62. Allen, H. K., Moe, L. A., Rodbum- OXA β-lactamase genes have been on Bonomo, R. A. (2011). New Delhi Chatterji, M., and Nagaraja, V. (2002). rer, J., Gaarder, A., and Handelsman, plasmids for millions of years. J. Mol. metallo-b-lactamase and multidrug GyrI: a counter-defensive strat- J. (2009). Functional metagenomics Evol. 55, 314–321. resistance: a global SOS? Clin. Infect. egy against proteinaceous inhibitors reveals diverse beta-lactamases in a Barlow, M., and Hall, B. G. (2002c). Dis. 52, 485–487. of DNA gyrase. EMBO Rep. 3, remote Alaskan soil. ISME J. 3, Predicting evolutionary potential: in Brown, N. G., Pennington, J. M., 261–267. 243–251. vitro evolution accurately reproduces Huang, W., Ayvaz, T., and Palzkill T. Codoñer, F. M., Daròs, J. A., Solé, Archetti, M. (2009). Survival of the natural evolution of the TEM beta- (2010). Multiple global suppressors R. V., and Elena, S. F. (2006). The steepest: hypersensitivity to muta- lactamase. Genetics 160, 823–832. of protein stability defects facilitate fittest versus the flattest: experimen- tionsasanadaptationtosoftselec- Bauernfeind, A., Grimm, H., and the evolution of extended-spectrum tal confirmation of the quasispecies β tion. J. Evol. Biol. 22, 740–750. Schweighart, S. A. (1990). New plas- TEM -lactamases. J. Mol. Biol. 404, effect with subviral pathogens. PLoS Arifuzzaman, M., Maeda, M., Itoh, A., midic cefotaximase in a clinical iso- 832–846. Pathog. 2:e136. doi: 10.1371/jour- β Nishikata, K., Takita, C., Saito, R., late of Escherichia coli. Infection 18, Bush, K. (1997). The evolution of - nal.ppat.0020136 et al. (2006). Large-scale identifica- 294–298. lactamases. Ciba Found. Symp. 207, Cornaglia, G., Giamarellou, H., and tion of protein–protein interaction of Bauernfeind, A., Schweighart, S., and 152–163. Rossolini, G. M. (2011). Metallo- β β β Escherichia coli K-12. Genome Res. 16, Chong, Y. (1989). Extended broad Bush, K. (2010). Alarming -lactamase- -lactamases: a last frontier for - 686–691. spectrum b-lactamase in Klebsiella mediated resistance in multidrug- lactams? Lancet Infect. Dis. 11, Bagge, N., Schuster, M., Hentzer, M., pneumoniae including resistance to resistant Enterobacteriaceae. Curr. 381–393. Ciofu, O., Givskov M., Greenberg, E. cephamycins. Infection 17, 316–321. Opin. Microbiol. 13, 558–564. Couture, F., Lachapelle, J., and P., et al. (2004). Pseudomonas aerug- Belda-Ferre, P., Alcaraz, L. D., Cabrera- Bush, K., and Jacoby, G. A. (2010). Levesque, R. C. (1992). Phylogeny of inosa biofilms exposed to imipenem Rubio, R., Romero, H., Simón-Soro, Updated functional classification of LCR-1 and OXA-5 with class A and β exhibit changes in global gene expres- A, Pignatelli, M., et al. (2012). The -lactamases. Antimicrob. Agents class D b-lactamases. Mol. Microbiol. sion and beta-lactamase and algi- oral metagenome in health and dis- Chemother. 54, 969–976. 6, 1693–1705. nate production. Antimicrob. Agents ease. ISME J. 6, 46–56. Camps, M., Herman, A., Loh, E., and D’Andrea, M. M., Literacka, E., Zioga, Chemother. 48, 1175–1187. Benveniste, R., and Davies, J. Loeb, L. A. (2007). Genetic con- A., Giani, T., Baraniak, A., Fiett, Baquero, F. (2012). Metagenomic epi- (1973). Aminoglycoside anti- straints on protein evolution. Crit. J., et al. (2011). Evolution and demiology: a public health need for biotic-inactivating enzymes in Rev. Biochem. Mol. Biol. 42, 313–326. spread of a multidrug-resistant Pro- the control of antimicrobial resis- actinomycetes similar to those Cantón, R, González-Alba, J. M., teus mirabilis clone with chromo- tance. Clin. Microbiol. Infect. 18, present in clinical isolates of and Galán, J. C. (2012). CTX- somal AmpC-type cephalosporinases 67–73 antibiotic-resistant bacteria. Proc. M enzymes: origin and diffusion. in Europe. Antimicrob. Agents Baquero, F., and Blázquez, J. (1997). Natl. Acad. Sci. U.S.A. 70, 2276–2280. Front. Microbiol. 3:110. doi: 10.3389/ Chemother. 55, 2735–2742. Evolution of antibiotic resistance. Bhullar, K., Waglechner, N., Pawlowski, fmicb.2012.00110 D’Costa, V. M., King, C. E., Kalan, L., Trends Ecol. Evol. 12, 482–487. A., Koteva, K., Banks, E. D., Johnston, Cantón, R. (2007). “Epidemiology and Morar, M., Sung, W. W., Schwarz, C., Baquero, F., Martinez, J. L., and Cantón, M. D., et al. (2012). Antibiotic resis- evolution of beta-lactactamases,” in et al. (2011). Antibiotic resistance is R. (2008). Antibiotics and antibiotic tance is prevalent in an isolated cave Evolutionary Biology of Bacteria and ancient. Nature 477, 457–461. resistance in water environments. microbiome. PLoS ONE 7:e34953. Fungal Pathogens, eds F. Baquero, C. D’Costa, V. M., McGrann, K. M., Curr. Opin. Biotechnol. 19, 260–265. doi: 10.1371/journal.pone.0034953 Nombela, G. H. Cassell, and Jose Hughes, D. W., and Wright, G. D.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 52

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 14 — #14 Galán et al. Exploring the diversity of β-lactamases

(2006). Sampling the antibiotic resis- Kutilova, I., et al. (2011). CTX-M- Members of the genera Paenibacil- Jacoby, G. A. (2009). AmpC β- tome. Science 311, 374–377. 15-producing Escherichia coli clone lus and Rhodococcus harbor genes lactamases. Clin. Microbiol. Rev. 22, da Silva, V. L., Cacador, N. C., da B2-O25b-ST131 and Klebsiella spp. homologous to enterococcal gly- 161–182. Silva, C. S., Fontes, C. O., García, isolates in municipal wastewater copeptide resistance genes vanA and Ji, G., Beavis, R., and Novick, R. P. G. D., Nicoli, J. R., et al. (2012). treatment plant effluents. J. Antimi- vanB. Antimicrob. Agents Chemother. (1997). Bacterial interference caused Occurrence of multidrug-resistant crob. Chemother. 66, 2784–2790. 48, 4915–4918. by autoinducing peptide variants. and toxic-metal tolerant enterococci Edgar, R. C. (2004a). MUSCLE: a multi- Guardabassi, L., Schwarz, S., and Lloyd, Science 276, 2027–2030. in fresh feces from urban pigeons ple sequence alignment method with D. H. (2004). Pet animals as reser- Kather, I., Jakob, R. P., Dobbek, H., and in Brazil. Microbes Environ. 27, reduced time and space complex- voirs of antimicrobial-resistant bac- Schmid, F. X. (2008). Increased fold- 179–185. ity. BMC Bioinformatics 5:113. doi: teria. J. Antimicrob. Chemother. 54, ing stability of TEM-1 β-lactamase by Dantas, G., and Sommer, M. O. (2012). 10.1186/1471-2105-5-113 321–332. in vitro selection. J. Mol. Biol. 383, Context matters – the complex inter- Edgar, R. C. (2004b). MUSCLE: Gullberg, E., Cao, S., Berg, O. G., Ilbäck, 238–251. play between resistome genotypes multiple sequence alignment with C., Sandegren, L., Hughes, D., et al. Kempf, M., Rolain, J. M., Diatta, G., and resistance phenotypes. Curr. high accuracy and high throughput. (2011). Selection of resistant bacte- Azza, S., Samb, B., Mediannikov, O., Opin. Microbiol. 15, 577–582. Nucleic Acids Res. 32, 1792–1797. ria at very low antibiotic concentra- et al. (2012). Carbapenem resistance Dantas, G., Sommer, M. O., Oluwase- Endimiani, A., Doi, Y., Bethel, C. R., tions. PLoS Pathog. 7:e1002158. doi: and Acinetobacter baumannii in Sene- gun, R. D., and Church, G. M. Taracila, M., Adams-Haduch, J. M., 10.1371/journal.ppat.1002158 gal: the paradigm of a common phe- (2008). Bacteria subsisting on antibi- O’Keefe, A., et al. (2010). Enhancing Guthrie, V. B., Allen, J., Camps, M., and nomenon in natural reservoirs. PLoS otics. Science 320, 100–103. resistance to cephalosporins in class C Karchin, R. (2011). Network mod- ONE 7:e39495. doi: 10.1371/jour- Datta, N., and Kontomichalou, P. β-lactamases: impact of Gly214Glu in els of TEM β-lactamase mutations nal.pone.0039495 (1965). Penicillinase synthesis con- CMY-2. Biochemistry 49, 1014–1023. coevolving under antibiotic selec- Knapp, C. W., Dolfing, J., Ehlert, P. trolled by infectious R factors Fajardo, A., Martínez-Martín, N., Mer- tion show modular structure and A., and Graham, D. W. (2010). Evi- in Enterobacteriaceae. Nature 208, cadillo, M., Galán, J. C., Ghysels, anticipate evolutionary trajectories. dence of increasing antibiotic resis- 239–241. B., Matthijs, S., et al. (2008). The PLoS Comput. Biol. 7:e1002184. doi: tance gene abundances in archived Davies, J. (2007). Microbies have the neglected intrinsic resistome of bac- 10.1371/journal.pcbi.1002184 soils since 1940. Environ. Sci. Technol. last word. A drastic re-evaluation terial pathogens. PLoS ONE 3:e1619. Handelsman, J. (2004). Metagenomics: 44, 580–587. of antimicrobial treatment is needed doi: 10.1371/journal.pone.0001619 application of genomics to uncul- Kosakovsky Pond, S. L., Frost, S. D. to overcome the treat of antibiotic- Fajardo, A., and Martínez, J. L. (2008). tured microorganisms. Microbiol. W., and Muse, S. V. (2005). HyPhy: resistant bacteria. EMBO Rep. 8, Antibiotics as signals that trigger spe- Mol. Biol. Rev. 68, 669–685. hypothesis testing using phylogenies. 616–621. cific bacterial responses. Curr. Opin. Hedges, R. W., and Jacob, A. E. (1974). Bioinformatics 21, 676–679. Davies, J. E. (1997). Origins, acquisi- Microbiol. 11, 161–167. Transposition of ampicillin resistance Lartigue, M. F., Poirel, L., Aubert, tion and dissemination of antibiotic Ferrada, E., and Wagner, A. (2008). from RP4 to other replicons. Mol. D., and Nordmann, P. (2006). In resistance determinants. Ciba Found. Protein robustness promotes evolu- Gen. Genet. 132, 31–40. vitro analysis of ISEcp1B-mediated Symp. 207, 15–27. tionary innovations on large evolu- Heeb, S., Fletcher, M. P., Chhabra, mobilization of naturally occur- Davies, J., and Davies, D. (2010). Ori- tionary time-scales. Proc. Biol. Soc. S. R., Diggle, S. P., Williams, P., ring beta-lactamase gene blaCTX- gins and evolution of antibiotic resis- 275, 1595–1602. and Cámara, M. (2011). Quinolones: MofKluyvera ascorbata. Antimi- tance. Microbiol. Mol. Biol. Rev. 74, Fischbach, M. A. (2009). Antibiotics from antibiotics to autoinducers. crob. Agents Chemother. 50, 1282– 417–433. from microbes: converging to kill. FEMS Microbiol. Rev. 35, 247–274. 1286. Deng, Z., Huang, W., Bakkalbasi, E., Curr. Opin. Microbiol. 12, 520–507. Henderson, T. A., Young, K. D., Leclercq, R., Cantón, R., Brown, Brown, N. G., Adamski, C. J., Rice, Forsberg, K. J., Reyes, A., Wang, B., Sell- Denome, S. A., and Elf, P. K. (1997). D. F., Giske, C. G., Heisig, P., K., et al. (2012). Deep sequencing eck, E. M., Sommer, M. O., and Dan- AmpC and AmpH, proteins related Macgowan, A. P., et al. (2011). of systematic combinatorial libraries tas, G. (2012). The shared antibiotic to the class C beta-lactamases, bind EUCAST expert rules in antimi- reveals β-lactamase sequence con- resistome of soil bacteria and human penicillin and contribute to the nor- crobial susceptibility testing. Clin. straints at high resolution. J. Mol. pathogens. Science 337, 1107–1111. mal morphology of Escherichia coli. J. Microbiol. Infect. doi: 10.1111/j.1469- Biol. 424, 150–167. Fukigai, S., Alba, J., Kimura, S., Bacteriol. 179, 6112–6121. 0691.2011.03703.x [Epub ahead of Didelot, X., Bowden, R., Wilson, D. J., Iida, T., Nishikura, N., Ishii, Y., Huang, H., Yang, Z. L., Wu, X. M., print]. Peto, T. E., and Crook, D. W. (2012). et al. (2007). Nosocomial outbreak Wang, Y., Liu, Y. J., Luo, H., et al. Leclercq, R., Derlot, E., Duval, J., Transforming clinical microbiology of genetically related IMP-1 β- (2012). Complete genome sequence and Courvalin, P. (1988). Plasmid- with bacterial genome sequencing. lactamase-producing Klebsiella pneu- of Acinetobacter baumannii MDR-TJ mediated resistance to vancomycin Nat. Rev. Genet. 13, 601–612. moniae in a general hospital in and insights into its mechanism of and teicoplanin in Enterococcus fae- Diene, S. M., Merhej, V., Henry, Japan. Int. J. Antimicrob. Agents 29, antibiotic resistance. J. Antimicrob. cium. N.Engl.J.Med.319, 157–161. M., El, F. A., Roux, V., Robert, 306–310. Chemother. 67, 2825–2832. Li, X. Z., Livermore, D. M., and Nikaido, C., et al. (2012). The rhizome of Galán, J. C., Morosini, M. I., Baquero, Huang, W., and Palzkill, T. (1997). H. (1994a). Role of efflux pump(s) the multidrug-resistant Enterobacter M. R., Reig, M., and Baquero, A natural polymorphism in β- in intrinsic resistance of Pseudomonas aerogenes genome reveals how new F. (2003). Haemophilus influenzae lactamase is a global suppressor. Proc. aeruginosa: resistance to tetracycline, “killer bugs” are created because of bla(ROB-1) mutations in hyper- Natl. Acad. Sci. U.S.A. 94, 8801–8806. chloramphenicol, and norfloxacin. a sympatric lifestyle. Mol. Biol. Evol. mutagenic ΔampC Escherichia coli Isozumi, R., Yoshimatsu, K., Yamashiro, Antimicrob. Agents Chemother. 38, doi: 10.1093/molbev/mss236 [Epub conferring resistance to cefotaxime T., Hasebe, F., Nguyen, B. M., Ngo, 1732–1741. ahead of print]. and beta-lactamase inhibitors and T. C., et al. (2012). bla(NDM-1)- Li, X. Z., Ma, D., Livermore, D. M., Dimou, V., Dhanji, H., Pike, R., Liv- increased susceptibility to cefaclor. positive Klebsiella pneumoniae from and Nikaido, H. (1994b). Role of ermore, D. M., and Woodford, N. Antimicrob. Agents Chemother. 47, environment, Vietnam. Emerg. Infect. efflux pump(s) in intrinsic resistance (2012). Characterization of Enter- 2551–2557. Dis. 18, 1383–1385. of Pseudomonas aeruginosa:active obacteriaceae producing OXA-48- Gilliver, M. A., Bennett, M., Begon, M., Jacobs, C., Huang, L. J., Bartowsky, E., efflux as a contributing factor to beta- like carbapenemases in the UK. J. Hazel, S. M., and Hart, C. A. (1999). Normark, S., and Park, J. T. (1994). lactam resistance. Antimicrob. Agents Antimicrob. Chemother. 67, 1660– Antibiotic resistance found in wild Bacterial cell wall recycling provides Chemother. 38, 1742–1752. 1665. rodents. Nature 401, 233–234. cytosolic muropeptides as effectors Linares, J. F., Gustafsson, I., Baquero, F., Dolejska, M., Frolkova, P., Florek, Guardabassi, L., Christensen, H., Has- for beta-lactamase induction. EMBO and Martinez, J. L. (2006). Antibiotics M., Jamborova, I., Purgertova, M., man, H., and Dalsgaard, A. (2004). J. 13, 4684–4694. as intermicrobial signaling agents

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 53

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 15 — #15 Galán et al. Exploring the diversity of β-lactamases

instead of weapons. Proc. Natl. Acad. for therapeutic intervention. Future Osbourn, A. (2010). Secondary Poirel, L., Rodriguez-Martinez, J. M., Sci. U.S.A. 103, 19484–19489. Med. Chem. 4, 347–359. metabolic gene clusters: evolutionary Mammeri, H., Liard, A., and Literak, I., Dolejska, M., Radimersky, T., Martínez, J. L., Baquero, F., and Ander- toolkits for chemical innovation. Nordmann, P. (2005). Origin of Klimes, J., Friedman, M., Aarestrup, sson, D. I. (2011). Beyond serial Trends Genet. 26, 449–457. plasmid-mediated quinolone resis- F. M., et al. (2010). Antimicrobial- passages: new methods for predicting Piddock, L. J. (2006). Multidrug- tance determinant QnrA. Antimicrob. resistant faecal Escherichia coli in wild the emergence of resistance to novel resistance efflux pumps – not just Agents Chemother. 49, 3523–3525. mammals in central Europe: mul- antibiotics. Curr. Opin. Pharmacol. for resistance. Nat. Rev. Microbiol. 4, Poirel, L., Naas, T., Le Thomas, tiresistant Escherichia coli producing 11, 439–445. 629–636. I., Karim, A., Bingen, E., and extended-spectrum beta-lactamases Martínez, J. L. (2009). The role of natu- Pitout, J. D., and Laupland, K. Nordmann, P. (2001). CTX-M-type in wild boars. J. Appl. Microbiol. 108, ral environments in the evolution of B. (2008). Extended-spectrum β- extended-spectrum β-lactamase that 1702–1711. resistance traits in pathogenic bacte- lactamase-producing Enterobacteri- hydrolyzes ceftazidime through a Literak, I., Dolejska, M., Rybarikova, J., ria. Proc. Biol. Sci. 276, 2521–2530. aceae: an emerging public-health single amino acid substitution in Cizek, A., Strejckova, P., Vyskocilova, Martínez, J. L. (2008). Antibiotics and concern. Lancet Infect. Dis. 8, the omega loop. Antimicrob. Agents M., et al. (2009). Highly variable antibiotic resistance genes in natural 159–166. Chemother. 45, 3355–3361. patterns of antimicrobial resistance environments. Science 321, 365–367. Poeta, P., Radhouani, H., Pinto, L., Pollini, S., Maradei, S., Pecile, P., in commensal Escherichia coli iso- Martínez, J. L, Baquero, F., and Ander- Martinho, A., Rego, V., Rodrigues, Olivo, G., Luzzaro, F., Docquier, lates from pigs, sympatric rodents, sson, D. I. (2007). Predicting antibi- R., et al. (2009). Wild boars J. D., et al. (2012). FIM-1, a new and flies. Microb. Drug Resist. 15, otic resistance. Nat. Rev. Microbiol. 5, as reservoirs of extended-spectrum acquired metallo-β-lactamase from a 229–237. 958–965. beta-lactamase (ESBL) producing Pseudomonas aeruginosa clinical iso- Liu, E. M., Pegg, K. M., and Martínez, J. L., Fajardo, A., Garmen- Escherichia coli of different phyloge- late from Italy. Antimicrob. Agents Oelschlaeger, P. (2012). The dia, L., Hernandez, A., Linares, J. F., netic groups. J. Basic Microbiol. 49, Chemother. 57, 410–416. sequence-activity relationship bet- Martínez-Solano, L., et al. (2009). A 584–588. Potron, A., Poirel, L., and Nordmann, ween metallo-β-lactamases IMP-1, global view of antibiotic resistance. Poirel, L., Bercot, B., Millemann, P. (2011). Origin of OXA-181, an IMP-6, and IMP-25 suggests FEMS Microbiol. Rev. 33, 44–65. Y., Bonnin, R. A., Pannaux, emerging carbapenem-hydrolyzing an evolutionary adaptation to Martiny, A. C., Martiny, J. B., G., and Nordmann, P. (2012). oxacillinase, as a chromosomal gene meropenem exposure. Antimi- Weihe, C., Field, A., and Ellis, Carbapenemase-producing Acineto- in Shewanella xiamenensis. Antimi- crob. Agents Chemother. 56, 6403– J. C. (2011). Functional metage- bacter spp. in cattle, France. Emerg. crob. Agents Chemother. 55, 4405– 6406. nomics reveals previously unrecog- Infect. Dis. 18, 523–525. 4407. Lloyd, D. H. (2007). Reservoirs of nized diversity of antibiotic resistance Poirel, L., Decousser, J. W., and Nord- Radimersky, T., Frolkova, P., Janos- antimicrobial resistance in pet ani- genes in gulls. Front. Microbiol. 2:238. mann, P. (2003). Insertion sequence zowska, D., Dolejska, M., Svec, mals. Clin. Infect. Dis. 45(Suppl. 2), doi: 10.3389/fmicb.2011.00238. ISEcp1B is involved in expression and P., Roubalova, E., et al. (2010). S148–S152. Massova, I., and Mobashery, S. (1998). mobilization of a bla(CTX-M) beta- Antibiotic resistance in faecal bacteria Lupo, A., Coyne, S., and Beren- Kinship and diversification of bacte- lactamase gene. Antimicrob. Agents (Escherichia coli, Enterococcus spp.) in donk, T. U. (2012). Origin and rial penicillin-binding proteins and Chemother. 47, 2938–2945. feral pigeons. J. Appl. Microbiol. 109, evolution of antibiotic resistance: beta-lactamases. Antimicrob. Agents Poirel, L., Gerome, P., De Champs, 1687–1695. the common mechanisms of emer- Chemother. 42, 1–17. C., Stephanazzi, J., Naas, T., and Ripoll, A., Baquero, F., Novais, A., gence and spread in water bod- Muse, S. V., and Gaut, B. S. (1994). Nordmann, P. (2002). Integron- Rodríguez-Domínguez, M. J., Tur- ies. Front. Microbiol. 3:18. doi: A likelihood approach for compar- located oxa-32 gene cassette encod- rientes, M. C., Cantón, R., et al. 10.3389/fmicb.2012.00018 ing synonymous and nonsynony- ing an extended-spectrum variant of (2011). In vitro selection of vari- Mallon, D. J., Corkill, J. E., Hazel, S. mous nucleotide substitution rates, OXA-2 beta-lactamase from Pseu- ants resistant to beta-lactams plus M., Wilson, J. S., French, N. P., Ben- with application to the chloroplast domonas aeruginosa. Antimicrob. β-lactamase inhibitors in CTX-M nett, M., et al. (2002). Excretion of genome. Mol. Biol. Evol. 11, 715–724. Agents Chemother. 46, 566–569. beta-lactamases: predicting the in vancomycin-resistant enterococci by Nessar, R., Cambau, E., Reyrat, J. Poirel, L., Héritier, C., and Nordmann, vivo scenario? Antimicrob. Agents wild mammals. Emerg. Infect. Dis. 8, M., Murray, A., and Gicquel, B. P. (2005). Genetic and biochemical Chemother. 55, 4530–4536. 636–638. (2012). Mycobacterium abscessus:a characterization of the chromosome- Rodriguez-Martinez, J. M., Conejo, M. Marciano, D. C., Pennington, J. M., new antibiotic nightmare. J. Antimi- encoded class B beta-lactamases from C., Martínez-Martínez, L., Cano, M. Wang, X., Wang, J., Chen, Y., crob. Chemother. 67, 810–818. Shewanella livingstonensis (SLB-1) E., Velasco, C, and Pascual, A. (2005). Thomas, V. L., et al. (2008). Genetic Novais, A., Cantón, R., Coque, T. M., and Shewanella frigidimarina (SFB- Evaluation of antimicrobial suscepti- and structural characterization of an Moya, A., Baquero, F., and Galán, J. 1). J. Antimicrob. Chemother. 55, bility of bacteria containing the qnr L201P global suppressor substitution C. (2008). Mutational events in cefo- 680–685. gene and FOX-5 beta-lactamase by in TEM-1 beta-lactamase. J. Mol. taximase extended-spectrum beta- Poirel, L., Kampfer, P., and Nordmann, four automated systems. Clin. Micro- Biol. 384, 151–164. lactamases of the CTX-M-1 cluster P. (2002). Chromosome-encoded biol. Infect. 11, 402–404. Marshall, C. G., Broadhead, G., Leskiw, involved in ceftazidime resistance. Ambler class A beta-lactamase of Rodriguez-Martinez, J. M., Poirel, L., B. K., and Wright, G. D. (1997). Antimicrob. Agents Chemother. 52, Kluyvera georgiana, a probable pro- Cantón, R., and Nordmann, P. D-Ala-D-Ala ligases from glycopep- 2377–2382. genitor of a subgroup of CTX- (2006). Common region CR1 for tide antibiotic-producing organisms Novais, A., Comas, I., Baquero, F., M extended-spectrum β-lactamases. expression of antibiotic resistance are highly homologous to the entero- Cantón, R., Coque, T. M., Moya, Antimicrob. Agents Chemother. 46, genes. Antimicrob. Agents Chemother. coccal vancomycin-resistance ligases A., et al. (2010). Evolutionary tra- 4038–4040. 50, 2544–2546. VanA and VanB. Proc. Natl. Acad. Sci. jectories of β-lactamase CTX-M-1 Poirel, L., Nass, T., and Nordmann, Rolain, J. M., Cantón, R., and Cor- U.S.A. 94, 6480–6483. cluster enzymes: predicting antibiotic P. (2010). Diversity, epidemiology naglia, G. (2012). Emergence of Marshall, C. G., Lessard, I. A., Park, resistance. PLoS Pathog. 6:e1000735. and genetics of class D β-lactamases. antibiotic resistance: need for a new I., and Wright, G. D. (1998). doi: 10.1371/journal.ppat.1000735 Antimicrob. Agents Chemother. 54, paradigm. Clin. Microbiol. Infect. 18, Glycopeptide antibiotic resistance Orencia, M. C., Yoon, J. S., Ness, J. E., 24–38. 615–616. genes in glycopeptide-producing Stemmer, W. P., and Stevens, R. C. Poirel, L., Potron, A., and Nordmann, Rolain, J. M., Parola, P., and Cornaglia, organisms. Antimicrob. Agents (2001). Predicting the emergence of P. (2012). OXA-48-like carbapen- G. (2010). New Delhi metallo-beta- Chemother. 42, 2215–2220. antibiotic resistance by directed evo- emases: the phantom menace. J. lactamase (NDM-1): towards a new Martínez, J. L. (2012). The antibiotic lution and structural analysis. Nat. Antimicrob. Chemother. 67, 1597– pandemia? Clin. Microbiol. Infect. 16, resistome: challenge and opportunity Struct. Biol. 8, 238–242. 1606. 1699–1701.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 9 | 54

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 16 — #16 Galán et al. Exploring the diversity of β-lactamases

Romero, D., Traxler, M. F., López, D., Tamura, K., Peterson, D., Peterson, soil samples. Environ. Microbiol. 13, Lee, K., et al. (2009). Characteriza- and Kolter, R. (2011). Antibiotics as N., Stecher, G., Nei, M., and 1101–1114. tion of a new metallo-beta-lactamase signal molecules. Chem. Rev. 111, Kumar, S. (2011). MEGA5: molecular Van der Bij, A. K., Van Mans- gene, bla(NDM-1), and a novel ery- 5492–5505. evolutionary genetics analysis using feld, R., Peirano, G., Goessens, thromycin esterase gene carried on a Salmond, G. P., and Welch, M. (2008). maximum likelihood, evolutionary W. H. F., Severin, J. A., Pitout, unique genetic structure in Klebsiella Antibiotic resistance: adaptive evolu- distance, and maximum parsimony J. D. D., et al. (2011). First out- pneumoniae sequence type 14 from tion. Lancet 372, S97–S103. methods. Mol. Biol. Evol. 28, 2731– break of VIM-2 metallo-β-lactamase- India. Antimicrob. Agents Chemother. Salverda, M. L. M., Dellus, E., Gorter, 2739. producing Pseudomonas aeruginosa 53, 5046–5054. F. A., Debets, A. J. M., Van der Tanaka, Y., Nishida, K., Nakamura, T., in The Netherlands: microbiol- Zhang, X. X., Zhang, T., and Fang, H. H. Oost, J., Hoekstra, R. F., et al. Chapagain, S. K., Inoue, D., Sei, ogy, epidemiology and clinical out- (2009). Antibiotic resistance genes in (2011). Initial mutations direct alter- K., et al. (2012). Characterization of comes. Int. J. Antimicrob. Agents 37, water environment. Appl. Microbiol. native pathways of protein evolu- microbial communities distributed 513–518. Biotechnol. 82, 397–414. tion. PLoS Genet. 7:e1001321. doi: in the groundwater pumped from Walsh, T. R. (2008). Clinically sig- Zhao, W. H., and Hu, Z. Q. (2012). 10.1371/journal.pgen.1001321 deep tube wells in the Kathmandu nificant carbapenemases: an update. Epidemiology and genetics of CTX- Salverda, M. L., De Visser, J. A. G., and Valley of Nepal. J. Water Health 10, Curr. Opin. Infect. Dis. 21, 367–371. M extended-spectrum β-lactamases Barlow, M. (2010). Natural evolution 170–180. Walsh,T. R., Weeks, J., Livermore, D. M., in Gram-negative bacteria. Crit. Rev. of TEM-1 β-lactamase: experimental Tausova, D., Dolejska, M., Cizek, A., and Toleman, M. A. (2011). Dissem- Microbiol. 39, 79–101. reconstruction and clinical relevance. Hanusova, L., Hrusakova, J., Svo- ination of NDM-1 positive bacteria Zhao, W. H., and Hu, Z. Q. (2010). FEMS Microbiol. Rev. 34, 1015–1036. boda, O., et al. (2012). Escherichia coli in the New Delhi environment and β-lactamases identified in clini- Sanschagrin, F., Couture, F., and with extended-spectrum β-lactamase its implications for human health: cal isolates of Pseudomonas aerug- Levesque, R. C. (1995). Pri- and plasmid-mediated quinolone an environmental point prevalence inosa. Crit. Rev. Microbiol. 36, mary structure of OXA-3 and phy- resistance genes in great cormorants study. Lancet Infect. Dis. 11, 245–258. logeny of oxacillin-hydrolyzing class and mallards in Central Europe. J. 355–362. D β-lactamases. Antimicrob. Agents Antimicrob. Chemother. 67, 1103– Wang, X., Minasov, G., and Shoichet, Chemother. 39, 887–893. 1107. B. K. (2002). Evolution of an antibi- Sarkar, S. K., Dutta, M., Chowdhury, C., Taverna, D. M., and Goldstein, R. A. otic resistance constrained by stabil- Kumar, A., and Ghosh, A. S. (2011). (2002). Why are proteins so robust ity and activity trade-offs. J. Mol. Biol. Conflict of Interest Statement: The PBP5, PBP6 and DacD play different to site mutations? J. Mol. Biol. 315, 320, 85–95. authors declare that the research was roles in intrinsic β-lactam resistance 479–484. Weinreich, D. M., Delaney, N. F., conducted in the absence of any com- of Escherichia coli. Microbiology 157, Tokuriki, N., and Tawfik, D. S. (2009). Depristo, M. A., and Hartl, D. mercial or financial relationships that 2702–2707. Stability effects of mutations and pro- L. (2006). Darwinian evolution can could be construed as a potential con- Schmieder, R., and Edwards, R. (2012). tein evolvability. Curr. Opin. Struct. follow only very few mutational flict of interest. Insights into antibiotic resistance Biol. 19, 596–604. paths to fitter proteins. Science 312, through metagenomic approaches. Tokuriki, N., Stricher, F., Serrano, L., 111–114. Received: 15 December 2012; accepted: Future Microbiol. 7, 73–89. and Tawfik, D. S. (2008). How pro- Widmann, M., Pleiss, J., and 09 January 2013; published online: 08 Shahid, M., Khan, F., Shah, M. S., tein stability and new functions trade Oelschlaeger, P. (2012). Systematic February 2013. Shukla, I., Shujatullah, F., Khan, off. PLoS Comput. Biol. 4:e1000002. analysis of metallo-β-lactamases Citation: Galán J-C, González-Candelas H. M., et al. (2012). NDM-1 in doi: 10.1371/journal.pcbi.1000002 using an automated database. F, Rolain J-M and Cantón R (2013) the Indian environment: hitherto the Toleman, M. A., Spencer, J., Jones, L., Antimicrob. Agents Chemother. 56, Antibiotics as selectors and accelerators of problem is not disquieting. Asian Pac. and Walsh, T. R. (2012). blaNDM- 3481–3491. diversity in the mechanisms of resistance: J. Trop. Med. 5, 335–336. 1 is a chimera likely constructed Wright, G. D. (2007). The antibiotic from the resistome to genetic plasticity in Soskine, M., and Tawfik, D. S. (2010). in Acinetobacter baumannii. Antimi- resistome: the nexus of chemical and the β-lactamases world. Front. Microbio. Mutational effects and the evolution crob. Agents Chemother. 56, 2773– genetic diversity. Nat. Rev. Microbiol. 4:9. doi: 10.3389/fmicb.2013.00009 of new protein functions. Nat. Rev. 2776 5, 175–186. This article was submitted to Fron- Genet. 11, 572–582. Tomatis, P.E., Fabiane, S. M., Simona, F., Wright, G. D. (2010). Antibiotic resis- tiers in Antimicrobials, Resistance and Tamae, C., Liu, A., Kim, K., Sitz, D., Carloni, P., Sutton, B. J., and Vila, A. tance in the environment: a link to Chemotherapy, a specialty of Frontiers in Hong, J., Becket, E., et al. (2008). J. (2008). Adaptive protein evolution the clinic? Curr. Opin. Microbiol. 13, Microbiology. Determination of antibiotic hyper- grants organismal fitness by improv- 589–594. Copyright © 2013 Galán, González- sensitivity among 4,000 single-gene- ing catalysis and flexibility. Proc. Wright, G. D. (2010). The antibiotic Candelas, Rolain and Cantón. This is knockout mutants of Escherichia coli. Natl. Acad. Sci. U.S.A. 105, 20605– resistome. Expert Opin. Drug Discov. an open-access article distributed under J. Bacteriol. 190, 5981–5988. 20610. 5, 779–788. the terms of the Creative Commons Tamura, K., and Nei, M. (1993). Esti- Torres-Cortés, G., Millán, V., Ramírez- Yim, G., Wang, H. H., and Davies, Attribution License, which permits use, mation of the number of nucleotide Saad, H. C., Nisa-Martínez, R., J. (2007). Antibiotics as signalling distribution and reproduction in other substitutions in the control region of Toro, N., and Martínez-Abarca, F. molecules. Philos. Trans. R. Soc. Lond. forums, provided the original authors and mitochondrial DNA in humans and (2011). Characterization of novel B Biol. Sci. 362, 1195–1200. source are credited and subject to any chimpanzees. Mol. Biol. Evol. 10, antibiotic resistance genes identi- Yong, D., Toleman, M. A., Giske, copyright notices concerning any third- 512–526. fied by functional metagenomics on C. G., Cho, H. S., Sundman, K., party graphics etc.

www.frontiersin.org February 2013 | Volume 4 | Article 9 | 55

“fmicb-04-00009” — 2013/2/7 — 12:09 — page 17 — #17 REVIEW ARTICLE published: 22 January 2013 doi: 10.3389/fmicb.2013.00004 Evolutionary consequences of antibiotic use for the resistome, mobilome, and microbial pangenome

Michael R. Gillings* Department of Biological Sciences, Macquarie University, Sydney, NSW, Australia

Edited by: The widespread use and abuse of antibiotic therapy has evolutionary and ecological con- Fiona Walsh, Agroscope Changins sequences, some of which are only just beginning to be examined. One well known Wädenswil, Switzerland consequence is the fixation of mutations and lateral gene transfer (LGT) events that confer Reviewed by: antibiotic resistance. Sequential selection events, driven by different classes of antibiotics, Antonio Oliver, Hospital Son Dureta, Spain have resulted in the assembly of diverse resistance determinants and mobile DNAs into Veljo Kisand, University of Tartu, novel genetic elements of ever-growing complexity and flexibility. These novel plasmids, Estonia integrons, and genomic islands have now become fixed at high frequency in diverse cell *Correspondence: lineages by human antibiotic use. Consequently they can be regarded as xenogenetic Michael R. Gillings, Department pollutants, analogous to xenobiotic compounds, but with the critical distinction that they of Biological Sciences, Macquarie University, Sydney, NSW replicate rather than degrade when released to pollute natural environments. Antibiotics 2109, Australia themselves must also be regarded as pollutants, since human production overwhelms e-mail: [email protected] natural synthesis, and a major proportion of ingested antibiotic is excreted unchanged into waste streams. Such antibiotic pollutants have non-target effects, raising the general rates of mutation, recombination, and LGT in all the microbiome, and simultaneously providing the selective force to fix such changes. This has the consequence of recruiting more genes into the resistome and mobilome, and of increasing the overlap between these two components of microbial genomes. Thus the human use and environmental release of antibiotics is having second order effects on the microbial world, because these small molecules act as drivers of bacterial evolution. Continued pollution with both xenogenetic elements and the selective agents that fix such elements in populations has potentially adverse consequences for human welfare.

Keywords: metagenomics, evolvability, pollution, pangenome, resistome, parvome, mobilome

INTRODUCTION Mackie, 2007; Baquero et al., 2009; Fajardo et al., 2009; Stokes The discovery of antibiotics and their use in the treatment of bac- and Gillings, 2011). terial infections was one of the major scientific achievements of There is evidence that the antibiotic revolution may have sec- the 20th century. However, over the last 60 years, there has been ond order effects, and is influencing the evolution of the entire a spectacular and rapid evolution of antibiotic resistant strains microbial biosphere (Martinez, 2008; Baquero, 2009; Couce and of bacteria. This has culminated in the appearance of pathogens Blázquez, 2009; Gillings and Stokes, 2012). This paper attempts to with resistance to a wide range of antibiotics, and a rise of sim- place antibiotics, resistance genes, their vectors, and their hosts ilarly resistant opportunistic organisms (Davies, 2007; Davies into an evolutionary perspective. By understanding the evolu- and Davies, 2010). Antibiotic resistance is a critical problem for tionary history of these genes and molecules, we place ourselves humans and their domestic animals, and is recognized as such by in a better position to predict their future (Martinez et al., 2007; workers in government, clinical practice, research, and industry Courvalin, 2008; Conway Morris, 2010). (Bush et al., 2011). The focus of research and thinking about this problem has THE NATURAL HISTORY OF ANTIBIOTICS mainly been from an anthropogenic viewpoint. However, at its The term “antibiotic” reflects our anthropocentric viewpoint. heart, antibiotic resistance is an environmental and evolution- Antibiotics are not a discrete class of molecules, but rather, encom- ary problem. Humans are now the greatest evolutionary force pass a broad range of structural and molecular families, united by on the planet (Palumbi, 2001), and it would help us understand their ability to inhibit microbial growth at high concentrations. and manage the resistance problem to investigate resistance from The original use of the word “antibiotic” was a generic term that a broader perspective, most notably in terms of the interplay simply reflected the outcome of a laboratory test (Waksman, 1973; between ecology, evolutionary dynamics, and natural selection. Davies and Davies, 2010). In modern terms, an antibiotic has A number of authors are now thinking about the ecology and nat- broadly come to mean any synthetic or naturally occurring low ural history of antibiotics and resistance genes. This approach molecular weight molecule that inhibits bacterial growth. will inform clinical and veterinary practices and will improve It is clear that there are millions of low molecular weight our understanding of evolutionary processes (Aminov and compounds in natural environments, and that in high enough

www.frontiersin.org January 2013 | Volume 4 | Article 4 | 56

“fmicb-04-00004” — 2013/1/19 — 14:54 — page1—#1 Gillings Humans, antibiotics and evolution

concentrations some of these could exhibit an antibiotic effect. subset of the diverse secondary metabolites used in the microbial However, it is unlikely that such compounds ever reach inhibitory world for signaling and other cell–cell interactions. Their clinical concentrations in nature, and there is a growing realization that use is based on a serendipitous ability to inhibit growth at unnat- their primary role is not necessarily in cross-species warfare, but urally high concentrations. There is a growing realization that the may largely lie in cell–cell communication (Linares et al., 2006). discovery, clinical use, and evolution of antibiotics must be consid- This change in viewpoint began with the discovery of bacterial ered against the background of their roles in natural environments quorum sensing, a system that allows communication between (Davies et al., 2006; Yim et al., 2007; Fajardo and Martínez, 2008; cells of the same or different species via signaling molecules called Ryan and Dow, 2008). auto-inducers. Quorum sensing is found in diverse organisms, and the scale and extent of signaling systems in general has led A GENERIC MODEL OF SMALL MOLECULE SIGNALING to the revelation that the microbial world is in a state of constant Thinking about antibiotics as a subset of signaling molecules and complex communication (Lyon and Muir, 2003; Henke and allows us to construct simple conceptual models that might Bassler, 2004; Stevens et al., 2012). Consequently, many of the establish some general principles about antibiotics and antibi- small molecules produced by bacteria may be involved in addi- otic resistance (Figure 1). A signaling molecule must be made tional forms of communication between cells. The compounds by a metabolic pathway, whose enzymes are encoded by genes (A, we know as “antibiotics” are a minor subset of this world of small B, and C in Figure 1). Any intermediate small molecule in this molecules. pathway can be exported as a signaling molecule, most probably The growth-inhibiting concentrations of antibiotics used in using an efflux pump. Small molecules then diffuse into envi- clinical practice are unlikely to ever be reached via natural syn- ronmental space, where they can bind to receptors on the cell thesis, and consequently, antibiotics probably have significant surface of the producing species (intra-species signaling), or to effects at sub-inhibitory concentrations, without affecting bac- cell surface receptors of different species (inter-species signaling). terial growth rates. It has now been shown that sub-inhibitory Alternatively, small molecules can be imported into cells via a concentrations of various antibiotic classes have effects on gene membrane transport protein, and then bind to a target within the transcription. Up regulation and down regulation of diverse genes cell (Figure 1). Binding of signaling molecules can influence tran- has been demonstrated, with some estimates suggesting that as scription or biochemical pathways, and thus affect the phenotypic many as 5% of gene promoters might be affected (Goh et al., 2002; attributes of the receptor cell. Linares et al., 2006). Some antibiotic classes modulate expres- This conceptual model (Figure 1) allows us to consider antibi- sion of particular genes (Tsui et al., 2004). The types of functions otics and resistance from a different and broader perspective. affected are diverse, including genes for protein synthesis, carbo- Antibiotics can be thought of as a subset of small biosynthetic hydrate metabolism, transport/binding proteins and genes of as molecules, albeit those which happen to have the property of yet unknown function (Goh et al., 2002; Lin et al., 2005). inhibiting bacterial growth at high concentrations. Given the fact The cascade of gene expression induced by sub-inhibitory that bacteria have been evolving and interacting for some 3.8 bil- concentrations of antibiotics leads to phenotypes of adaptive sig- lion years, it should not be surprising that the genes encoding nificance. These include flagella biosynthesis and the ability to biosynthetic and catabolic pathways for small molecules have colonize biotic and abiotic surfaces (Seshasayee et al., 2006). As an ancient evolutionary history. Pathways for synthesis of ery- might be expected, some of these phenotypes have direct rele- thromycin and streptomycin date to more than 600 million years vance for species–species interactions and responses to host cells, ago (Baltz, 2005), and it has been estimated that the serine β- such as virulence, biofilm formation, and motility (Goh et al., lactamases date back 2 billion years (Hall and Barlow,2004). Under 2002; Hoffman et al., 2005; Linares et al., 2006; Marr et al., 2007; this view “antibiotics” predate humans by some billions of years. Shank and Kolter, 2009). Collectively, these observations strongly Note that the use of an intermediate in the biosynthetic path- suggest that the antibiotics used in medical treatment are a small way as a signaling molecule (Figure 1, triangle) means that

FIGURE 1 | Conceptual schematic illustrating the production, receptors, or enter a second cell via a membrane transport protein. export, and target sites of a small molecule biosynthetic cluster. Inside the second cell there may be binding sites on additional The metabolic pathway for synthesis of a small molecule is encoded target molecules. Most of the molecules we know as antibiotics by genes A, B, and C. One of the intermediates (triangle) is exported may be a subset of this more general class of signal-receptor from the cell via an efﬂux pump, where it can then bind to cell surface systems.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy January 2013 | Volume 4 | Article 4 | 57

“fmicb-04-00004” — 2013/1/19 — 14:54 — page2—#2 Gillings Humans, antibiotics and evolution

the producing cell already encodes an enzyme capable of mod- gene clusters for small molecules (together with their associated ifying the signal molecule. Under other circumstances, such efflux pumps and catabolic functions) explains the penetration of a gene might confer resistance to high concentrations of the efflux pumps and degradative enzymes into species unrelated to agent. Many biosynthetic gene clusters that make “antibiotics” are the original “antibiotic” producer. also known contain genes that confer “resistance” to those same antibiotics (Pootoolal et al., 2002; Huang et al., 2005). Given that ANTIBIOTICS, RESISTANCE GENES, AND THE GLOBAL gene clusters for secreted molecules are frequently subject to lat- MICROBIOME eral gene transfer (LGT; Fischbach et al., 2008; Nogueira et al., In thinking about the ecology and evolution of antibiotics and 2009), it should not be surprising that there is clear evidence resistance genes, we can summarize the sub-components upon for lateral transfer of antibiotic resistance genes from environ- which selection can act in a series of Venn diagrams (Figure 2). mental bacteria into pathogens (Gillings et al., 2008a,b; Forsberg The largest category is the Global Microbiome, encompassing all et al., 2012). the prokaryotic cells in the biosphere. This may contain 4–6 × 1030 Small molecules can exit the producing cell via a wide diver- cells and hold a significant proportion of the carbon, nitrogen, and sity of membrane bound efflux pumps (Figure 1; Paulsen et al., phosphorus found in living things (Whitman et al., 1998). The 1996; Desvaux et al., 2004; Poole, 2005; Bay and Turner, 2009), phenotypes exhibited by the global microbiome are encoded by each of which, in turn, may be able to export diverse molec- the microbial Pangenome, that is, the set of genes present in all ular species (Zgurskaya and Nikaido, 2000; Kuete et al., 2011). the genomes of all the prokaryotes in the biosphere (Medini et al., Such pumps have evolved as mechanisms to export natural sub- 2005; Tettelin et al., 2008; Lapierre and Gogarten, 2009). Estimates stances produced internally, or those that are produced by other of the composition of the pangenome have been made, based cells (Martinez et al., 2009b). Efflux pumps may also be essen- on the rapid accumulation of bacterial genome and metagenome tial for colonizing and persisting in eukaryotic hosts (Piddock, sequences. Some 250 gene families are common to all bacterial 2006). It is thus clear that antibiotic resistance mediated by genomes (the extended core genome) and about 8,000 gene fam- efflux pumps is a side-effect of a more general molecular export ilies are niche-specific genes essential for survival in particular system. environments (the character genome). The bulk of pangenomic Genes for efflux pumps are commonly found in bacteria diversity comprises more than 139,000 gene families that occur that produce antibiotics (Petković et al., 2006), and these genes as accessory genes, these being found dispersed amongst single are often found embedded within biosynthetic gene clusters for strains, serovars or species (Lapierre and Gogarten, 2009). The antibiotics, thus conferring efflux ability (and “resistance”) on combined coding capacity of the pangenome is expressed as the the producing organism (Méndez and Salas, 2001). Again, the Panproteome, this being the sum of all the proteins encoded and “resistance” gene is preexistent, and located within a gene cluster produced by the microbial realm. characterized by LGT (Nogueira et al., 2009). A proportion of the pangenome is highly mobile, and able Once exported from the cell, a signaling molecule is free to bind to move comparatively freely between prokaryote species in the with receptors on the surface of neighboring cells (Figure 1). Point process known as LGT. Elements that specialize in moving DNA mutations in the genes for these receptors might alter the bind- within and between genomes include plasmids, transposons, inte- ing site and thus generate a “resistance” phenotype. Alternatively, grons, insertion sequences, and integrative conjugative elements. the signaling molecule may be taken up by a membrane trans- These elements and the genes they carry are collectively known as port protein and imported into a new cell. A surprising diversity the Mobilome (Figure 2; Siefert, 2009; Leplae et al., 2010). In prin- of bacteria make use of such transport proteins to import small ciple, and over evolutionary timescales, any part of the pangenome molecules such as antibiotics, which they can then use as a sole can be mobilized by LGT. The mobilome helps to generate enor- carbon source (Dantas et al., 2008). Lateral transfer of the genes mous diversity within prokaryote genomes by recombination for such catabolic processes would confer resistance on the recip- between various mobile elements to create a series of complex ient cell. Resistance could also be generated by mutation to the mosaic structures (Garriss et al., 2009; Toleman and Walsh, 2011; gene encoding the membrane transport protein, preventing entry Toussaint and Chandler, 2012). of the signaling molecule. Once inside a recipient cell, a signaling The Resistome is defined as the collection of all genes that could molecule may bind to a target site, and thus exert an effect. Muta- contribute to a phenotype of antibiotic resistance (D’Costa et al., tion of the gene encoding the target site may abolish binding and 2006; Wright, 2007, 2010; Forsberg et al., 2012). This is a purely prevent the effect. In the case of an antibiotic, such a change would functional definition made from a human and medical point of be called a resistance mutation. view, for as we have seen, the resistome encompasses diverse Although the model I have presented above is simplistic, nev- genes whose original functions were not simply for avoiding the ertheless it has good explanatory power for understanding the effects of antibiotics. Nevertheless, it is a useful concept, because ecology and evolution of both antibiotics and their correspond- it underscores the point that resistance genes originate from envi- ing resistance genes. Antibiotics are seen as a subset of a diverse ronmental bacteria. A database of antibiotic resistance genes has group of small molecules whose primary function is in cell–cell been developed (Liu and Pop, 2009), but the 20,000 genes already interactions. Genes for synthesizing, transporting, and cataboliz- listed are only a small proportion of the resistome, since inves- ing these small molecules can be co-opted as resistance genes when tigation of soil metagenomes has revealed diverse and divergent cells are exposed to unnaturally high concentrations of these com- resistance genes capable of dealing with multiple classes of antibi- pounds. The high frequency of lateral exchange of biosynthetic otic (Riesenfeld et al., 2004; Allen et al., 2008). Genes encoding

www.frontiersin.org January 2013 | Volume 4 | Article 4 | 58

“fmicb-04-00004” — 2013/1/19 — 14:54 — page3—#3 Gillings Humans, antibiotics and evolution

EVOLUTIONARY CONSEQUENCES OF THE ANTIBIOTIC AGE Having examined the relationships between the key components of relevance to antibiotic production and resistance (Figure 2), we can now examine how each might be affected by human activity. There are several potential dimensions to these effects, including changes to the abundance and diversity of each component, and whether the effects are transient or permanent.

THE PARVOME Antibiotics are released into the environment via human waste streams because a signiﬁcant proportion of prophylactic antibiotics are excreted essentially unchanged (Sarmah et al., 2006; Le-Minh et al., 2010). Although it is difﬁcult to obtain precise estimates for commercial production of antibiotics, it probably amounts to millions of metric tons per year (Segura et al., 2009). Because of the volume of antibiotics generated by human activity, the majority of the environmental load of antibiotics now orig- inates from this commercial production (Figure 2; Davies and

FIGURE 2 | Conceptual representation of the biological molecules of Davies, 2010). Certainly the local concentrations of this subset relevance to antibiotic resistance. The small cross-hatched boxes of the parvome have markedly increased due to human activity. represent the antibiotics and resistance genes of relevance to clinical Further, the diversity of small bioactive molecules has increased, practice. Respectively, these are a small subset of the world of small because many antibiotics are synthetic modifications of natural bioactive molecules (the parvome), and the world of potential resistance determinants (the resistome). The resistome comprises the genes that structures. There is now a zone of influence around all human potentially encode resistance to antibiotics. The mobilome comprises the activities where the abundance and diversity of the parvome is mobile proportion of bacterial genomes. The mobilome and resistome increased. Such zones increase selective pressures on the local overlap, since many resistance genes are located on mobile elements. Both the resistome and mobilome are a subset of the total coding capacity microbiome, not just the intended targets of antibiotic therapy of prokaryotic cells, the pangenome, which is expressed as the (Martinez et al., 2009a; Taylor et al., 2011), and may also interfere panproteome. Note that only a small proportion of the parvome is utilized with cell–cell communication. by humans for antibiotic purposes, and that the scale of commercial antibiotic production probably overwhelms the natural production of these molecules by the entire global microbiota. THE RESISTOME Increasing the concentration of antibiotics in the environment has effects on both the diversity and the abundance of genes antibiotic resistance are ancient components of the pangenome, belonging to the resistome. Selection for cells that carry resis- since they have been recovered from 30,000 year old permafrost tance determinants increases their relative abundance, and thus (D’Costa et al., 2011), and from a cave microbiome that has been increases the abundance of genes that confer resistance. Further, isolated for 4 million years (Bhullar et al., 2012). Note that the the ability of many elements of the resistome to undergo LGT, resistome overlaps with the mobilome (Figure 2), because many means that identical resistance genes can now be found in diverse antibiotic resistance genes are found on mobile elements, allow- bacterial species, from clinical contexts, from domestic animals, ing the resistome to be widely disseminated by LGT (Norman wild animals, and in locations apparently distant from the influ- et al., 2009; Fondi and Fani, 2010; Skippington and Ragan, 2011). ence of developed societies, such as the Arctic, Antarctica, and Also note that the resistance genes and mobile elements that are the Amazonian jungle (Pallecchi et al., 2008; Sjolund et al., 2008; of concern for clinical antibiotic resistance (cross-hatched box, Bartoloni et al., 2009; Stokes and Gillings, 2011). That humans left hand side Figure 2) are just a fraction of the resistome and are responsible for this phenomenon is demonstrated by the pos- mobilome. itive correlation between the abundance of resistome elements of The Parvome is the world of small bioactive molecules pro- clinical significance and proximity to human activity (Skurnik duced by cells (also sometimes called secondary metabolites). The et al., 2006; Hardwick et al., 2008; Thaller et al., 2010; Nardelli parvome includes important classes of molecules such as polyke- et al., 2012). tides, aminoglycosides, terpenoids, alkaloids, and non-ribosomal Selection pressure is particularly acute in human waste streams, peptides, many of which have antimicrobial activity (Davies, 2011; where resistance genes are shed, mixed with high concentrations Davies and Ryan, 2011). The parvome overlaps with the proteome, of antibiotics and other selective agents (Baquero et al., 2008; because some of these small bioactive molecules are peptides. Note Schlüter et al., 2008; Moura et al., 2010). Exposure of environmen- however, that only a small proportion of the parvome is utilized tal microorganisms to this mixture encourages fixation of LGT by humans for its antimicrobial properties (cross-hatched box, events, spreading elements of the resistome into diverse strains right hand side Figure 2). Commercial production of antibiotics and species, thus further increasing their abundance, and their overwhelms their natural synthesis, such that humans are now the penetration into new hosts and niches. major source of antibiotics in the general environment (Figure 2; Exposure to sub-inhibitory concentrations of antibiotics Sarmah et al., 2006; Davies and Davies, 2010). induces expression of error-prone DNA polymerases, increasing

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy January 2013 | Volume 4 | Article 4 | 59

“fmicb-04-00004” — 2013/1/19 — 14:54 — page4—#4 Gillings Humans, antibiotics and evolution

the basal mutation rate (Kohanski et al., 2010; Thi et al., 2011). heavy metals, arsenic, and disinfectants. Exposure to any one This has the effect of generating additional genomic diversity, and of these agents selects for lineages containing mobile elements of co-opting additional genes into the resistome via mutational with appropriate resistance genes, and simultaneously fixes all changes to housekeeping or accessory genes whose original func- the other genes in physical linkage on that element, including tion was not antibiotic resistance (Dantas and Sommer, 2012). antibiotic resistance determinants. Such co-selection of antibiotic As a second order effect, constant low level antibiotic exposure resistance has long been thought to arise as a consequence of pol- selects for lineages with inherently higher rates of mutation, gen- lution with heavy metals (Baker-Austin et al., 2006; Salyers and erating additional diversity across the entire pangenome via drift Shoemaker, 2006; Stepanauskas et al., 2006; Wright et al., 2008; and selection (Earl and Deem, 2004; Pigliucci, 2008; Couce and Rosewarne et al., 2010; Drudge et al., 2012). Disinfectant use also Blázquez, 2009; Gillings and Stokes, 2012). results in co-selection of antibiotic resistance (Gaze et al., 2005; Human activity has increased the abundance of resistome ele- Hegstad et al., 2010), and probably had a role in the origin of the ments by selection, as shown by their increase in frequency in soils class 1 integrons responsible for the widespread dissemination of collected over the last 70 years (Knapp et al.,2009), and by their lat- antibiotic resistance amongst gram negative organisms (Gillings eral transfer to diverse species. We have also increased the diversity et al., 2008b). and membership of the resistome by fixation of de novo mutations High rates of LGT occur between organisms in similar Phyla, and by the co-option of genes as resistance determinants. Thus organisms with similar GC content, and organisms that occupy the size of the resistome, as a proportion of the pangenome, is the same environmental niches (Thomas and Nielsen, 2005; Popa probably becoming larger (Figure 2). and Dagan, 2011). This activity creates lateral exchange communities, where DNA can be exchanged with relative ease (Beiko THE MOBILOME et al., 2005; Kloesges et al., 2010; Skippington and Ragan, 2011). The clinical use and environmental dissemination of antibiotics Shared niches, and in particular, biofilms, promote such exchanges has had significant effects on the abundance and diversity of ele- (Sorensen et al., 2005), and both conjugation and natural trans- ments within the mobilome. Selection for antibiotic resistance formation appear to be important in the process (Kloesges et al., has fixed lineages carrying diverse resistance determinants on a 2010; Domingues et al., 2012). While evolutionary distance and similarly diverse array of mobile elements. Such complex mobile difference in GC content can restrict LGT, there are mechanisms elements now occur at high frequency in human-dominated sys- for bypassing these barriers (Popa et al., 2011), and genes can make tems, and in more natural ecosystems, where they can move by their way between distantly related organisms via a sequential LGT into environmental organisms (Chee-Sanford et al., 2001; series of lateral transfers. Nagachinta and Chen, 2008; Schlüter et al., 2008; Gillings et al., Human use of antibiotics is therefore having multiple effects 2009a; Pellegrini et al., 2009). on the mobile components of the bacterial pangenome. One The very use of antibiotics may itself increase the frequency would predict that the size of the mobilome as a proportion of of LGT, and thus the penetration of elements of the mobilome the pangenome is becoming larger, as more genes are recruited into new bacterial hosts (Beaber et al., 2004; Úbeda et al., 2005; onto mobile elements (Figure 2). The number of resistome ele- Prudhomme et al., 2006). This effect is driven by the bacterial SOS ments now residing on mobile DNA is also increasing, given response, which temporarily increases both the basal rate of LGT that plasmids collected before the antibiotic era had no resis- and of recombination (Tenaillon et al., 2004; Schlacher and Good- tance determinants (Datta and Hughes, 1983; Hughes and Datta, man, 2007). There is also evidence that human activities actually 1983), and modern plasmids of similar structure have acquired select for bacteria with a permanently increased propensity for a wide range of resistance genes dealing with various selective LGT (Gillings and Stokes, 2012). Consequently, antibiotic pollu- agents in addition to antibiotics. Consequently, there is now a tion creates hotspots for the assembly of complex, mosaic mobile greater overlap between the resistome and mobilome, particularly elements from diverse sources, and provides a selective force for with respect to genes of concern for human health and welfare their subsequent fixation in diverse lineages (Szczepanowski et al., (Figure 2). 2005; Schlüter et al., 2008; Gillings et al., 2009b). The accumulation of diverse mobile elements within single THE PANGENOME AND GLOBAL MICROBIOME plasmids or at single loci provides opportunities for complex Human antibiotic use has pervasive effects on both the pangenome rearrangements and recombination events that in turn, gener- and the local composition of the microbiome, largely because atemorediversity(Garriss et al., 2009). The emergent properties antibiotics have non-target effects at gene, cell, and population that arise as mobile elements gain more components means that levels. Exposure of cells to antibiotics induces an SOS response they can effectively increase their own complexity (Krizova et al., that has widespread effects on bacterial genomes, including rais- 2011; Toleman and Walsh, 2011). This phenomenon is evident in ing the rates of mutation, recombination, and LGT (Tenaillon the increasing complexity and phenotypic plasticity of genomic et al., 2004; Aertsen and Michiels, 2006; Schlacher and Goodman, islands and integrative conjugative elements in emerging nosoco- 2007). This increase in basal rates of evolution applies to all the mial pathogens such as Pseudomonas aeruginosa and Acinetobacter genes and cells in the exposed environment, not just the targets baumannii (Fournier et al., 2006; Klockgether et al., 2011). of antibiotic therapy. Consequently, the release of antibiotics into The selective forces acting upon complex mobile elements are natural environments will affect the diversity of the pangenome not restricted to antibiotic pressure. Mobile DNAs often carry and the composition and ecology of the global microbiota (Couce genes conferring resistance to other selective agents, such as and Blázquez, 2009; Martinez, 2009a; Gillings and Stokes, 2012).

www.frontiersin.org January 2013 | Volume 4 | Article 4 | 60

“fmicb-04-00004” — 2013/1/19 — 14:54 — page5—#5 Gillings Humans, antibiotics and evolution

Effects in particular environments have been demonstrated environmental bacteria (Schlüter et al., 2008; Moura et al., 2010). experimentally. Short term antibiotic treatments lead to changes Resistome and mobilome elements are not necessarily removed in abundance, richness, and diversity of the human gut micro- by water treatment (Graham et al., 2011; Drudge et al., 2012), biota (Dethlefsen et al., 2008), which can remain perturbed for allowing resistance genes to be used as markers of human influ- years after treatment (Jakobsson et al., 2010; Sommer and Dan- ence on aquatic ecosystems (Pei et al., 2006; Pruden et al., 2006; tas, 2011). The composition and functional diversity of soil Storteboom et al., 2010). and sediment communities is affected by exposure to antibiotics Domestic and agricultural animals are a source of significant (Kong et al., 2006; Cordova-Kreylos and Scow, 2007), and persis- quantities of antibiotics and resistance genes. Animal waste and tence of antibiotic residues in sediments or in the water column pig slurry are used to manure soils, with the consequent introduc- leads to alteration of microbiota and in situ selection for antibi- tion of resistance genes and resistant bacteria (Binh et al., 2009; otic resistance (Cabello, 2006; Knapp et al., 2008). While pulses Byrne-Bailey et al., 2009; Gaze et al., 2011).Thelongtermfateof of exposure to antibiotics may only produce transient selection antibiotics and resistance genes is difficult to predict without quan- events, alterations to community composition, and the fixation of titative measurements over appropriate timescales (Chee-Sanford resistance genes and their mobile vectors may be permanent, with et al., 2009). However, it is clear that antibiotic resistant bacteria unpredictable consequences for the whole microbiome (Martinez, will increase in abundance, lateral transfers to soil organisms will 2009b; Gillings and Stokes, 2012). occur, and resistance genes will be sequestered by diverse elements of the mobilome (Heuer et al., 2011). ANTIBIOTICS AND RESISTANCE GENES AS POLLUTANTS Antibiotics and their resistance genes originated from natural environments, but human use of antibiotics has perturbed the CONCLUSION dynamics of this natural system. The high concentration of Human use of antibiotics for medicine and agriculture may have antibiotics used prophylactically by humans has two main con- consequences beyond their intended applications. Large quan- sequences. Firstly, it leads to antibiotic contamination of human tities of antibiotics now emanate from human waste streams, waste streams, and secondly, the selection imposed by antibiotic as do the xenogenetic elements fixed in human ecosystems by use has fixed ever more complex genetic elements in commensals antibiotic selection. Much more attention needs to be paid to the and pathogens. These new“xenogenetic”elements are also released origins and fates of such pollutants (Allen et al., 2010). The antibi- via human waste streams. While antibiotics might be treated as otic revolution may be having effects across the entire microbial simple pollutants, xenogenetic mobile elements are capable of biosphere (Martinez, 2009b), changing the basal rate of bac- replication, and are thus more akin to invasive species (Gillings terial evolution, altering the composition of the resistome and and Stokes, 2012). mobilome, and promoting lateral transfer of mobile genetic ele- Between 30 and 90% of the antibiotics given for human or ments (Couce and Blázquez, 2009; Gillings and Stokes, 2012). veterinary use are excreted essentially unchanged (Sarmah et al., Antibiotic contamination promotes the fixation and mobilization 2006). These compounds can be both persistent and mobile, and of resistance genes between environmental and clinical microbiota are often not removed during sewage treatment (Watkinson et al., (Kristiansson et al., 2011), and resistance genes are now widely 2007; Le-Minh et al., 2010; Zuccato et al., 2010). Antibiotics are spread through the biosphere (Martinez, 2009a; Stokes and also released in high concentrations from facilities where antibi- Gillings, 2011). otics are produced (Li et al., 2009, 2010), and are disseminated Above all, we need to address the antibiotic resistance problem during application of manure to agricultural land (Chee-Sanford from a broader evolutionary and ecological perspective (Aminov et al., 2009; Heuer et al., 2011). The consequences of pollution and Mackie, 2007; Baquero et al., 2009; Fajardo et al., 2009). The with antibiotics, particularly for aquatic systems, are being actively ability of natural selection to shape species and communities is examined (Taylor et al., 2011; Lupo et al., 2012), and there is a call the same for microorganisms as it is for larger species (Gillings for development of policies to reduce the release of antibiotics and and Stokes, 2012), and the ecological theory of community assem- bacteria via human waste streams (Baquero et al., 2008). bly developed for multicellular organisms can be applied to the Waste streams that release antibiotics into the environment microbiome (Costello et al., 2012). The risk associated with the also disseminate antibiotic resistance genes and mobile DNA ele- environmental spread of resistance genes with known adverse con- ments, which should similarly be regarded as pollutants emanating sequences for human welfare has had little attention, nor has the from human activity (Pruden et al., 2006; Martinez, 2009a; Stor- potential for pollution with antibiotics to widely affect the global teboom et al., 2010; Graham et al., 2011). The abundance of microbiome. In comparison, the potential escape of resistance resistance genes in soils has been increasing since the introduc- gene markers used in the generation of genetically modified plants tion of antibiotics in the 1940s (Knapp et al., 2009), in parallel has been the subject of considerable research (Martinez,2012). It is with the increasing concentrations of more conventional chemical time to pay more attention to the bioactive molecules that humans pollutants. release into the environment. Resistance genes and complex mobile elements are commonly reported from wastewater and sewage treatment plants (Tennstedt ACKNOWLEDGMENTS et al., 2003; Zhang et al., 2009; Pellegrini et al., 2011). Wastewater Aspects of the author’s work are supported by the Australian is regarded as a hotspot for interactions between mobile elements Research Council, the National Health and Medical Research and for their lateral transfer between pathogens, commensals, and Council of Australia and Macquarie University.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy January 2013 | Volume 4 | Article 4 | 61

“fmicb-04-00004” — 2013/1/19 — 14:54 — page6—#6 Gillings Humans, antibiotics and evolution

REFERENCES Binh, C. T. T., Heuer, H., Kaupenjo- Dantas, G., and Sommer, M. O. Fajardo, A., and Martínez, J. L. (2008). Aertsen, A., and Michiels, C. W. (2006). hann, M., and Smalla, K. (2009). A. (2012). Context matters—the Antibiotics as signals that trigger spe- Upstream of the SOS response: figure Diverse aadA gene cassettes on class complex interplay between resis- cific bacterial responses. Curr. Opin. out the trigger. Trends Microbiol. 14, 1 integrons introduced into soil via tome genotypes and resistance phe- Microbiol. 11, 161–167. 421–423. spread manure. Res. Microbiol. 160, notypes. Curr. Opin. Microbiol. 15, Fischbach, M. A., Walsh, C. T., and Allen, H. K., Donato, J., Wang, H. 427–433. 577–582. Clardy, J. (2008). The evolution of H., Cloud-Hansen, K. A., Davies, J., Bush, K., Courvalin, P., Dantas, G., Dantas, G., Sommer, M. O. A., Oluwase- gene collectives: how natural selec- and Handelsman, J. (2010). Call of Davies, J., Eisenstein, B., Huovinen, gun, R. D., and Church, G. M. tion drives chemical innovation. Proc. the wild: antibiotic resistance genes P., et al. (2011). Tackling antibiotic (2008). Bacteria subsisting on antibi- Natl. Acad. Sci. U.S.A. 105, 4601– in natural environments. Nat. Rev. resistance. Nat. Rev. Microbiol. 9, otics. Science 320, 100–103. 4608. Microbiol. 8, 251–259. 894–896. Datta, N., and Hughes, V. M. (1983). Fondi, M., and Fani, R. (2010). The Allen, H. K., Moe, L. A., Rodbum- Byrne-Bailey, K. G., Gaze, W. H., Kay, P., Plasmids of the same Inc groups in horizontal flow of the plasmid resis- rer, J., Gaarder, A., and Handelsman, Boxall, A. B. A., Hawkey, P. M., and Enterobacteria before and after the tome: clues from inter-generic sim- J. (2008). Functional metagenomics Wellington, E. M. H. (2009). Preva- medical use of antibiotics. Nature ilarity networks. Environ. Microbiol. reveals diverse β-lactamases in a lence of sulfonamide resistance genes 306, 616–617. 12, 3228–3242. remote Alaskan soil. ISME J. 3, in bacterial isolates from manured Davies, J. (2007). Microbes have the last Forsberg, K. J., Reyes, A., Wang, B., 243–251. agricultural soils and pig slurry in the word. EMBO Rep. 8, 616–621. Selleck, E. M., Sommer, M. O. A., Aminov, R. I., and Mackie, R. I. (2007). United Kingdom. Antimicrob. Agents Davies, J. (2011). How to discover new and Dantas, G. (2012). The shared Evolution and ecology of antibiotic Chemother. 53, 696–702. antibiotics: harvesting the parvome. antibiotic resistome of soil bacteria resistance genes. FEMS Microbiol. Cabello, F. C. (2006). Heavy use of pro- Curr. Opin. Chem. Biol. 15, 5–10. and human pathogens. Science 337, Lett. 271, 147–161. phylactic antibiotics in aquaculture: a Davies, J., and Davies, D. (2010). Ori- 1107–1111. Baker-Austin, C., Wright, M. S., growing problem for human and ani- gins and evolution of antibiotic resis- Fournier, P.-E., Vallenet, D., Barbe, Stepanauskas, R., and McArthur, J. V. mal health and for the environment. tance. Microbiol. Mol. Biol. Rev. 74, V., Audic, S., Ogata, H., Poirel, L., (2006). Co-selection of antibiotic and Environ. Microbiol. 8, 1137–1144. 417–433. et al. (2006). Comparative genomics metal resistance. Trends Microbiol. 14, Chee-Sanford, J. C., Aminov, R. I., Kra- Davies, J., and Ryan, K. S. (2011). of multidrug resistance in Acinetobac- 176–182. pac, I. J., Garrigues-Jeanjean, N., and Introducing the parvome: bioactive ter baumannii. PLoS Genet. 2:e7. doi: Baltz, R. H. (2005). Antibiotic discovery Mackie, R. I. (2001). Occurrence and compounds in the microbial world. 10.1371/journal.pgen.0020007 from actinomycetes: will a renais- diversity of tetracycline resistance ACS Chem. Biol. 7, 252–259. Garriss, G., Waldor, M. K., and sance follow the decline and fall? SIM genes in lagoons and groundwa- Davies, J., Spiegelman, G. B., and Burrus, V. (2009). Mobile antibi- News 55, 186–196. ter underlying two swine production Yim, G. (2006). The world of subin- otic resistance encoding elements Baquero, F. (2009). Environmental facilities. Appl. Environ. Microbiol. 67, hibitory antibiotic concentrations. promote their own diversity. PLoS stress and evolvability in microbial 1494–1502. Curr. Opin. Microbiol. 9, 445–453. Genet. 5:e1000775. doi: 10.1371/ systems. Clin. Microbiol. Infect. 15, Chee-Sanford, J. C., Mackie, R. I., Desvaux, M., Parham, N. J., Scott- journal.pgen.1000775 5–10. Koike, S., Krapac, I. G., Lin, Y.-F., Tucker, A., and Henderson, I. R. Gaze, W. H., Abdouslam, N., Hawkey, P. Baquero, F., Alvarez-Ortega, C., and Yannarell,A. C., et al. (2009). Fate and (2004). The general secretory path- M., and Wellington, E. M. H. (2005). Martinez, J. L. (2009). Ecology and transport of antibiotic residues and way: a general misnomer? Trends Incidence of class 1 integrons in a evolution of antibiotic resistance. antibiotic resistance Ggenes follow- Microbiol. 12, 306–309. quaternary ammonium compound- Environ. Microbiol. Rep. 1, 469–476. ing land application of manure waste. Dethlefsen, L., Huse, S. M., Sogin, M. L., polluted environment. Antimicrob. Baquero, F.,Martínez, J.-L., and Cantón, J. Environ. Qual. 38, 1086–1108. and Relman, D. A. (2008). The per- Agents Chemother. 49, 1802–1807. R. (2008). Antibiotics and antibi- Conway Morris, S. (2010). Evolution: vasive effects of an antibiotic on the Gaze, W. H., Zhang, L., Abdouslam, otic resistance in water environments. like any other science it is predictable. human gut microbiota, as revealed N. A., Hawkey, P. M., Calvo-Bado, Curr. Opin. Biotechnol. 19, 260–265. Philos. Trans. R. Soc. Lond. B Biol. Sci. by deep 16S rRNA sequencing. PLoS L., Royle, J., et al. (2011). Impacts of Bartoloni, A., Pallecchi, L., Rodríguez, 365, 133–145. Biol. 6:e280. doi: 10.1371/jour- anthropogenic activity on the ecology H., Fernandez, C., Mantella, A., Bar- Cordova-Kreylos, A. L., and Scow, K. nal.pbio.0060280 of class 1 integrons and integron- talesi, F., et al. (2009). Antibiotic M. (2007). Effects of ciprofloxacin Domingues, S., Harms, K., Fricke, associated genes in the environment. resistance in a very remote Amazonas on salt marsh sediment microbial W. F., Johnsen, P. J., da Silva, ISME J. 5, 1253–1261. community. Int. J. Antimicrob. Agents communities. ISME J. 1, 585–595. G. J., and Nielsen, K. M. (2012). Gillings, M., Boucher, Y., Labbate, M., 33, 125–129. Costello, E. K., Stagaman, K., Dethlef- Natural transformation facilitates Holmes, A., Krishnan, S., Holley, Bay, D., and Turner, R. (2009). Diversity sen, L., Bohannan, B. J. M., and Rel- transfer of transposons, integrons M., et al. (2008a). The evolution and evolution of the small multidrug man, D. A. (2012). The application and gene cassettes between bacte- of class 1 integrons and the rise of resistance protein family. BMC Evol. of ecological theory toward an under- rial species. PLoS Pathog. 8:e1002837. antibiotic resistance. J. Bacteriol. 190, Biol. 9:140. doi: 10.1186/1471-2148- standing of the human microbiome. doi: 10.1371/journal.ppat.1002837 5095–5100. 9-140 Science 336, 1255–1262. Drudge, C. N., Elliott, A. V. C., Plach, Gillings, M. R., Xuejun, D., Hardwick, Beaber, J. W., Hochhut, B., and Waldor, Couce, A., and Blázquez, J. (2009). J. M., Ejim, L. J., Wright, G. D., S. A., Holley, M. P., and Stokes, H. M. K. (2004). SOS response promotes Side effects of antibiotics on genetic Droppo, I. G., et al. (2012). Diversity W. (2008b). Gene cassettes encoding horizontal dissemination of antibi- variability. FEMS Microbiol. Rev. 33, of integron- and culture-associated resistance to quaternary ammonium otic resistance genes. Nature 427, 531–538. antibiotic resistance genes in fresh- compounds: a role in the origin of 72–74. Courvalin, P. (2008). Predictable and water floc. Appl.Env.Microbiol.78, clinical class 1 integrons? ISME J.3, Beiko, R. G., Harlow, T. J., and Ragan, unpredictable evolution of antibiotic 4367–4372. 209–215. M. A. (2005). Highways of gene shar- resistance. J. Intern. Med. 264, 4–16. Earl, D. J., and Deem, M. W. (2004). Gillings, M. R., Holley, M. P., and ing in prokaryotes. Proc. Natl. Acad. D’Costa, V. M., King, C. E., Kalan, L., Evolvability is a selectable trait. Proc. Stokes, H. W. (2009a). Evidence Sci. U.S.A. 102, 14332–14337. Morar, M., Sung, W. W. L., Schwarz, Natl. Acad. Sci. U.S.A. 101, 11531– for dynamic exchange of qac gene Bhullar, K., Waglechner, N., Pawlowski, C., et al. (2011). Antibiotic resistance 11536. cassettes between class 1 integrons A., Koteva, K., Banks, E. D., Johnston, is ancient. Nature 477, 457–461. Fajardo, A., Linares, J. F., and Martínez, and other integrons in freshwater M. D., et al. (2012). Antibiotic resis- D’Costa, V. M., McGrann, K. M., J. L. (2009). Towards an ecological biofilms. FEMS Microbiol. Lett. 296, tance is prevalent in an isolated cave Hughes, D. W., and Wright, G. D. approach to antibiotics and antibi- 282–288. microbiome. PLoS ONE 7:e34953. (2006). Sampling the antibiotic resis- otic resistance genes. Clin. Microbiol. Gillings, M. R., Labbate, M., Sajjad, A., doi: 10.1371/journal.pone.0034953 tome. Science 311, 374–377. Infect. 15, 14–16. Giguere, N. J., Holley, M. P., and

www.frontiersin.org January 2013 | Volume 4 | Article 4 | 62

“fmicb-04-00004” — 2013/1/19 — 14:54 — page7—#7 Gillings Humans, antibiotics and evolution

Stokes, H. W. (2009b). Mobilization the ‘pre-antibiotic’ era. Nature 302, a multidrug-resistant phenotype. Int. Martinez, J. L. (2009a). Environmen- of a Tn402-like class 1 integron with 725–726. J. Antimicrob. Agents 37, 156–161. tal pollution by antibiotics and by a novel cassette array via flanking Jakobsson, H. E., Jernberg, C., Ander- Lapierre, P., and Gogarten, J. P. (2009). antibiotic resistance determinants. miniature inverted-repeat transpos- sson, A. F., Sjölund-Karlsson, M., Estimating the size of the bacte- Environ. Pollut. 157, 2893–2902. able element-like structures. Appl. Jansson, J. K., and Engstrand, L. rial pan-genome. Trends Genet. 25, Martinez, J. L. (2009b). The role of nat- Environ. Microbiol. 75, 6002–6004. (2010). Short-term antibiotic treat- 107–110. ural environments in the evolution Gillings, M. R., and Stokes, H. W. ment has differing long-term impacts Le-Minh, N., Khan, S. J., Drewes, J. of resistance traits in pathogenic bac- (2012). Are humans increasing bac- on the human throat and gut micro- E., and Stuetz, R. M. (2010). Fate teria. Proc. R. Soc. B Biol. Sci. 276, terial evolvability? Trends Ecol. Evol. biome. PLoS ONE 5:e9836. doi: of antibiotics during municipal water 2521–2530. 27, 346–352. 10.1371/journal.pone.0009836 recycling treatment processes. Water Martinez, J. L. (2012). Natural antibi- Goh, E.-B., Yim, G., Tsui, W., McClure, Klockgether, J., Cramer, N., Res. 44, 4295–4323. otic resistance and contamination J., Surette, M. G., and Davies, J. Wiehlmann, L., Davenport, C. F., and Leplae, R., Lima-Mendez, G., and Tous- by antibiotic resistance determi- (2002). Transcriptional modulation Tummler, B. (2011). Pseudomonas saint, A. (2010). ACLAME: a CLAssi- nants: the two ages in the evolu- of bacterial gene expression by subin- aeruginosa genomic structure and fication of Mobile genetic Elements, tion of resistance to antimicrobials. hibitory concentrations of antibi- diversity. Front. Microbiol. 2:150. doi: update 2010. Nucleic Acids Res. 38, Front. Microbiol. 3:1. doi: 10.3389/ otics. Proc. Natl. Acad. Sci. U.S.A. 99, 10.3389/fmicb.2011.00150 D57–D61. fmicb.2012.00001 17025–17030. Kloesges, T., Popa, O., Martin, W., and Li, D., Yang, M., Hu, J., Zhang, J., Liu, Martinez, J. L., Baquero, F., and Ander- Graham, D. W., Olivares-Rieumont, S., Dagan, T. (2010). Networks of gene R., Gu, X., et al. (2009). Antibiotic- sson, D. I. (2007). Predicting antibi- Knapp, C. W., Lima, L., Werner, sharing among 329 Proteobacterial resistance profile in environmental otic resistance. Nat. Rev. Microbiol. 5, D., and Bowen, E. (2011). Antibi- genomes reveal differences in lateral bacteria isolated from penicillin pro- 958–965. otic resistance gene abundances asso- gene transfer frequency at different duction wastewater treatment plant Martinez, J. L., Fajardo, A., Garmen- ciated with waste discharges to phylogenetic depths. Mol. Biol. Evol. and the receiving river. Environ. dia, L., Hernandez, A., Linares, J. F., the Almendares River near Havana, 28, 1057–1074. Microbiol. 11, 1506–1517. Martínez-Solano, L., et al. (2009a). A Cuba. Environ. Sci. Technol. 45, Knapp, C. W., Dolfing, J., Ehlert, P. A. Li, D., Yu, T., Zhang, Y., Yang, global view of antibiotic resistance. 418–424. I., and Graham, D. W. (2009). Evi- M., Li, Z., Liu, M., et al. (2010). FEMS Microbiol. Rev. 33, 44–65. Hall, B. G., and Barlow, M. (2004). dence of increasing antibiotic resis- Antibiotic resistance characteristics Martinez, J. L., Sánchez, M. B., Evolution of the serine β-lactamases: tance gene abundances in archived of environmental bacteria from an Martínez-Solano, L., Hernandez, A., past, present and future. Drug Resist. soils since 1940. Environ. Sci. Technol. oxytetracycline production wastewa- Garmendia, L., Fajardo, A., et al. Updat. 7, 111–123. 44, 580–587. ter treatment plant and the receiving (2009b). Functional role of bacterial Hardwick, S. A., Stokes, H. W., Find- Knapp, C. W., Engemann, C. A., Han- river. Appl. Environ. Microbiol. 76, multidrug efflux pumps in microbial lay, S., Taylor, M., and Gillings, M. son, M. L., Keen, P. L., Hall, K. J., and 3444–3451. natural ecosystems. FEMS Microbiol. R. (2008). Quantification of class 1 Graham, D. W. (2008). Indirect evi- Lin, J. T., Connelly, M. B., Amolo, Rev. 33, 430–449. integron abundance in natural envi- dence of transposon-mediated selec- C., Otani, S., and Yaver, D. Medini, D., Donati, C., Tettelin, ronments using real-time quantita- tion of antibiotic resistance genes in S. (2005). Global transcriptional H., Masignani, V., and Rappuoli, tive PCR. FEMS Microbiol. Lett. 278, aquatic systems at low-level oxyte- response of Bacillus subtilis to treat- R. (2005). The microbial pan- 207–212. tracycline exposures. Environ. Sci. ment with subinhibitory concen- genome. Curr. Opin. Genet. Dev. 15, Hegstad, K., Langsrud, S., Lunestad, Technol. 42, 5348–5353. trations of antibiotics that inhibit 589–594. B. T., Scheie, A. A., Sunde, M., and Kohanski, M. A., DePristo, M. A., protein synthesis. Antimicrob. Agents Méndez, C., and Salas, J. A. Yazdankhah, S. P. (2010). Does the and Collins, J. J. (2010). Sublethal Chemother. 49, 1915–1926. (2001). The role of ABC transporters wide use of quaternary ammonium antibiotic treatment leads to mul- Linares, J. F., Gustafsson, I., Baquero, F., in antibiotic-producing organisms: compounds enhance the selection tidrug resistance via radical-induced and Martinez, J. L. (2006). Antibiotics drug secretion and resistance mech- and spread of antimicrobial resis- mutagenesis. Mol. Cell 37, 311–320. as intermicrobial signaling agents anisms. Res. Microbiol. 152, 341–350. tance and thus threaten our health? Kong, W. D., Zhu, Y. G., Fu, B. instead of weapons. Proc. Natl. Acad. Moura, A., Henriques, I., Smalla, K., Microb. Drug Resist. 16, 91–104. J., Marschner, P., and He, J. Sci. U.S.A. 103, 19484–19489. and Correia, A. (2010). Wastewater Henke, J. M., and Bassler, B. L. (2004). Z. (2006). The veterinary antibi- Liu, B., and Pop, M. (2009). ARDB – bacterial communities bring together Bacterial social engagements. Trends otic oxytetracycline and Cu influence antibiotic resistance genes database. broad-host range plasmids, integrons Cell Biol. 14, 648–656. functional diversity of the soil micro- Nucleic Acids Res. 37, D443–D447. and a wide diversity of uncharacter- Heuer, H., Schmitt, H., and Smalla, bial community. Environ. Pollut. 143, Lupo, A., Coyne, S., and Beren- ized gene cassettes. Res. Microbiol. K. (2011). Antibiotic resistance gene 129–137. donk, T. U. (2012). Origin and 161, 58–66. spread due to manure application Kristiansson, E., Fick, J., Janzon, A., evolution of antibiotic resistance: Nagachinta, S., and Chen, J. (2008). on agricultural fields. Curr. Opin. Grabic, R., Rutgersson, C., Wei- the common mechanisms of emer- Transfer of class 1 integron-mediated Microbiol. 14, 236–243. jdegard, B., et al. (2011). Pyrose- gence and spread in water bodies. antibiotic resistance genes from shiga Hoffman, L. R., D’Argenio, D. A., Mac- quencing of antibiotic-contaminated Front. Microbiol. 3:18. doi: 10.3389/ toxin-producing Escherichia coli to Coss, M. J., Zhang, Z., Jones, R. river sediments reveals high levels fmicb.2012.00018 a susceptible E. coli K-12 strain A., and Miller, S. I. (2005). Amino- of resistance and gene transfer ele- Lyon, G. J., and Muir, T. W. (2003). in storm water and bovine feces. glycoside antibiotics induce bacterial ments. PLoS ONE 6:e17038. doi: Chemical signaling among bacteria Appl. Environ. Microbiol. 74, 5063– biofilm formation. Nature 436, 1171– 10.1371/journal.pone.0017038 and its inhibition. Chem. Biol. 10, 5067. 1175. Krizova, L., Dijkshoorn, L., and Nemec, 1007–1021. Nardelli, M., Scalzo, P. M., Ramírez, Huang, F., Haydock, S. F., Mironenko, A. (2011). Diversity and evolution Marr, A. K., Overhage, J., Bains, M., M. S., Quiroga, M. P., Cassini, M. T., Spiteller, D., Li, Y., and Spencer, J. of AbaR genomic resistance islands and Hancock, R. E. W. (2007). The H., and Centrón, D. (2012). Class 1 B. (2005). The neomycin biosynthetic in Acinetobacter baumannii strains of Lon protease of Pseudomonas aerug- integrons in environments with dif- gene cluster of Streptomyces fradiae European clone I. Antimicrob. Agents inosa is induced by aminoglycosides ferent degrees of urbanization. PLoS NCIMB 8233: characterisation of an Chemother. 55, 3201–3206. and is involved in biofilm forma- ONE 7:e39223. doi: 10.1371/jour- aminotransferase involved in the for- Kuete, V., Alibert-Franco, S., Eyong, tion and motility. Microbiology 153, nal.pone.0039223 mation of 2-deoxystreptamine. Org. K. O., Ngameni, B., Folefoc, G. 474–482. Nogueira, T., Rankin, D. J., Touchon, Biomol. Chem. 3, 1410–1418. N., Nguemeving, J. R., et al. (2011). Martinez, J. L. (2008). Antibiotics and M., Taddei, F., Brown, S. P., and Hughes, V. M., and Datta, N. (1983). Antibacterial activity of some natural antibiotic resistance genes in natural Rocha, E. P. C. (2009). Horizontal Conjugative plasmids in bacteria of products against bacteria expressing environments. Science 321, 365–367. gene transfer of the secretome drives

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy January 2013 | Volume 4 | Article 4 | 63

“fmicb-04-00004” — 2013/1/19 — 14:54 — page8—#8 Gillings Humans, antibiotics and evolution

the evolution of bacterial coopera- toyocaensis NRRL15009. Proc. Natl. Seshasayee, A. S. N., Bertone, P., Fraser, Szczepanowski, R., Braun, S., Riedel, tion and virulence. Curr. Biol. 19, Acad. Sci. U.S.A. 99, 8962–8967. G. M., and Luscombe, N. M. (2006). V., Schneiker, S., Krahn, I., Puh- 1683–1691. Popa, O., and Dagan, T. (2011). Trends Transcriptional regulatory networks ler, A., et al. (2005). The 120592bp Norman, A., Hansen, L. H., and and barriers to lateral gene transfer in bacteria: from input signals to out- IncF plasmid pRSB107 isolated from Sorensen, S. J. (2009). Conjugative in prokaryotes. Curr. Opin. Microbiol. put responses. Curr. Opin. Microbiol. a sewage-treatment plant encodes plasmids: vessels of the communal 14, 615–623. 9, 511–519. nine different antibiotic-resistance gene pool. Philos. Trans. R. Soc. Lond. Popa, O., Hazkani-Covo, E., Landan, G., Shank, E. A., and Kolter, R. (2009). New determinants, two iron-acquisition B Biol. Sci. 364, 2275–2289. Martin, W., and Dagan, T. (2011). developments in microbial inter- systems and other putative virulence- Pallecchi, L., Bartoloni, A., Paradisi, Directed networks reveal genomic species signaling. Curr. Opin. Micro- associated functions. Microbiology F., and Rossolini, G. M. (2008). barriers and DNA repair bypasses to biol. 12, 205–214. 151, 1095–1111. Antibiotic resistance in the absence of lateral gene transfer among prokary- Siefert, J. L. (2009). “Defining the Taylor, N. G. H., Verner-Jeffreys, D. W., antimicrobial use: mechanisms and otes. Genome Res. 21, 599–609. Mobilome,” in Horizontal Gene and Baker-Austin, C. (2011). Aquatic implications. Expert Rev. Anti Infect. Pruden, A., Pei, R., Storteboom, H., Transfer: Genomes in Flux, eds M. systems: maintaining, mixing and Ther. 6, 725–732. and Carlson, K. H. (2006). Antibiotic B. Gogarten, J. P. Gogarten, and L. mobilising antimicrobial resistance? Palumbi, S. R. (2001). Humans as the resistance genes as emerging contam- C. Olendzenski (New York: Humana Trends Ecol. Evol. 26, 278–284. world’s greatest evolutionary force. inants: studies in Northern Colorado. Press), 13–27. Tenaillon, O., Denamur, E., and Matic, I. Science 293, 1786–1790. Environ. Sci. Technol. 40, 7445– Sjolund, M., Bonnedahl, J., Hernan- (2004). Evolutionary significance of Paulsen, I. T., Skurray, R. A., Tam, R., 7450. dez, J., Bengtsson, S., Cederbrant, G., stress-induced mutagenesis in bacte- Saier, M. H., Turner, R. J., Weiner, Prudhomme, M., Attaiech, L., Sanchez, Pinhassi, J., et al. (2008). Dissemina- ria. Trends Microbiol. 12, 264–270. J. H., et al. (1996). The SMR fam- G., Martin, B., and Claverys, J.-P. tion of multidrug-resistant bacteria Tennstedt, T., Szczepanowski, R., Braun, ily: a novel family of multidrug efflux (2006). Antibiotic stress induces into the Arctic. Emerg. Infect. Dis. 14, S., Pühler, A., and Schlüter, A. (2003). proteins involved with the efflux of genetic transformability in the 70–72. Occurrence of integron-associated lipophilic drugs. Mol. Microbiol. 19, human pathogen Streptococcus Skippington, E., and Ragan, M. A. resistance gene cassettes located on 1167–1175. pneumoniae. Science 313, 89–92. (2011). Lateral genetic transfer and antibiotic resistance plasmids iso- Pei, R., Kim, S.-C., Carlson, K. H., Riesenfeld, C. S., Goodman, R. M., and the construction of genetic exchange lated from a wastewater treatment and Pruden, A. (2006). Effect of Handelsman, J. (2004). Uncultured communities. FEMS Microbiol. Rev. plant. FEMS Microbiol. Ecol. 45, river landscape on the sediment con- soil bacteria are a reservoir of new 35, 707–735. 239–252. centrations of antibiotics and corre- antibiotic resistance genes. Environ. Skurnik, D., Ruimy, R., Andremont, A., Tettelin, H., Riley, D., Cattuto, C., sponding antibiotic resistance genes Microbiol. 6, 981–989. Amorin, C., Rouquet, P., Picard, B., and Medini, D. (2008). Comparative (ARG). Water Res. 40, 2427–2435. Rosewarne, C. P., Pettigrove, V., Stokes, et al. (2006). Effect of human vicin- genomics: the bacterial pan-genome. Pellegrini, C., Celenza, G., Segatore, H. W., and Parsons, Y. M. (2010). ity on antimicrobial resistance and Curr. Opin. Microbiol. 11, 472–477. B., Bellio, P., Setacci, D., Ami- Class 1 integrons in benthic bacte- integrons in animal faecal Escherichia Thaller, M. C., Migliore, L., Mar- cosante, G., et al. (2011). Occurrence rial communities: abundance, asso- coli. J. Antimicrob. Chemother. 57, quez, C., Tapia, W., CedeÃ±o, of class 1 and 2 integrons in resistant ciation with Tn402-like transposition 1215–1219. V., Rossolini, G. M., et al. (2010). Enterobacteriaceae collected from a modules and evidence for coselection Sommer, M. O. A., and Dantas, G. Tracking acquired antibiotic resis- urban wastewater treatment plant: with heavy-metal resistance. FEMS (2011). Antibiotics and the resistant tance in commensal bacteria of Gala- first report from Central Italy. Microb. Microbiol. Ecol. 72, 35–46. microbiome. Curr. Opin. Microbiol. pagos land iguanas: no man, no Drug Resist. 17, 229–234. Ryan, R. P., and Dow, J. M. (2008). Dif- 14, 556–563. resistance. PLoS ONE 5:e8989. doi: Pellegrini, C., Mercuri, P.S., Celenza, G., fusible signals and interspecies com- Sorensen, S. J., Bailey, M., Hansen, L. 10.1371/journal.pone.0008989 Galleni, M., Segatore, B., Sacchetti, munication in bacteria. Microbiology H., Kroer, N., and Wuertz, S. (2005). Thi, T. D., Lopez, E., Rodriguez-Rojas, E., et al. (2009). Identification of 154, 1845–1858. Studying plasmid horizontal transfer A., Rodriguez-Beltran, J., Couce, A., blaIMP-22 in Pseudomonas spp. in Salyers, A., and Shoemaker, N. B. in situ: a critical review. Nat. Rev. Guelfo, J. R., et al. (2011). Effect urban wastewater and nosocomial (2006). Reservoirs of antibiotic resis- Microbiol. 3, 700–710. of recA inactivation on mutagenesis environments: biochemical charac- tance genes. Anim. Biotechnol. 17, Stepanauskas, R., Glenn, T. C., Jagoe, of Escherichia coli exposed to sub- terization of a new IMP metallo- 137–146. C. H., Tuckfield, R. C., Lindell, A. lethal concentrations of antimicro- enzyme variant and its genetic Sarmah, A. K., Meyer, M. T., and H., King, C. J., et al. (2006). Coselec- bials. J. Antimicrob. Chemother. 66, location. J. Antimicrob. Chemother. Boxall, A. B. A. (2006). A global tion for microbial resistance to metals 531–538. 63, 901–908. perspective on the use, sales, expo- and antibiotics in freshwater micro- Thomas, C. M., and Nielsen, K. M. Petković, H., Cullum, J., Hranueli, D., sure pathways, occurrence, fate and cosms. Environ. Microbiol. 8, 1510– (2005). Mechanisms of, and barriers Hunter, I. S., Perić-Concha, N., Pigac, effects of veterinary antibiotics (VAs) 1514. to, horizontal gene transfer between J., et al. (2006). Genetics of Strep- in the environment. Chemosphere 65, Stevens, A. M., Schuster, M., and Rum- bacteria. Nat. Rev. Microbiol. 3, tomyces rimosus, the oxytetracycline 725–759. baugh, K. P.(2012). Working together 711–721. producer. Microbiol. Mol. Biol. Rev. Schlacher, K., and Goodman, M. F. for the common good: cell–cell com- Toleman,M. A., and Walsh,T. R. (2011). 70, 704–728. (2007). Lessons from 50 years of SOS munication in bacteria. J. Bacteriol. Combinatorial events of insertion Piddock, L. J. V. (2006). Multidrug- DNA-damage-induced mutagenesis. 194, 2131–2141. sequences and ICE in Gram-negative resistance efflux pumps? not just for Nat. Rev. Mol. Cell Biol. 8, 587–594. Stokes, H. W., and Gillings, M. bacteria. FEMS Microbiol. Rev. 35, resistance. Nat. Rev. Microbiol. 4, Schlüter, A., Krause, L., Szczepanowski, R. (2011). Gene flow, mobile 912–935. 629–636. R., Goesmann, A., and Pühler, A. genetic elements and the recruitment Toussaint, A., and Chandler, M. (2012). Pigliucci, M. (2008). Is evolvability (2008). Genetic diversity and com- of antibiotic resistance genes into “Prokaryote genome fluidity: toward evolvable? Nat. Rev. Genet. 9, 75–82. position of a plasmid metagenome Gram-negative pathogens. FEMS a system approach of the mobilome,” Poole, K. (2005). Efflux-mediated from a wastewater treatment plant. J. Microbiol. Rev. 35, 790–819. in Bacterial Molecular Networks, eds J. antimicrobial resistance. J. Antimi- Biotechnol. 136, 65–76. Storteboom, H., Arabi, M., Davis, Helden, A. Toussaint and D. Thieffry crob. Chemother. 56, 20–51. Segura, P. A., Francois, M., Gagnon, C., J. G., Crimi, B., and Pruden, A. (New York: Springer), 57–80. Pootoolal, J., Thomas, M. G., Marshall, and Sauve, S. (2009). Review of the (2010). Identification of antibiotic- Tsui, W. H. W., Yim, G., Wang, H. C. G., Neu, J. M., Hubbard, B. K., occurrence of anti-infectives in con- resistance-gene molecular signatures H., McClure, J. E., Surette, M. G., Walsh, C. T., et al. (2002). Assembling taminated wastewaters and natural suitable as tracers of pristine river, and Davies, J. (2004). Dual effects the glycopeptide antibiotic scaffold: and drinking waters. Environ. Health urban, and agricultural sources. Env- of MLS antibiotics: transcriptional the biosynthesis of from Streptomyces Perspect. 117, 675–684. iron. Sci. Technol. 44, 1947–1953. modulation and interactions on the

www.frontiersin.org January 2013 | Volume 4 | Article 4 | 64

“fmicb-04-00004” — 2013/1/19 — 14:54 — page9—#9 Gillings Humans, antibiotics and evolution

ribosome. Chem. Biol. 11, 1307– Natl. Acad. Sci. U.S.A. 95, 6578– mechanisms: drug efflux across two Received: 25 October 2012; paper pending 1316. 6583. membranes. Mol. Microbiol. 37, published: 11 December 2012; accepted: Úbeda, C., Maiques, E., Knecht, E., Wright, G. D. (2007). The antibiotic 219–225. 03 January 2013; published online: 22 Lasa, Í., Novick, R. P., and Penadés, resistome: the nexus of chemical and Zhang, X.-X., Zhang, T., Zhang, M., January 2013. J. R. (2005). Antibiotic-induced genetic diversity. Nat. Rev. Microbiol. Fang, H., and Cheng, S.-P. (2009). SOS response promotes horizontal 5, 175–186. Characterization and quantification dissemination of pathogenicity Wright, G. D. (2010). The antibiotic of class 1 integrons and associated Citation: Gillings MR (2013) Evolution- island-encoded virulence factors in resistome. Expert Opin. Drug Discov. gene cassettes in sewage treatment ary consequences of antibiotic use for staphylococci. Mol. Microbiol. 56, 5, 779–788. plants. Appl. Microbiol. Biotechnol. the resistome, mobilome, and microbial 836–844. Wright, M. S., Baker-Austin, C., Lin- 82, 1169–1177. pangenome. Front. Microbio. 4:4. doi: Waksman, S. A. (1973). History of the dell, A. H., Stepanauskas, R., Stokes, Zuccato, E., Castiglioni, S., Bagnati, 10.3389/fmicb.2013.00004 word ‘Antibiotic’. J. Hist. Med. Allied H. W., and McArthur, J. V. (2008). R., Melis, M., and Fanelli, R. This article was submitted to Fron- Sci. 28, 284–286. Influence of industrial contamina- (2010). Source, occurrence and fate tiers in Antimicrobials, Resistance and Watkinson, A. J., Murby, E. J., and tion on mobile genetic elements: of antibiotics in the Italian aquatic Chemotherapy, a specialty of Frontiers in Costanzo, S. D. (2007). Removal class 1 integron abundance and environment. J. Hazard. Mater. 179, Microbiology. of antibiotics in conventional and gene cassette structure in aquatic 1042–1048. Copyright © 2013 Gillings. This is advanced wastewater treatment: bacterial communities. ISME J. 2, an open-access article distributed under implications for environmen- 417–428. the terms of the Creative Commons tal discharge and wastewater Yim, G., Huimi Wang, H., and Davies, Conflict of Interest Statement: The Attribution License, which permits use, recycling. Water Res. 41, 4164– J. (2007). Antibiotics as signalling author declares that the research was distribution and reproduction in other 4176. molecules. Philos. Trans. R. Soc. Lond. conducted in the absence of any com- forums, provided the original authors and Whitman, W. B., Coleman, D. C., B Biol. Sci. 362, 1195–1200. mercial or financial relationships that source are credited and subject to any and Wiebe, W. J. (1998). Prokary- Zgurskaya, H. I., and Nikaido, could be construed as a potential con- copyright notices concerning any third- otes: the unseen majority. Proc. H. (2000). Multidrug resistance flict of interest. party graphics etc.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy January 2013 | Volume 4 | Article 4 | 65

“fmicb-04-00004” — 2013/1/19 — 14:54 — page 10 — #10 REVIEW ARTICLE published: 14 March 2013 doi: 10.3389/fmicb.2013.00048 “Stormy waters ahead”: global emergence of carbapenemases

Gopi Patel1 and Robert A. Bonomo2,3,4,5,6* 1 Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA 2 Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, USA 3 Division of Infectious Diseases and HIV Medicine, University Hospitals Case Medical Center, Cleveland, OH, USA 4 Department of Medicine, Case Western Reserve School of Medicine, Cleveland, OH, USA 5 Department of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, OH, USA 6 Department of Pharmacology, Case Western Reserve School of Medicine, Cleveland, OH, USA

Edited by: Carbapenems, once considered the last line of defense against of serious infections Fiona Walsh, Agroscope with Enterobacteriaceae, are threatened with extinction. The increasing isolation of Changins-Wädenswil, Switzerland carbapenem-resistant Gram-negative pathogens is forcing practitioners to rely on uncertain Reviewed by: alternatives. As little as 5 years ago, reports of carbapenem resistance in Enterobacte- Charles W. Knapp, University of Strathclyde, UK riaceae, common causes of both community and healthcare-associated infections, were Yoshikazu Ishii, Toho University School sporadic and primarily limited to case reports, tertiary care centers, intensive care units, and of Medicine, Japan outbreak settings. Carbapenem resistance mediated by β-lactamases, or carbapenemases, *Correspondence: has become widespread and with the paucity of reliable antimicrobials available or in Robert A. Bonomo, Research Service, development, international focus has shifted to early detection and infection control. Louis Stokes Cleveland Department of Veteran Affairs Medical Center, However, as reports of Klebsiella pneumoniae carbapenemases, New Delhi metallo-β- 10701 East Boulevard, Cleveland, OH lactamase-1, and more recently OXA-48 (oxacillinase-48) become more common and with 44106, USA. the conveniences of travel, the assumption that infections with highly resistant Gram- e-mail: [email protected] negative pathogens are limited to the inﬁrmed and the heavily antibiotic and healthcare exposed are quickly being dispelled. Herein, we provide a status report describing the increasing challenges clinicians are facing and forecast the “stormy waters” ahead.

Keywords: carbapenemases, NDM-1, KPC, OXA-48, metallo-β-lactamases, CHDL

Carbapenems are potent and broad-spectrum β-lactam antibi- Carbapenem resistance among Gram-negative bacteria results otics traditionally reserved for the treatment of the most serious from one or more of the following mechanisms: (i) hyperpro- infections (El-Gamal and Oh, 2010). The emergence and dis- duction or derepression of Ambler class C β-lactamases (AmpC semination of carbapenem-resistant Gram-negative pathogens β-lactamases) or ESBLs (e.g., sulfhydryl variable (SHV),temoneira including Pseudomonas aeruginosa, Acinetobacter baumannii, and (TEM),cefotaxime (CTX-M) type β-lactamases) with loss or alter- Enterobacteriaceae is a significant contributor to patient mor- ation in outer membrane porins; (ii) augmented drug efflux; (iii) bidity and mortality (Patel et al., 2008; Schwaber et al., 2008; alterations in penicillin binding proteins (PBPs); (iv) carbapen- Lautenbach et al., 2009, 2010; Marchaim et al., 2011). Despite emase production (Patel and Bonomo, 2011). Carbapenemases radical efforts in infection control (Schwaber et al., 2011) and belong to three molecular classes of β-lactamases, Ambler class improvements in rapid molecular diagnostics (Centers for Dis- A, B, and D (Ambler, 1980; Bush and Jacoby, 2010). Our aim ease Control and Prevention, 2009; Nordmann et al., 2012c), is to provide a status report of the molecular diversity and carbapenem-resistant Gram-negative bacilli remain a formidable epidemiology of carbapenemases as well as current and future threat as few antimicrobial agents are reliably active and very little therapeutics. The increasing public safety concerns associated with is expected to be available in the near future. organisms harboring these enzymes has created significant tur- Clinicians hold that the increasing prevalence of extended- moil. Regrettably, the situation is critical and our patients are in spectrum β-lactamases (ESBLs) among Klebsiella pneumoniae and peril. Escherichia coli in the 1980s and 1990s contributed to the increased consumption of carbapenems. Experience implied that delayed AMBLER CLASS A CARBAPENEMASES administration of carbapenems in at-risk patients led to poor Few Ambler class A β-lactamases demonstrate carbapenem- clinical outcomes (Paterson and Bonomo, 2005; Endimiani and hydrolyzing activity and, up until a decade ago, these were Paterson, 2007). Thus, carbapenems (i.e., imipenem, meropenem, rarely recovered. Class A carbapenemases include: K. pneumoniae ertapenem, and doripenem) became vital tools in the treatment of carbapenemase (KPC), Guiana extended-spectrum (GES), non- healthcare-associated and severe community-acquired infections. metallo-carbapenemase-A (Nmc-A)/imipenem-resistant (IMI), Despite heavy reliance on these agents, carbapenem resistance Serratia marcescens enzyme (SME), serratia fonticola carbapen- in Enterobacteriaceae, common causes of both community and emase (SFC), and BIC β-lactamases (Table 1; Walther-Rasmussen healthcare-associated infections, remained rare until the past and Høiby, 2007). With the notable exception of KPCs, the clinical decade. isolation of these types of carbapenemases is relatively limited.

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 66

“fmicb-04-00048” — 2013/3/13 — 9:48 — page1—#1 Patel and Bonomo The global emergence of carbapenemases

Table 1 | Class A carbapenemases*.

Enzyme Year isolated Organism(s) Origin and geographic Location Reference or described distribution

Nmc-A 1990 Enterobacter cloacae France, Argentina, USA Chromosomal Nordmann et al. (1993) IMI-1 1984 Enterobacter cloacae USA Chromosomal Rasmussen et al. (1996) IMI-2 1999 Enterobacter asburiae, Enterobacter USA†, China Plasmid Aubron et al. (2005), Yu et al. (2006) cloacae SME-1 1982 S. marcescens UK, USA Chromosomal Naas et al. (1994) SME-2 1992 S. marcescens USA, Canada, Chromosomal Deshpande et al. (2006a), Switzerland Poirel et al. (2007), Carrer et al. (2008) SME-3 2003 S. marcescens USA Chromosomal Queenan et al. (2006) SFC-1 2003 S. fonticola Portugal† Chromosomal Henriques et al. (2004) GES-2 2000 P.aeruginosa South Africa Plasmid Vourli et al. (2004) GES-4 2002 K. pneumoniae Japan Plasmid Wachino et al. (2004) GES-5 2001 K. pneumoniae, E. coli, P.aeruginosa Greece, Korea, worldwide Plasmid Jeong et al. (2005), Viau et al. (2012) GES-6 2003 K. pneumoniae Greece Plasmid Viau et al. (2012) GES-11 2008 Acinetobacter baumannii France Plasmid Moubareck et al. (2009) GES-14 2010 A. baumannii France Plasmid Bogaerts et al. (2010) KPC-1‡ 1996 K. pneumoniae USA Plasmid Yigit et al. (2001) KPC-2 1998 Enterobacteriaceae, P.aeruginosa, USA and worldwide Plasmid§ Yigit et al. (2001) Acinetobacter spp. KPC-3 2000 Enterobacteriaceae, Acinetobacter spp. USA and worldwide Plasmid Woodford et al. (2004) KPC-4 2003 Enterobacter cancerogenus, K. Scotland, Puerto Rico Plasmid Palepou et al. (2005), pneumoniae, Acinetobacter spp. Robledo et al. (2007) KPC-5 2006 P.aeruginosa Puerto Rico Plasmid Wolter et al. (2009) KPC-6 2003 K. pneumoniae Puerto Rico Plasmid Bartual et al. (2005), Robledo et al. (2008) KPC-7 2007 K. pneumoniae USA Plasmid Perez et al. (2010a) KPC-8 2008 K. pneumoniae Puerto Rico Plasmid Diancourt et al. (2010) KPC-9 2009 E. coli Israel Plasmid Grosso et al. (2011) KPC-10 2009 Acinetobacter spp. Puerto Rico Plasmid Robledo et al. (2010) KPC-11 2009 K. pneumoniae Greece Unknown Da Silva et al. (2004) KPC-12 2010 E. coli China Unknown KPC-13 2010 Enterobacter cloacae Thailand Unknown BIC-1 2009 P.ﬂuorescens France† Chromosomal Girlich et al. (2010)

* Adapted from Walther-Rasmussen and Høiby (2007). †Environmental isolates. ‡KPC-1 was later found to be the same enzyme as KPC-2 (Higgins et al., 2012a). § Chromosomal expression of blaKPC−2 has been described in P.aeruginosa (Villegas et al., 2007).

Non-metallo-carbapenemase-A is a chromosomal carbapen- SME-1 (S. marcescens enzyme) was originally identiﬁed in an emase originally isolated from Enterobacter cloacae in France isolate of S. marcescens from a patient in London in 1982 (Yang (Nordmann et al., 1993). Currently, reports of this particular et al., 1990). SME-2 and SME-3 were subsequently isolated in the β-lactamase are still rare (Pottumarthy et al., 2003; Castanheira USA, Canada, and Switzerland (Naas et al., 1994; Queenan et al., et al., 2008; Osterblad et al., 2012). IMI-1 was initially recov- 2000, 2006; Deshpande et al., 2006b; Poirel et al., 2007; Carrer ered from the chromosome of an Enterobacter cloacae isolate et al., 2008). Chromosomally encoded SME-type carbapenemases in the southwestern USA (Rasmussen et al., 1996). A variant of continue to be isolated at a low frequency in North America IMI-1, IMI-2, has been identiﬁed on plasmids isolated from en- (Deshpande et al., 2006a,b; Fairfax et al., 2011; Mataseje et al., vironmental strains of Enterobacter asburiae in USA rivers (Aubron 2012). Both SFC-1 and BIC-1 are chromosomal serine carbapen- et al., 2005). emases recovered from environmental isolates. The former from

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 67

“fmicb-04-00048” — 2013/3/13 — 9:48 — page2—#2 Patel and Bonomo The global emergence of carbapenemases

a S. fonticola isolate in Portugal (Henriques et al., 2004) and the Carbapenem resistance secondary to KPC production was first latter from Pseudomonas fluorescens isolates recovered from the described in a K. pneumoniae recovered in North Carolina in 1996 Seine River (Girlich et al., 2010). (Yigit et al., 2001). To date 12 KPC subtypes (KPC-2 to KPC-13; The GES-type β-lactamases are acquired β-lactamases recov- Robledo et al., 2008; Kitchel et al., 2009a; Navon-Venezia et al., ered from P. aeruginosa, Enterobacteriaceae, and A. baumannii 2009; Wolter et al., 2009; Gregory et al., 2010)havebeenreported (Poirel et al., 2000a; Castanheira et al., 2004a). The genes encoding with the vast majority of analyzed isolates expressing either KPC-2 these β-lactamase have often, but not exclusively, been identified or KPC-3. within class 1 integrons residing on transferable plasmids (Bon- The blaKPC gene has been mapped to a highly conserved nin et al., 2013; Walther-Rasmussen and Høiby, 2007). GES-1 has Tn3-based transposon, Tn4401 (Figure 1A), and five isoforms a similar hydrolysis profile to other ESBLs, although they essen- of Tn4401 are described (Naas et al., 2008; Cuzon et al., 2010; tially spare monobactams. Several GES β-lactamases are described Kitchel et al., 2010). Plasmids carrying blaKPC are of various with six (i.e., GES-2, GES-4, GES-5, GES-6, GES-11, and GES-14), sizes and many carry additional genes conferring resistance to demonstrating detectable carbapenemase activity in the setting of fluoroquinolones and aminoglycosides thus limiting the antibi- amino acid substitutions at their active sites (specifically at residue otics available to treat infections with KPC-producing pathogens 104 and 170; Walther-Rasmussen and Høiby, 2007; Kotsakis et al., (Endimiani et al., 2008; Rice et al., 2008). blaKPC has rarely 2010). These GES-type carbapenemases have been described in been mapped to a chromosomal location (Villegas et al., 2007; Europe, South Africa, Asia, and the Middle East (Poirel et al., 2002; Castanheira et al., 2009). Jeong et al., 2005; da Fonseca et al., 2007; Moubareck et al., 2009; A predominant strain of K. pneumoniae appears responsible for Bonnin et al., 2011, 2013). outbreaks and the international spread of KPC-producing K. pneu- Currently, most carbapenem resistance among Enterobacteri- moniae (Woodford et al., 2008; Kitchel et al., 2009a; Samuelsen aceae in the USA and Israel is attributed to plasmid-mediated et al., 2009). Congruent pulsed-field gel electrophoresis (PFGE) expression of a KPC-type carbapenemase (Endimiani et al., 2009b; patterns also suggest a clonal relationship between outbreak- Nordmann et al., 2009; Gupta et al., 2011; Schwaber et al., 2011). associated strains of KPC-producing K. pneumoniae recovered KPC-producing Enterobacteriaceae are considered endemic to from different areas that are endemic (Navon-Venezia et al., 2009; Greece along with other carbapenemases, specifically VIM-type Woodford et al., 2011). The Centers for Disease Control and Pre- [Verona integron-encoded metallo-β-lactamases (MBLs); Canton vention (CDC) performed PFGE and multilocus sequence typing et al., 2012]. KPCs efficiently hydrolyze carbapenems as well as (MLST) on isolates submitted to their reference laboratory from penicillins, cephalosporins, and aztreonam and are not overcome 1996 to 2008. A dominant PFGE pattern was observed and noted to in vitro by clinically available β-lactamase inhibitors (i.e., clavu- be of a specific MLST type, ST 258 (Kitchel et al., 2009a). A second lanic acid, sulbactam, tazobactam – in fact these are hydrolyzed). sequence type, ST 14, was common in institutions in the Midwest These enzymes have been identified in several genera of Enterobac- (Kitchel et al., 2009b). These findings implied that certain strains teriaceaeaswellasPseudomonas spp. and A. baumannii (Miriagou of K. pneumoniae may be more apt to obtain and retain the blaKPC et al., 2003; Yigit et al., 2003; Bratu et al., 2005; Villegas et al., 2007; gene. Another study, however, analyzing 16 KPC-2 producing K. Cai et al., 2008; Rasheed et al., 2008; Tibbetts et al., 2008; Robledo pneumoniae isolates from different geographic regions demon- et al., 2010; Mathers et al., 2011; Geffen et al., 2012). strated diverse PFGE patterns and MLST types. This included four

FIGURE 1 | Basic genetic construct of select carbapenemase genes. (A) of the blaNDM−1 and a novel bleomycin resistance gene, bleMBL, Schematic representation of Tn4401 type of transposon associated with downstream (Dortet et al., 2012). (C) blaOXA−48 is often mapped to a Tn1999 blaKPC which includes a transposase gene (tnpA), a resolvase gene (tnpR), as composite transposon where it is bracketed between two copies of the same well as insertion sequences, ISKpn6 and ISKpn7 (Cuzon et al., 2010). (B) The insertion sequence, IS1999. Downstream of blaOXA−48 lies a lysR gene blaNDM−1 construct demonstrates ISAba125 insertion sequence(s) upstream which encodes for a regulatory protein (Poirel et al., 2012b).

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 68

“fmicb-04-00048” — 2013/3/13 — 9:48 — page3—#3 Patel and Bonomo The global emergence of carbapenemases

different MLST types in Colombia (ST 14, ST 337, ST 338, and ST resistance to penicillins, cephalosporins, and carbapenems. MBLs 339) and two in Israel (ST 227 and ST 340). Although this study are not inhibited by the presence of commercially available analyzed a smaller number of isolates, these findings suggest that β-lactamase inhibitors and susceptibility to monobactams (e.g., the global propagation of KPC-2 is more complicated than the suc- aztreonam) appears to be preserved in the absence of con- cessful expansion of a fixed number of clones (Cuzon et al., 2010; comitant expression of other resistance mechanisms (e.g., ESBL Qi et al., 2011). More recently, a study evaluating the MLST types production). The more geographically widespread MBLs include associated with widespread KPC-2 production in K. pneumoniae IMP (imipenem-resistant), VIM, and New Delhi metallo-β-lacta in Greece suggested that although ST 258 predominates at least 10 mase (NDM). additional sequence types were found to carry blaKPC−2. Of note Chromosomal MBLs were the first to be identified and are three (i.e., ST 147, ST 323, and ST 383) carried both KPC-2 as well the cause of carbapenem resistance observed in Bacillus cereus, as genes encoding VIM-type MBLs (Giakkoupi et al., 2011; Wood- Aeromonas spp., and Stenotrophomonas maltophilia (Walsh et al., ford et al., 2011). A retrospective study in Cleveland documented 2005). However, of growing concern are the “mobile” MBLs that the presence of ST 36 in a long-term care facility for children have been reported since the mid-1990s. Although most frequently (Viau et al., 2012). found in carbapenem-resistant isolates of P. aeruginosa and occa- Klebsiella pneumoniae carbapenemases-production can confer sionally Acinetobacter spp., there is growing isolation of these variable levels of carbapenem resistance with reported minimum enzymes in Enterobacteriaceae. inhibitory concentrations (MICs) ranging from susceptible to Prior to the description of NDM-1, frequently detected MBLs ≥16 μg/mL. Analysis of isolates displaying high-level carbapenem include IMP-type and VIM-type with VIM-2 being the most resistance demonstrated that increased phenotypic resistance may prevalent. These MBLs are embedded within a variety of genetic be due to increased blaKPC gene copy number or the loss of an outer structures, most commonly integrons. When these integrons membrane porin, OmpK35 and/or OmpK36. The highest level of are associated with transposons or plasmids they can readily be imipenem resistance was seen with isolates lacking both porins and transferred between species. with augmented KPC enzyme production (Kitchel et al., 2010). In 1991, IMP-1, a plasmid-mediated MBL, was identified in an isolates of S. marcescens from Japan (Ito et al., 1995). Since AMBLER CLASS B CARBAPENEMASES: then plasmid-mediated carbapenem resistance secondary to IMP- METALLO-β-LACTAMASES 1 spread widely in Japan, Europe, Brazil, and other parts of Asia Class B β-lactamases (Table 2) are referred to as MBLs and require and in several species of Gram-negative bacilli including Acineto- a metal ion, usually zinc, for β-lactam hydrolysis (Walsh et al., bacter spp. and Enterobacteriaceae. At the present time, 42 variants 2005). Due to the dependence on Zn2+, catalysis is inhibited in the of IMP have been identified with most cases of IMP-mediated presence of metal-chelating agents like ethylenediaminetetraacetic carbapenem resistance being reported from Asia and among P. acid (EDTA). MBL expression in Gram-negative bacteria confers aeruginosa (Bush and Jacoby, 2010).

Table 2 | Metallo-β-lactamases.

Enzyme Year isolated Organism(s) Geographic Location Reference or described distribution

IMP-1 to IMP-42 1988 Enterobacteriaceae, Pseudomonas spp., Worldwide Plasmid or Osano et al. (1994), Acinetobacter spp. chromosomal Riccio et al. (2000) VIM-1 to VIM-37 1997 Enterobacteriaceae, Pseudomonas spp., Worldwide Plasmid or Lauretti et al. (1999), Acinetobacter spp. chromosomal Poirel et al. (2000b) SPM-1 2001 P.aeruginosa Brazil* Chromosomal Toleman et al. (2002) GIM-1 2002 P.aeruginosa Germany Plasmid Castanheira et al. (2004b) SIM-1 2003–2004 A. baumannii Korea Chromosomal Lee et al. (2005) NDM-1 to NDM-7 2006 Enterobacteriaceae, Acinetobacter spp., Worldwide Plasmid or Yong et al. (2009), Kaase et al. (2011), Vibrio cholerae chromosomal Nordmann et al. (2012a) AIM-1 2007 P.aeruginosa Australia Chromosomal Yong et al. (2007) KHM-1 1997 C. freundii Japan Plasmid Sekiguchi et al. (2008) DIM-1 2007 P.stutzeri Netherlands Plasmid Poirel et al. (2010c) SMB-1 2010 S. marcescens Japan Chromosomal Wachino et al. (2011) TMB-1 2011 Achromobacter xylosoxidans Libya Chromosomal El Salabi et al. (2012) FIM-1 2007 P.aeruginosa Italy Chromosomal Pollini et al. (2012)

*Single report of SPM-1 in Europe linked to healthcare exposure in Brazil (Salabi et al., 2010).

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 69

“fmicb-04-00048” — 2013/3/13 — 9:48 — page4—#4 Patel and Bonomo The global emergence of carbapenemases

A more commonly recovered MBL is the VIM-type enzyme. history of recent travel to the Indian subcontinent with the VIM-1 was first described in Italy in 1997 in P. aeruginosa (Lau- majority having been hospitalized in those countries (Kumarasamy retti et al., 1999). VIM-2 was next discovered in southern France et al., 2010). in P. aeruginosa cultured from a neutropenic patient in 1996 European reports suggest that horizontal transfer of blaNDM−1 (Poirel et al., 2000b). Although originally thought to be limited to exists within hospitals outside endemic areas. Of overwhelm- non-fermenting Gram-negative bacilli, VIM-type MBLs are being ing concern are the reported cases without specific contact with increasingly identified in Enterobacteriaceae as well (Giakkoupi the healthcare system locally or in endemic areas suggesting et al., 2003; Kassis-Chikhani et al., 2006; Morfin-Otero et al., 2009; autochthonous acquisition (Kumarasamy et al., 2010; Kus et al., Canton et al., 2012). To date, 37 variants of VIM have been 2011; Arpin et al., 2012; Borgia et al., 2012; Nordmann et al., described with VIM-2 being the most common MBL recovered 2012b). worldwide. Surveillance of public water supplies in India indicates that Other more geographically restricted MBLs include SPM- exposure to NDM-1 may be environmental. Walsh et al. (2011) 1 (Sao Paulo MBL), which has been associated with hospital analyzed samples of public tap water and seepage water from outbreaks in Brazil (Toleman et al., 2002; Rossi, 2011); GIM-1 sites around New Delhi. The results were disheartening in that (German imipenemase) isolated in carbapenem-resistant P.aerug- blaNDM−1 was detected by PCR in 4% of drinking water samples inosa isolates in Germany (Castanheira et al.,2004b); SIM-1 (Seoul and 30% of seepage samples. In this survey, carriage of blaNDM−1 imipenemase) isolated from A. baumannii isolates in Korea (Lee was noted in 11 species of bacteria not previously described, et al., 2005); KHM-1 (Kyorin Health Science MBL) isolated from including virulent ones like Shigella boydii and Vibrio cholerae. a C. freundii isolate in Japan (Sekiguchi et al., 2008); AIM-1 The rapid spread of NDM-1 highlights the fluidity and rapid- (Australian imipenemase) isolated from P. aeruginosa in Aus- ity of gene transfer between bacterial species. Although blaNDM−1 tralia (Yong et al., 2007); DIM-1 (Dutch imipenemase) isolated was initially and repeatedly mapped to plasmids isolated from from a clinical P. stutzeri isolate in the Netherlands (Poirel et al., carbapenem-resistant E. coli and K. pneumoniae, reports of both 2010c); SMB-1 (S. marcescens MBL) in S. marcescens in Japan plasmid and chromosomal expression of blaNDM−1 has been (Wachino et al., 2011); TMB-1 (Tripoli MBL) in Achromobacter noted in other species of Enterobacteriaceae as well as Acineto- xylosoxidans in Libya (El Salabi et al., 2012), and FIM-1 (Flo- bacter spp. and P. aeruginosa (Moubareck et al., 2009; Bogaerts rence imipenemase) from a clinical isolate of P. aeruginosa in Italy et al., 2010; Bonnin et al., 2011; Nordmann et al., 2011; Patel and (Pollini et al., 2012). With the notable exception of SPM-1, these Bonomo, 2011). Recently, bacteremia with a NDM-1 expressing MBLs have remained confined to their countries of origin (Salabi V. cholerae has been described in a patient previously hospitalized et al., 2010). in India colonized with a variety of Enterobacteriaceae previously NDM-1 was first identified in 2008. Due to its rapid interna- known to be capable of carrying plasmids with blaNDM−1 (Darley tional dissemination and its ability to be expressed by numerous et al., 2012). Gram-negative pathogens, NDM is poised to become the most In contrast to KPCs, the presence of a dominant clone among commonly isolated and distributed carbapenemase worldwide. blaNDM−1 carrying isolates remains elusive (Poirel et al., 2011c). Initial reports frequently demonstrated an epidemiologic link NDM-1 expression in E. coli has been noted among sequence types to the Indian subcontinent where these MBLs are endemic previously associated with the successful dissemination of other (Kumarasamy et al., 2010). Indeed, retrospective analyses of stored β-lactamases including ST 101 and ST 131 (Mushtaq et al., 2011). isolates suggest that NDM-1 may have been circulating in the sub- Mushtaq et al. (2011) analyzed a relatively large group of blaNDM−1 continent as early as 2006 (Castanheira et al., 2011). Despite initial expressing E. coli from the UK, Pakistan, and India in order to controversy, the Balkans may be another area of endemicity for potentially identify a predominant strain responsible for the rapid NDM-1 (Struelens et al., 2010; Jovcic et al., 2011; Livermore et al., and successful spread of NDM-1. The most frequent sequence 2011c; Halaby et al., 2012). Sporadic recovery of NDM-1 in the type identified was ST 101. Another study examining a collection Middle East suggests that this region may be an additional reser- of carbapenem-resistant Enterobacteriaceae from India demon- voir (Poirel et al., 2010a, 2011d; Nordmann et al., 2011; Ghazawi strates the diversity of strains capable of harboring blaNDM−1. et al., 2012). Carriage of blaNDM−1 was confirmed in 10 different sequence Like KPCs, the conveniences of international travel and medi- types of K. pneumoniae and 5 sequence types of E. coli (Lascols cal tourism have quickly propelled this relatively novel MBL into et al., 2011). This multiplicity was confirmed in a study looking a formidable public health threat. Gram-negative bacilli harbor- at a collection of blaNDM−1 expressing Enterobacteriaceae from ing blaNDM have been identified worldwide with the exception of around the world (Poirel et al., 2011c). Of most concern is that Central and South America. NDM-1 has been identified in E. coli ST 131, the strain of E. coli NDM-1 was first identified in Sweden in a patient of Indian credited with the global propagation of CTX-M-15 ESBLs (Mush- descent previously hospitalized in India (Yong et al., 2009). The taq et al., 2011; Peirano et al., 2011; Pfeifer et al., 2011b; Woodford patient was colonized with a K. pneumoniae and an E. coli et al., 2011). Similar to KPCs, NDM-1 expression portends vari- carrying blaNDM−1 on transferable plasmids. In the UK, an able levels of carbapenem resistance and there is often concomitant increase in the number of clinical isolates of carbapenem-resistant carriage of a myriad of resistance determinants including other β- Enterobacteriaceae was seen in both 2008 and 2009. A UK ref- lactamases and carbapenemases as well as genes associated with erence laboratory reported that at least 17 of 29 patients found resistance to fluoroquinolones and aminoglycosides (Nordmann to be harboring NDM-1 expressing Enterobacteriaceae had a et al., 2011).

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 70

“fmicb-04-00048” — 2013/3/13 — 9:48 — page5—#5 Patel and Bonomo The global emergence of carbapenemases

NDM-1 shares the most homology with VIM-1 and VIM-2. It extended-spectrum cephalosporins. Carbapenem hydrolysis effi- is a 28-kDa monomeric protein that demonstrates tight binding to ciency is lower than that of other carbapenemases, including the both penicillins and cephalosporins (Zhang and Hao, 2011). Bind- MBLs, and often additional resistance mechanisms are expressed ing to carbapenems does not appear to be as strong as other MBLs, in organisms demonstrating higher levels of phenotypic car- but hydrolysis rates appear to be similar. Using ampicillin as a bapenem resistance. These include expression of other carbapen- substrate, allowed for detailed characterization of the interactions emases, alterations in outer membrane proteins (e.g., CarO, between NDM’s active site and β-lactams as well as improved eval- OmpK36; Perez et al., 2007; Gülmez et al., 2008; Pfeifer et al., uation of MBLs unique mechanism of β-lactam hydrolysis. More 2012), increased transcription mediated by IS elements function- recent crystal structures of NDM-1 reveal the molecular details of ing as promoters, increased gene copy number, and amplified drug how carbapenem antibiotics are recognized by dizinc-containing efflux (Poirel and Nordmann, 2006; Perez et al., 2007). Many sub- MBLs (King et al., 2012). groups of CHDLs have been described. We will focus on those To date, NDM-1 remains the most common NDM variant iso- found in A. baumannii and Enterobacteriaceae: OXA-23 and OXA- lated. Seven variants (NDM-1 to NDM-7) exist (Kaase et al., 2011; 27; OXA-24/40, OXA-25, and OXA-26; OXA-48 variants; OXA-51, Nordmann et al., 2012a). It is currently held that blaNDM−1 is a OXA-66, OXA-69; OXA-58, and OXA-143. chimeric gene that may have evolved from A. baumannii (Toleman CHDLs can be intrinsic or acquired. A. baumannii does have et al., 2012). Contributing to this theory is the presence of com- naturally occurring but variably expressed chromosomal CHDLs, plete or variations of the insertion sequence, ISAba125, upstream OXA-51, OXA-66, and OXA-69 (Brown et al., 2005; Héritier et al., to the blaNDM−1 gene in both Enterobacteriaceae and A. bauman- 2005b). For the most part, in isolation the phenotypic carbapenem nii (Pfeifer et al., 2011a; Poirel et al., 2011a; Dortet et al., 2012; resistance associated with these oxacillinases is low. However, levels Toleman et al., 2012). This insertion sequence has primarily been of carbapenem resistance appear to be increased in the pres- found in A. baumannii. ence of specific insertion sequences promoting gene expression A recent evaluation of the genetic construct associated with (Figueiredo et al., 2009; Culebras et al., 2010). Additional resis- blaNDM−1 (Figure 1B) has lead to the discovery of a new tance to extended-spectrum cephalosporins can be seen in the bleomycin resistance protein, BRPMBL. Evaluation of 23 isolates setting of co-expression of ESBLs and/or other carbapenemases of blaNDM−1/2 harboring Enterobacteriaceae and A. bauman- (Castanheira et al., 2011; Mathers et al., 2012; Pfeifer et al., 2012; nii noted that the overwhelming majority of them possessed a Voulgari et al., 2012; Potron et al., 2013). novel bleomycin resistance gene, bleMBL (Dortet et al., 2012). The first reported “acquired” oxacillinase with appreciable Co-expression of blaNDM−1 and bleMBL appear to be mediated carbapenem-hydrolyzing activity was OXA-23. OXA-23, or ARI- by a common promoter (PNDM−1)whichincludesportionsof 1, was identified from an A. baumannii isolate in Scotland in ISAba125. It is postulated that BRPMBL expression may contribute 1993 (the isolate was first recovered in 1985; Paton et al., 1993). some sort of selective advantage allowing NDM-1 to persist in the Subsequently, OXA-23 expression has been reported worldwide environment. (Mugnier et al., 2010) and both plasmid and chromosomal car- A contemporary evaluation of recently recovered NDM-1 pro- riage of blaOXA−23 are described. The OXA-23 group includes ducing A. baumannii isolates from Europe demonstrates that OXA-27, found in a single A. baumannii isolate from Singapore blaNDM−1 and blaNDM−2 genes are situated on the same chro- (Afzal-Shah et al.,2001). With the exception of an isolate of Proteus mosomally located transposon, Tn125 (Bonnin et al., 2012). mirabilis identified in France in 2002, this group of β-lactamases Dissemination of blaNDM in A. baumannii seems be due to dif- has been exclusively recovered from Acinetobacter species (Bonnet ferent strains carrying Tn125 or derivatives of Tn125 rather than et al., 2002). Increased expression of OXA-23 has been associated plasmid-mediated or clonal (Bonnin et al., 2013; Poirel et al., with the presence of upstream insertion sequences (e.g., ISAba1 2012a). and ISAba4) acting as strong promoters (Corvec et al., 2007). Another group of CHDLs include OXA-24/40, OXA-25, and CARBAPENEM-HYDROLYZING CLASS D β-LACTAMASES OXA-26 (Bou et al., 2000b; Afzal-Shah et al., 2001). OXA-24 and Oxacillinases comprise a heterogeneous group of class D β- OXA-40 differ by a few amino acid substitutions and OXA-25 and lactamases which are able to hydrolyze amino- and carboxypeni- OXA-26 are point mutation derivatives of OXA-40 (Afzal-Shah cillins (Poirel et al., 2010b). The majority of class D β-lactamases et al., 2001). Although primarily linked with clonal outbreaks in are not inhibited by commercially available β-lactamase inhibitors Spain and Portugal (Bou et al., 2000a; Lopez-Otsoa et al., 2002; Da but are inhibited in vitro by NaCl. Over 250 types of oxacilli- Silva et al., 2004; Acosta et al., 2011), OXA-24/40 β-lactamases has nases are reported with a minority demonstrating low levels of been isolated in other European countries and the USA (Lolans carbapenem-hydrolyzing activity. This select group of enzymes et al., 2006). OXA-40 was in fact the first CHDL documented in is also referred to as the carbapenem-hydrolyzing class D β- the USA (Lolans et al., 2006). lactamases (CHDLs; Table 3). CHDLs have been identified most OXA-58 has also only been detected in Acinetobacter spp. ini- frequently in Acinetobacter spp., however, there has been increas- tially identified in France (Héritier et al., 2005a; Poirel et al., 2005), ing isolation among Enterobacteriaceae, specifically OXA-48 OXA-58 has been associated with institutional outbreaks and has (oxacillinase-48; Lascols et al., 2012; Mathers et al., 2012). been recovered from clinical isolates of A. baumannii worldwide With the exception of OXA-163 (Poirel et al., 2011b), CHDLs (Coelho et al., 2006; Mendes et al., 2009; Gales et al., 2012). efficiently inactivate penicillins, first generations cephalosporins, As civilian and military personnel began returning from and β-lactam/β-lactamase inhibitor combinations, but spare Afghanistan and the Middle East, practitioners noted increasing

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 71

“fmicb-04-00048” — 2013/3/13 — 9:48 — page6—#6 Patel and Bonomo The global emergence of carbapenemases

Table 3 | Carbapenem-hydrolyzing class D β-lactamases.

Enzyme group Year isolated Organism(s) Geographic distribution Location Reference or described

OXA-23/27 1985/– Acinetobacter baumannii, Europe, USA, Middle East, Plasmid, chromosomal Afzal-Shah et al. (2001), Proteus mirabilis* Asia, Australia Gogou et al. (2011) OXA-24/40 1997 A. baumannii Europe and USA Plasmid, chromosomal Bou et al. (2000b), Lopez-Otsoa et al. (2002) OXA-25 – A. baumannii Spain Chromosomal Afzal-Shah et al. (2001) OXA-26 1996 A. baumannii Belgium Chromosomal Afzal-Shah et al. (2001) OXA-48 2001 K. pneumoniae, Turkey, Middle East, Plasmid Poirel et al. (2004b) Enterobacteriaceae Northern Africa, Europe, India, USA OXA-51/66/69 1993 A. baumannii Worldwide Chromosomal Brown et al. (2005), Evans et al. (2007) OXA-58 2003 A. baumannii Europe, USA, Middle East, Plasmid Poirel et al. (2005) South America OXA-143 2004 A. baumannii Brazil Plasmid Higgins et al. (2009) OXA-162 2008 Enterobacteriaceae Germany Plasmid Pfeifer et al. (2012) OXA-163 2008 K. pneumoniae, E. coli Argentina and Egypt Plasmid Poirel et al. (2011b), Abdelaziz et al. (2012) OXA-181 2006 K. pneumoniae, E. coli India Plasmid Castanheira et al. (2011) OXA-204 2012 K. pneumoniae Tunisia Plasmid Potron et al. (2013) OXA-232 2012 K. pneumoniae France Plasmid Poirel et al. (2012c)

*Single isolate described in France. recovery of A. baumannii from skin and soft tissue infections. in Portugal and Spain appear are incorporated in international Drug resistance was associated with expression of both OXA-23 clone II (Da Silva et al., 2004; Grosso et al., 2011) and ST 56 and OXA-58 (Hujer et al., 2006; Scott et al., 2007; Perez et al., (PubMLST; Acosta et al., 2011). OXA-58 expressing A. baumannii 2010b). Many isolates carrying the blaOXA−58 gene concurrently have been associated with international clones I, II, and II and a carry insertion sequences (e.g., ISaba1,ISAba2,orISAba3) asso- variety of unrelated sequence types (Di Popolo et al., 2011; Gogou ciated with increased carbapenemase production and thus higher et al., 2011). levels of carbapenem resistance. In one report increased gene copy OXA-48 was originally identified in a carbapenem-resistant iso- number was also associated with a higher level of enzyme pro- late of K. pneumoniae in Turkey (Poirel et al., 2004c). Early reports duction and increased phenotypic carbapenem resistance (Bertini suggested that this enzyme was geographically restricted to Turkey. et al., 2007). In the past few years, however, the enzyme has been recovered from Spread of OXA-type carbapenemases among A. baumannii variety of Enterobacteriaceae and has successfully circulated out- appears to be clonal and in depth reviews of the molecular side of Turkey with reports of isolation in the Middle East, North epidemiology and successful dissemination of these clones have Africa, Europe (Carrer et al., 2010), and most recently the USA been published (Woodford et al., 2011; Zarrilli et al., 2013). Two (Lascols et al., 2012; Mathers et al., 2012). The Middle East and MLST schemes with three loci in common exist for A. bauman- North Africa may be secondary reservoirs for these CHDLs (Hays nii – the PubMLST scheme (Bartual et al., 2005) and the Pasteur et al., 2012; Poirel et al., 2012c). Indeed, the introduction of OXA- scheme (Diancourt et al., 2010). Both schemes assign different 48 expressing Enterobacteriaceae in some countries has been from sequence types into clonal complexes (CC). Sequence types and patients from the Middle East or Northern Africa (Decre et al., CC from both schemes can be further categorized into the inter- 2010; Adler et al., 2011; Poirel et al., 2011e; Canton et al., 2012). In national (European) clones I, II, and III. It should be noted, the USA, the first clinical cases were associated with ST 199 and however, that the molecular taxonomy of A. baumannii con- ST 43 (Mathers et al., 2012). tinues to evolve (Higgins et al., 2012a). OXA-23 producing A. At least six OXA-48 variants (e.g., OXA-48, OXA-162, OXA- baumannii predominantly belong to international clones I and 163, OXA-181, OXA-204, and OXA-232) have been identified. II with a notable proportion being part of CC92 (PubMLST; OXA-48 is by far the most globally dispersed and its epidemiology Mugnier et al., 2010; Adams-Haduch et al., 2011). Similarly, A. has been recently reviewed (Poirel et al., 2012c). Unlike KPCs and baumannii isolates associated with epidemic spread of OXA-24/40 NDM-1 which have been associated with a variety of plasmids, a

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 72

“fmicb-04-00048” — 2013/3/13 — 9:48 — page7—#7 Patel and Bonomo The global emergence of carbapenemases

single 62 kb self-conjugative IncL/M-type plasmid has contributed OXA-143 is a novel plasmid-borne carbapenem-hydrolyzing to a large proportion of the distribution of blaOXA−48 in Europe oxacillinase recovered from clinical A. baumannii isolates in Brazil (Potron et al., 2011a). Sequencing of this plasmid (pOXA-48a) (Higgins et al., 2009). Information regarding its significance and notes that blaOXA−48 had been integrated through the acquisi- prevalence continues to evolve (Antonio et al.,2010; Werneck et al., tion of a Tn1999 composite transposon (Figure 1C; Poirel et al., 2011; Mostachio et al., 2012). 2012b) blaOXA−48 appears to be associated with a specific insertion sequence, IS1999 (Poirel et al., 2004c, 2012b). A variant of Tn1999, AVAILABLE AGENTS AND DRUGS IN DEVELOPMENT Tn1999.2, has been identified among isolates from Turkey and in Few antimicrobials are currently available to treat infections with Europe (Carrer et al., 2010; Potron et al., 2011a). Tn1999.2 har- carbapenemase-producing Gram-negative bacteria. Carriage of bors an IS1R element within the IS1999. OXA-48 appears to have concurrent resistance determinants can result in decreased sus- the highest affinity for imipenem of the CHDLs specifically those ceptibility non-β-lactams including the fluoroquinolones and harboring blaOXA−48 within a Tn1999.2 composite transposon aminoglycosides thus further compromising an already limited (Docquier et al., 2009). Three isoforms of the Tn1999 transposon antimicrobial arsenal. What frequently remains available are have been described (Giani et al., 2012). the polymyxins (including colistin), tigecycline, and fosfomy- Although much of the spread of OXA-48 is attributed to a spe- cin but susceptibilities to these agents are unpredictable (Falagas cific plasmid, outbreak evaluations demonstrate that a variety of et al., 2011). strains have contributed to dissemination of this emerging car- The reintroduction of polymyxins, both polymyxin B and bapenemase in K. pneumoniae. The same K. pneumoniae sequence colistin overlaps with the evolution of carbapenem resistance type, ST 395, harboring blaOXA−48 was identified in Morocco, among Gram-negative bacilli. The clinical “resurgence” of these France, and the Netherlands (Cuzon et al., 2011; Potron et al., agents is well documented (Falagas and Kasiakou, 2005; Li et al., 2011a). ST 353 was associated with an outbreak of OXA-48 pro- 2006a; Landman et al., 2008). Some experts advocate for the use ducing K. pneumoniae in London (Woodford et al., 2011) and ST of polymyxins in combination with other agents like rifampicin 221 with an outbreak of OXA-48 in Ireland (Canton et al., 2012). (Hirsch and Tam, 2010; Urban et al., 2010). In vitro evaluations OXA-48 production in K. pneumoniae, like KPC-expressing K. of different combinations including carbapenems, rifamycins, pneumoniae, has also been associated with ST 14 (Poirel et al., and/or tigecycline demonstrate variable results (Bercot et al., 2004c) and a recent outbreak in Greece was associated with ST 11 2011; Biswas et al., 2012; Deris et al., 2012; Jernigan et al., 2012). (Voulgari et al., 2012). Most evaluations of the clinical outcomes or “effectiveness” of blaOXA−48 is remarkably similar to blaOXA−54,aβ-lactamase combination therapies have been retrospective (Qureshi et al., gene intrinsic to Shewanella oneidensis (Poirel et al., 2004a). She- 2012; Tumbarello et al., 2012). Prospective clinical trials evalu- wanella spp. are relatively ubiquitous waterborne Gram-negative ating the superiority of colistin-based combination therapy over bacilli and are proving to be a potential environmental reservoir monotherapy are in their infancy. A real interest in combination for OXA-48 like carbapenemases as well as other resistance deter- therapy persists due to the concern of hetero-resistance (Li et al., minants (Héritier et al., 2004; Poirel et al., 2004b; Potron et al., 2006b; Poudyal et al., 2008; Lee et al., 2009; Yau et al., 2009; Meletis 2011b). et al., 2011). OXA-163, a single amino acid variant of OXA-48, was iden- Early evaluations of the glycylcycline, tigecycline, demonstrated tified in isolates of K. pneumoniae and Enterobacter cloacae from favorable in vitro activity against ESBL-producing Enterobacteri- Argentina and is unique in that it has activity against extended- aceae and specific isolates of carbapenem-resistant A. baumannii spectrum cephalosporins (Poirel et al., 2011b). OXA-163 also has and Enterobacteriaceae (Bratu et al., 2005; Fritsche et al., 2005; been identified in Egypt, which has a relatively prevalence of OXA- Noskin, 2005; Castanheira et al., 2008; Wang and Dowzicky, 2010). 48, in patients without epidemiologic links toArgentina (Abdelaziz Tigecycline remains untested in prospective trials and reports of et al., 2012). resistance are increasing (Navon-Venezia et al., 2007; Anthony OXA-181 was initially identified among carbapenem-resistant et al.,2008; Wangand Dowzicky,2010; Sun et al.,2012). The role of Enterobacteriaceae collected from India (Castanheira et al., 2011). tigecycline in treating primary bloodstream infections or urinary OXA-181 differs from OXA-48 by four amino acids, however, tract infections remains undefined due less than therapeutic con- appears to be nestled in an entirely different genetic platform. The centrations of drug achieved in the serum (Rodvold et al., 2006) blaOXA−181 gene has been mapped to a different group of plas- and urine (Satlin et al., 2011). We also note that meta-analyses mids, the ColE family, and has been associated with an alternative of pooled data from trials evaluating the use of tigecycline for a insertion sequence, ISEcp1. The latter insertion sequence has been variety of indications suggest there is a excess mortality associ- associated with the acquisition of other β-lactamases including ated with the use of tigecycline over comparator regimens (Cai CTX-M-like ESBLs. Like, OXA-48, it appears that OXA-181 may et al., 2011; Tasina et al., 2011; Yahav et al., 2011; Verde and Curcio, have evolved from a waterborne environmental species Shewanella 2012). However, in the absence of other tested regimens tige- xiamenensis (Potron et al., 2011b). cycline may be an appropriate or perhaps the only therapeutic OXA-204 differs from OXA-48 by a two amino acid substitu- option. tion. It was recently identified in a clinical K. pneumoniae isolate Growing resistance to both the polymyxins and tigecycline has from Tunisia (Potron et al., 2013). Its genetic construct appears to resulted the revisiting of older drugs including chloramphenicol, be similar to that of OXA-181. OXA-232 was recently identified nitrofurantoin, and temocillin (Livermore et al., 2011d). Fos- among K. pneumoniae isolates in France (Poirel et al., 2012c). fomycin is also one of these earlier antibiotics being reassessed

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 73

“fmicb-04-00048” — 2013/3/13 — 9:48 — page8—#8 Patel and Bonomo The global emergence of carbapenemases

(Falagas et al., 2008). In an in vitro evaluation of 68 KPC- contemporary pre-clinical evaluation of ME1071 in combination expressing K. pneumoniae isolates, fosfomycin demonstrated in with various type 2 carbapenems (i.e., biapenem, doripenem, vitro activity against 87% of tigecycline and/or polymyxin non- meropenem, imipenem) confirms remarkable decreases in the susceptible isolates and 83% of isolates that were resistant to both carbapenem MICs for Enterobacteriaceae and A. baumannii har- (Endimiani et al., 2010b). Fosfomycin may be a potential thera- boring IMP,VIM, and NDM-type MBLs (Livermore et al., 2013). peutic option for patients infected with carbapenemase-producing Irrespective of the candidate carbapenem, ME1071 activity against Enterobacteriaceae if the infection is localized to the genitourinary NDM MBLs was less than that of VIM-type and IMP-type MBLs. tract. Unfortunately, fosfomycin does not demonstrate reliable Of note, biapenem was the carbapenem with the lowest baseline activity against non-urinary pathogens. Fosfomycin demonstrated MICs to the MBLs, but it is commercially unavailable in many activity against only 30.2% of 1693 multidrug-resistant (MDR) countries including the USA. Other MBL-specific inhibitors are in P. aeruginosa isolates and 3.5% of 85 MDR A. baumannii isolates pre-clinical development (Chen et al., 2012). (Falagas et al.,2009). The individual studies included in this review Plazomicin (ACHN-490) is an aminoglycoside derivative with did not employ uniform MDR definitions or consistent suscepti- potent activity against some carbapenem-resistant Gram-negative bility breakpoints. Moreover, access to the parenteral fosfomycin bacilli (Zhanel et al., 2012). Studies have noted that susceptibilities is limited and the threshold for resistance is low (Rodriguez-Rojas to aminoglycosides vary among KPC-producing K. pneumoniae. et al.,2010; Karageorgopoulos et al.,2012). Concerns regarding the In one evaluation, 48% of 25 tested isolates were susceptible emergence of resistance have lead to an increasing interest in the to amikacin, 44% to gentamicin, and 8% to tobramycin. Pla- utility of combination therapy (Michalopoulos et al., 2010; Bercot zomicin demonstrated an MIC90 significantly lower than that et al., 2011; Souli et al., 2011). of amikacin (Endimiani et al., 2009c). In vitro studies also indi- Few agents are in the advanced stages of development with cate that depending on the aminoglycoside resistance mechanisms demonstrable in vitro activity against carbapenemase-producing present, Plazomicin may have activity against select isolates organisms. These include β-lactamase inhibitors, aminoglycoside of P. aeruginosa and A. baumannii (Aggen et al., 2010; Land- derivatives, polymyxin derivatives, and novel monobactams and man et al., 2011). Susceptibility to plazomicin in the setting monobactams-β-lactamase inhibitor combinations. of resistance to other aminoglycosides appears to be depen- Avibactam, or NXL104, is a β-lactamase inhibitor which has dent on the mechanism of aminoglycoside resistance (Livermore been tested in combination with ceftazidime, ceftaroline, and et al., 2011a). aztreonam against several carbapenemase-producing Enterobac- NAB739 and NAB7061 are polymyxin derivatives that may teriaceae with impressive decreases in MICs (Livermore et al., be less nephrotoxic than commercially available polymyxins. 2008, 2011b; Endimiani et al., 2009a; Mushtaq et al., 2010c). In a small in vitro study, NAB739 displayed activity against Cephalosporin–avibactam combinations do not inhibit MBLs. nine carbapenemase-producing polymyxin-susceptible isolates of Avibactam in combination with aztreonam, however, does seem Enterobacteriaceae (Vaara et al., 2010). A contemporary eval- to demonstrate activity against isolates harboring a variety of uation of NAB739 demonstrated higher MICs compared to carbapenem resistance mechanisms including MBLs (Livermore those of polymyxin B in a collection of polymyxin-susceptible et al., 2011b). Regrettably, the avibactam and aztreonam com- and non-susceptible Enterobacteriaceae, P. aeruginosa, and A. bination is not currently in clinical trials. The combination baumannii (Vaara et al., 2012). NAB7061 when used in combina- of ceftazidime–avibactam has been evaluated against collection with rifampicin or clarithromycin demonstrated synergistic tions of non-fermenting Gram-negative pathogens and its role activity against seven strains of carbapenemase-producing Gram- remains undefined (Mushtaq et al., 2010b). In some evalua- negative bacilli including one polymyxin-resistant strain (Vaara tions of ceftazidime non-susceptible isolates of P. aeruginosa et al., 2010). It remains unclear what role these agents will decrease MICs were noted with the addition of avibactam play in the setting the increasing burden of infections with (Mushtaq et al., 2010b; Walkty et al., 2011; Crandon et al., carbapenemase-producing Enterobacteriaceae. 2012; Levasseur et al., 2012). The combinations of ceftaroline– The activity of the siderophore monosulfactam, BAL30072, has avibactam and ceftazidime–avibactam are currently in clinical been against non-fermenting carbapenemase-producing Gram- trials. negative bacilli (Page et al., 2010). In one study, susceptibility to Methylidene penems (penem-1 and penem-2) are β-lactamase BAL30072 was noted in 73% of 200 isolates of carbapenemase- inhibitors and appear to be potent inhibitors of KPC-2 (Papp- producing A. baumannii, the majority of which were of the Wallace et al., 2010). The combination of cefepime with penem-1 same OXA-23 producing clone (Mushtaq et al., 2010a). In that demonstrated lower cefepime MICs in 88.1% of the 42 KPC- same study, smaller percentages of susceptibility were noted in producing K. pneumoniae isolates evaluated (Endimiani et al., a selection of carbapenem-resistant Burkholderia cepacia and P. 2010a). MK-7655 is a novel β-lactamase being evaluated in aeruginosa isolates. Recent evaluations of BAL30072 confirm that combination with imipenem against carbapenem-resistant Gram- there may be a role for this agent in the treatment of resis- negative bacilli (Hirsch et al., 2012). tant A. baumannii infections (Russo et al., 2011; Higgins et al., ME1071, formerly CP3242 (Bassetti et al., 2011), is a maleic 2012b). BAL 30376 is a combination of a siderophore monobac- acid derivative that competitively inhibits MBLs. Earlier studies tam with clavulanic acid. In two studies, this combination demonstrated concentration-dependent decreases in carbapenem demonstrated reasonable in vitro activity against CHDL, including MICs in MBL-producing P. aeruginosa (Ishii et al., 2010), A. OXA-48, and MBLs but not KPCs (Livermore et al., 2010; Page baumannii, and select Enterobacteriaceae (Shahid et al., 2009)A et al., 2011).

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 74

“fmicb-04-00048” — 2013/3/13 — 9:48 — page9—#9 Patel and Bonomo The global emergence of carbapenemases

CONCLUDING REMARKS The prudent use of antibiotics is essential in combating the In the last 5 years, we have witnessed the global spread of car- continuing evolution of resistance (Marchaim et al., 2012). This bapenem resistance among Gram-negative organisms. The notion may be even more crucial in areas where non-prescription antimi- that multidrug resistance among these pathogens is limited to crobial use is common and continues to be unregulated. In an age isolated outbreaks among the critically ill has met the ultimate where multidrug resistance is so widespread, even the appropriate challenge with NDM-1 (Kumarasamy et al., 2010). The conve- use of broad-spectrum antibiotics has contributed to our current niences of travel and medical tourism have introduced resistance state. mechanisms across states, countries, and even continents at an Research funding and support for the description of resistance alarming rate (Rogers et al., 2011; van der Bij and Pitout, 2012). mechanisms, validation of current infection control practices, Rates of resistance in some countries may be underestimated due and antimicrobial development must be prioritized. Institutions to the lack of organized reporting structures and limited resources. supporting infection control, state of the art microbiology labo- Long-term healthcare facilities are now recognized reservoirs for ratories, and antimicrobial stewardship programs should receive the continued propagation of MDR organisms (Urban et al., 2008; recognition and incentives for their foresight. Despite these Aschbacher et al., 2010; Perez et al., 2010a; Ben-David et al., 2011; continuing challenges, considerable progress has been made to Prabaker et al., 2012; Viau et al., 2012). identify at-risk populations and to describe resistance determi- Until the introduction of accurate, affordable, and readily nants. Collaborative efforts (Kitchel et al., 2009a; Struelens et al., accessible diagnostics and reliably effective antimicrobials a major 2010; Canton et al., 2012) have led to a better understanding and focus remains containment and eradication of these organisms awareness of the epidemiology and the contribution of antimicro- within the healthcare environment. Many cite a “bundle” type bial use and the environment to the propagation of antimicrobial approach that includes administrative support, active surveillance, resistance. These joint efforts have proven crucial for the prop- antimicrobial stewardship, and augmented infection control prac- agation of information about carbapenemases. Continuing to tices (Centers for Disease Control and Prevention, 2009; Schwaber encourage these partnerships is imperative in the ongoing strug- et al., 2011; Snitkin et al., 2012). Just as with drug development gle against antimicrobial resistance and to prevent antimicrobials (Tillotson, 2010), the future savings of investing in prevention is from essentially becoming obsolete. not as tangible as the immediate capital investment required to allot appropriate resources including advanced laboratory plat- ACKNOWLEDGMENTS forms, experienced laboratory personnel, dedicated nursing staff, This work was supported in part by the Veterans Affairs Merit and infection control personnel (Bilavsky et al., 2010). Expanding Review Program (to Robert A. Bonomo), the National Institutes these efforts to non-acute healthcare settings is recommended to of Health (grants R01-A1063517 and RO3-A1081036 to Robert begin to stem the evolving pandemic of carbapenem resistance A. Bonomo), and the Geriatric Research Education and Clinical (Gupta et al., 2011). Center VISN 10 (to Robert A. Bonomo).

REFERENCES Characterization of OXA-25, OXA- gene in Brazilian hospitals. Antimi- baumannii. J. Clin. Microbiol. 43, Abdelaziz, M. O., Bonura, C., Aleo, 26, and OXA-27, molecular class crob. Agents Chemother. 55, 1322– 4382–4390. A., El-Domany, R. A., Fasciana, T., D beta-lactamases associated with 1323. Bassetti, M., Ginocchio, F., and and Mammina, C. (2012). OXA-163- carbapenem resistance in clinical Arpin, C., Noury, P., Boraud, D., Mikulska, M. (2011). New treat- producing Klebsiella pneumoniae in isolates of Acinetobacter baumannii. Coulange, L., Manetti, A., Andre, ment options against Gram-negative Cairo, Egypt, in 2009 and 2010. J. Antimicrob. Agents Chemother. 45, C., et al. (2012). NDM-1-producing organisms. Crit. Care 15, 215. Clin. Microbiol. 50, 2489–2491. 583–588. Klebsiella pneumoniae resistant to Ben-David, D. M. D., Masarwa, S. M. A., Acosta, J., Merino, M., Viedma, E., Aggen, J. B., Armstrong, E. S., Gold- colistin in a French community Navon-Venezia, S. P., Mishali, H. M. Poza, M., Sanz, F., Otero, J. R., et al. blum, A. A., Dozzo, P., Linsell, M. patient without history of foreign A., Fridental, I. M. D., Rubinovitch, (2011). Multidrug-resistant Acineto- S., Gliedt, M. J., et al. (2010). Syn- travel. Antimicrob. Agents Chemother. B. M. D., et al. (2011). Carbapenem- bacter baumannii harboring OXA-24 thesis and spectrum of the neoglyco- 56, 3432–3434. resistant Klebsiella pneumoniae in carbapenemase, Spain. Emerg. Infect. side ACHN-490. Antimicrob. Agents Aschbacher, R., Pagani, L., Doumith, post-acute-care facilities in Israel. Dis. 17, 1064–1067. Chemother. 54, 4636–4642. M., Pike, R., Woodford, N., Infect. Control Hosp. Epidemiol. 32, Adams-Haduch, J. M., Onuoha, E. Ambler, R. P. (1980). The structure of Spoladore, G., et al. (2010). Metallo- 845–853. O., Bogdanovich, T., Tian, G. B., beta-lactamases. Philos. Trans. R. Soc. β-lactamases among Enterobacteri- Bercot, B., Poirel, L., Dortet, L., and Marschall, J., Urban, C. M., et al. Lond. B Biol. Sci. 289, 321–331. aceae from routine samples in an Nordmann, P. (2011). In vitro evalu- (2011). Molecular epidemiology of Anthony, K. B., Fishman, N. O., Linkin, Italian tertiary-care hospital and ation of antibiotic synergy for NDM- carbapenem-nonsusceptible Acineto- D. R., Gasink, L. B., Edelstein, P. long-term care facilities during 2008. 1-producing Enterobacteriaceae. J. bacter baumannii in the United H., and Lautenbach, E. (2008). Clin- Clin. Microbiol. Infect. 17, 181–189. Antimicrob. Chemother. 66, 2295– States. J. Clin. Microbiol. 49, 3849– ical and microbiological outcomes Aubron, C., Poirel, L., Ash, R. 2297. 3854. of serious infections with multidrug- J., and Nordmann, P. (2005). Bertini, A., Poirel, L., Bernabeu, S., Adler, A., Shklyar, M., Schwaber, M. resistant Gram-negative organisms Carbapenemase-producing Enter- Fortini, D., Villa, L., Nordmann, J., Navon-Venezia, S., Dhaher, Y., treated with tigecycline. Clin. Infect. obacteriaceae, U.S. rivers. Emerg. P., et al. (2007). Multicopy blaOXA- Edgar, R., et al. (2011). Introduction Dis. 46, 567–570. Infect. Dis. 11, 260–264. 58 gene as a source of high-level of OXA-48-producing Enterobacteri- Antonio, C. S., Neves, P. R., Medeiros, Bartual, S. G., Seifert, H., Hippler, C., resistance to carbapenems in Acineto- aceae to Israeli hospitals by medical M., Mamizuka, E. M., Elmor Luzon, M. A., Wisplinghoff, H., and bacter baumannii. Antimicrob. Agents tourism. J. Antimicrob. Chemother. De Araujo, M. R., and Linco- Rodriguez-Valera, F. (2005). Devel- Chemother. 51, 2324–2328. 66, 2763–2766. pan, N. (2010). High prevalence of opment of a multilocus sequence Bilavsky, E., Schwaber, M. J., and Afzal-Shah, M., Woodford, N., carbapenem-resistant Acinetobacter typing scheme for characterization Carmeli, Y. (2010). How to stem and Livermore, D. M. (2001). baumannii carrying the blaOXA-143 of clinical isolates of Acinetobacter the tide of carbapenemase-producing

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 75

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 10 — #10 Patel and Bonomo The global emergence of carbapenemases

Enterobacteriaceae?: proactive versus Bratu, S., Landman, D., Alam, M., program, 2006–2007. Antimicrob. Culebras, E., Gonzalez-Romo, F., Head, reactive strategies. Curr. Opin. Infect. Tolentino, E., and Quale, J. (2005). Agents Chemother. 55, 1274–1278. J., Gomez, M., Morales, G., and Dis. 23, 327–331. Detection of KPC carbapenem- Castanheira, M., Mendes, R. E., Walsh, Picazo, J. J. (2010). Outbreak of Biswas, S., Brunel, J.-M., Dubus, J.-C., hydrolyzing enzymes in Enterobac- T. R., Gales, A. C., and Jones, R. N. Acinetobacter baumannii producing Reynaud-Gaubert, M., and Rolain, J.- ter spp. from Brooklyn, New York. (2004a). Emergence of the extended- OXA-66 in a Spanish hospital: epi- M. (2012). Colistin: an update on Antimicrob. Agents Chemother. 49, spectrum beta-lactamase GES-1 in a demiology and study of patient the antibiotic of the 21st century. 776–778. Pseudomonas aeruginosa strain from movements. Microb. Drug Resist. 16, Expert Rev. Anti Infect. Ther. 10, Brown, S., Young, H. K., and Amyes, Brazil: report from the SENTRY 309–315. 917–934. S. G. B. (2005). Characterisation of antimicrobial surveillance program. Cuzon, G., Naas, T., Truong, H., Vil- Bogaerts, P., Naas, T., El Garch, F., OXA-51, a novel class D carbapene- Antimicrob. Agents Chemother. 48, legas, M. V., Wisell, K. T., Carmeli, Cuzon, G., Deplano, A., Delaire, mase found in genetically unrelated 2344–2345. Y., et al. (2010). Worldwide diversity T., et al. (2010). GES extended- clinical strains of Acinetobacter bau- Castanheira, M., Toleman, M. A., Jones, of Klebsiella pneumoniae that pro- spectrum beta-lactamases in Acineto- mannii from Argentina. Clin. Micro- R. N., Schmidt, F. J., and Walsh, T. R. duce beta-lactamase blaKPC-2 gene. bacter baumannii isolates in Belgium. biol. Infect. 11, 15–23. (2004b). Molecular characterization Emerg. Infect. Dis. 16, 1349–1356. Antimicrob. Agents Chemother. 54, Bush, K., and Jacoby, G. A. (2010). of a beta-lactamase gene, blaGIM-1, Cuzon, G., Ouanich, J., Gondret, 4872–4878. Updated functional classiﬁcation of encoding a new subclass of metallo- R., Naas, T., and Nordmann, Bonnet, R., Marchandin, H., Chanal, C., beta-lactamases. Antimicrob. Agents beta-lactamase. Antimicrob. Agents P. (2011). Outbreak of OXA-48- Sirot, D., Labia, R., De Champs, C., Chemother. 54, 969–976. Chemother. 48, 4654–4661. positive carbapenem-resistant Kleb- et al. (2002). Chromosome-encoded Cai, J. C., Zhou, H. W., Zhang, R., Castanheira, M., Sader, H. S., siella pneumoniae isolates in France. class D beta-lactamase OXA-23 in and Chen, G.-X. (2008). Emergence Deshpande, L. M., Fritsche, T. Antimicrob. Agents Chemother. 55, Proteus mirabilis. Antimicrob. Agents of Serratia marcescens, Klebsiella R., and Jones, R. N. (2008). 2420–2423. Chemother. 46, 2004–2006. pneumoniae, and Escherichia coli Antimicrobial activities of tigecy- da Fonseca, É. L., Vieira, V. V., Cipri- Bonnin, R.A., Nordmann, P.,Potron,A., isolates possessing the plasmid- cline and other broad-spectrum ano, R., and Vicente, A. C. P. (2007). Lecuyer, H., Zahar, J. R., and Poirel, mediated carbapenem-hydrolyzing antimicrobials tested against serine Emergence of blaGES-5 in clinical L. (2011). Carbapenem-hydrolyzing beta-lactamase KPC-2 in intensive carbapenemase- and metallo-beta- colistin-only-sensitive (COS) Pseu- GES-type extended-spectrum beta- care units of a Chinese hospital. lactamase-producing Enterobacteri- domonas aeruginosa strain in Brazil. J. lactamase in Acinetobacter bauman- Antimicrob. Agents Chemother. 52, aceae: report from the SENTRY Antimicrob. Chemother. 59, 576–577. nii. Antimicrob. Agents Chemother. 2014–2018. antimicrobial surveillance program. Darley, E., Weeks, J., Jones, L., Daniels, 55, 349–354. Cai, Y., Wang, R., Liang, B., Bai, N., Antimicrob. Agents Chemother. 52, V., Wootton, M., Macgowan, A., Bonnin, R. A., Poirel, L., Naas, and Liu, Y. (2011). Systematic review 570–573. et al. (2012). NDM-1 polymicrobial T., Pirs, M., Seme, K., Schrenzel, and meta-analysis of the effectiveness Centers for Disease Control and Preven- infections including Vibrio cholerae. J., et al. (2012). Dissemination of and safety of tigecycline for treatment tion. (2009). Guidance for control of Lancet 380, 1358. New Delhi metallo-beta-lactamase- of infectious disease. Antimicrob. infections with carbapenem-resistant Da Silva, G. J., Quinteira, S., Bértolo, E., 1-producing Acinetobacter bauman- Agents Chemother. 55, 1162–1172. or carbapenemase-producing Enter- Sousa, J. C., Gallego, L., Duarte, A., nii in Europe. Clin. Microbiol. Infect. Canton, R., Akova, M., Carmeli, Y., obacteriaceae in acute care facilities. et al. (2004). Long-term dissemina- 18, E362–E365. Giske, C. G., Glupczynski, Y., Gni- MMWR Morb. Mortal. Wkly. Rep. 58, tion of an OXA-40 carbapenemase- Bonnin, R. A., Rotimi, V. O., Al Hubail, adkowski, M., et al. (2012). Rapid 256–260. producing Acinetobacter baumannii M., Gasiorowski, E., Al Sweih, N., evolution and spread of carbapene- Chen, P., Horton, L. B., Mikul- clone in the Iberian Peninsula. J. Nordmann, P.,et al. (2013). Wide dis- mases among Enterobacteriaceae in ski, R. L., Deng, L., Sundriyal, Antimicrob. Chemother. 54, 255–258. semination of GES-type carbapen- Europe. Clin. Microbiol. Infect. 18, S., Palzkill, T., et al. (2012). Decre, D., Birgand, G., Geneste, emases in Acinetobacter baumannii 413–431. 2-Substituted 4,5-dihydrothiazole-4- D., Maury, E., Petit, J. C., Bar- isolates in Kuwait. Antimicrob. Agents Carrer, A., Poirel, L., Pitout, J. D., carboxylic acids are novel inhibitors but, F., et al. (2010). Possible Chemother. 57, 183–188. Church, D., and Nordmann, P. of metallo-β-lactamases. Bioorg. importation and subsequent cross- Borgia, S., Lastovetska, O., Richard- (2008). Occurrence of an SME-2- Med. Chem. Lett. 22, 6229–6232. transmission of OXA-48-producing son, D., Eshaghi, A., Xiong, J., producing Serratia marcescens isolate Coelho, J., Woodford, N., Afzal-Shah, Klebsiella pneumoniae, France, 2010. Chung, C., et al. (2012). Out- in Canada. Int. J. Antimicrob. Agents M., and Livermore, D. (2006). Occur- Euro Surveill. 15, pii: 19718. break of carbapenem-resistant Enter- 31, 181–182. rence of OXA-58-like carbapene- Deris, Z. Z., Yu, H. H., Davis, K., obacteriaceae containing blaNDM-1, Carrer, A., Poirel, L., Yilmaz, M., Akan, mases in Acinetobacter spp. collected Soon, R. L., Jacob, J., Ku, C. K., Ontario, Canada. Clin. Infect. Dis. 55, O. A., Feriha, C., Cuzon, G., et al. over 10 years in three continents. et al. (2012). The combination of e109–e117. (2010). Spread of OXA-48-encoding Antimicrob. Agents Chemother. 50, colistin and doripenem is synergistic Bou, G., Cervero, G., Dominguez, plasmid in Turkey and Beyond. 756–758. against Klebsiella pneumoniae at mul- M. A., Quereda, C., and Martinez- Antimicrob. Agents Chemother. 54, Corvec, S., Poirel, L., Naas, T., tiple inocula and suppresses colistin Beltran, J. (2000a). Characterization 1369–1373. Drugeon, H., and Nordmann, P. resistance in an in vitro pharma- of a nosocomial outbreak caused Castanheira, M., Deshpande, L. M., (2007). Genetics and expression of cokinetic/pharmacodynamic model. by a multiresistant Acinetobacter Dipersio, J. R., Kang, J., Weinstein, the carbapenem-hydrolyzing oxacil- Antimicrob. Agents Chemother. 56, baumannii strain with a carbapenem- M. P., and Jones, R. N. (2009). First linase gene blaOXA-23 in Acineto- 5103–5112. hydrolyzing enzyme: high-level car- descriptions of blaKPC in Raoultella bacter baumannii. Antimicrob. Agents Deshpande, L. M., Jones, R. N., bapenem resistance in A. baumannii spp. (R. planticola and R. ornithi- Chemother. 51, 1530–1533. Fritsche, T. R., and Sader, H. S. is not due solely to the presence of nolytica): report from the SENTRY Crandon, J. L., Schuck, V. J., Banevi- (2006a). Occurrence and character- beta-lactamases. J. Clin. Microbiol. antimicrobial surveillance program. cius, M. A., Beaudoin, M. E., ization of carbapenemase-producing 38, 3299–3305. J. Clin. Microbiol. 47, 4129–4130. Nichols, W. W., Tanudra, M. A., Enterobacteriaceae: report from the Bou, G., Oliver, A., and Martinez- Castanheira, M., Deshpande, L. M., et al. (2012). Comparative in vitro SENTRY antimicrobial surveillance Beltran, J. (2000b). OXA-24, a Mathai, D., Bell, J. M., Jones, R. and in vivo efﬁcacies of human program (2000–2004). Microb. Drug novel class D beta-lactamase with N., and Mendes, R. E. (2011). Early simulated doses of ceftazidime Resist. 12, 223–230. carbapenemase activity in an Acine- dissemination of NDM-1- and OXA- and ceftazidime–avibactam against Deshpande, L. M., Rhomberg, P. R., tobacter baumannii clinical strain. 181-producing Enterobacteriaceae in Pseudomonas aeruginosa. Antimi- Sader, H. S., and Jones, R. N. Antimicrob. Agents Chemother. 44, Indian hospitals: report from the crob. Agents Chemother. 56, 6137– (2006b). Emergence of serine car- 1556–1561. SENTRY antimicrobial surveillance 6146. bapenemases (KPC and SME) among

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 76

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 11 — #11 Patel and Bonomo The global emergence of carbapenemases

clinical strains of Enterobacteriaceae plasmid-mediated quinolone resis- treatment of infections caused by Girlich, D., Poirel, L., and Nord- isolated in the United States Medi- tance in Klebsiella pneumoniae iso- multidrug-resistant non-fermenting mann, P. (2010). Novel ambler cal Centers: report from the MYS- lates possessing blaKPC in the United Gram-negative bacilli: a systematic class A carbapenem-hydrolyzing TIC Program (1999–2005). Diagn. States. Antimicrob. Agents Chemother. review of microbiological, animal beta-lactamase from a Pseudomonas Microbiol. Infect. Dis. 56, 367–372. 52, 2680–2682. and clinical studies. Int. J. Antimicrob. ﬂuorescens isolate from the Seine Diancourt, L., Passet, V., Nemec, Endimiani, A., Choudhary, Y., and Agents 34, 111–120. River, Paris, France. Antimicrob. A., Dijkshoorn, L., and Brisse, S. Bonomo, R. A. (2009a). In vitro Figueiredo, S., Poirel, L., Croize, Agents Chemother. 54, 328–332. (2010). The population structure of activity of NXL104 in combination J., Recule, C., and Nordmann, P. Gogou, V., Pournaras, S., Giannouli, Acinetobacter baumannii: expanding with beta-lactams against Klebsiella (2009). In vivo selection of reduced M., Voulgari, E., Piperaki, E.-T., multiresistant clones from an ances- pneumoniae isolates producing KPC susceptibility to carbapenems in Zarrilli, R., et al. (2011). Evolution tral susceptible genetic pool. PLoS carbapenemases. Antimicrob. Agents Acinetobacter baumannii related to of multidrug-resistant Acinetobacter ONE 5:e10034. doi: 10.1371/jour- Chemother. 53, 3599–3601. ISAba1-mediated overexpression of baumannii clonal lineages: a 10 year nal.pone.0010034 Endimiani, A., Hujer, A. M., Perez, F., the natural blaOXA-66 oxacillinase study in Greece (2000–09). J. Antimi- Di Popolo, A., Giannouli, M., Tri- Bethel, C. R., Hujer, K. M., Kroeger, gene. Antimicrob. Agents Chemother. crob. Chemother. 66, 2767–2772. assi, M., Brisse, S., and Zarrilli, R. J., et al. (2009b). Characterization of 53, 2657–2659. Gregory, C. J., Llata, E., Stine, (2011). Molecular epidemiological blaKPC-containing Klebsiella pneu- Fritsche, T. R., Strabala, P. A., Sader, H. N., Gould, C., Santiago, L. M., investigation of multidrug-resistant moniae isolates detected in different S., Dowzicky, M. J., and Jones, R. N. Vazquez, G. J., et al. (2010). Out- Acinetobacter baumannii strains institutions in the Eastern USA. J. (2005). Activity of tigecycline tested break of carbapenem-resistant Kleb- in four Mediterranean countries Antimicrob. Chemother. 63, 427–437. against a global collection of Enter- siella pneumoniae in Puerto Rico with a multilocus sequence typing Endimiani, A., Hujer, K. M., Hujer, A. obacteriaceae, including tetracycline- associated with a novel carbapene- scheme. Clin. Microbiol. Infect. 17, M., Armstrong, E. S., Choudhary, Y., resistant isolates. Diagn. Microbiol. mase variant. Infect. Control Hosp. 197–201. Aggen, J. B., et al. (2009c). ACHN- Infect. Dis. 52, 209–213. Epidemiol. 31, 476–484. Docquier, J.-D., Calderone, V., De 490, a neoglycoside with potent Gales, A. C., Castanheira, M., Jones, Grosso, F., Quinteira, S., and Peixe, L. Luca, F., Benvenuti, M., Giuliani, in vitro activity against multidrug- R. N., and Sader, H. S. (2012). (2011). Understanding the dynamics F., Bellucci, L., et al. (2009). Crystal resistant Klebsiella pneumoniae iso- Antimicrobial resistance among of imipenem-resistant Acinetobacter structure of the OXA-48 β-lactamase lates. Antimicrob. Agents Chemother. Gram-negative bacilli isolated from baumannii lineages within Portugal. reveals mechanistic diversity among 53, 4504–4507. Latin America: results from SENTRY Clin. Microbiol. Infect. 17, 1275–1279. class D carbapenemases. Chem. Biol. Endimiani, A., and Paterson, D. L. Antimicrobial Surveillance Pro- Gülmez, D., Woodford, N., Palepou, 16, 540–547. (2007). Optimizing therapy for infec- gram (Latin America, 2008–2010). M.-F. I., Mushtaq, S., Metan, G., Dortet, L., Nordmann, P., and Poirel, tions caused by enterobacteriaceae Diagn. Microbiol. Infect. Dis. 73, Yakupogullari, Y., et al. (2008). L. (2012). Association of the emerg- producing extended-spectrum beta- 354–360. Carbapenem-resistant Escherichia ing carbapenemase NDM-1 with lactamases. Semin. Respir. Crit. Care Geffen, Y., Adler, A., Paikin, S., coli and Klebsiella pneumoniae iso- a bleomycin resistance protein in Med. 28, 646–655. Khabra, E., Gorenshtein, S., lates from Turkey with OXA-48-like Enterobacteriaceae and Acinetobac- Evans, B. A., Brown, S., Hamouda, A., Aronov, R., et al. (2012). Detec- carbapenemases and outer mem- ter baumannii. Antimicrob. Agents Findlay, J., and Amyes, S. G. B. (2007). tion of the plasmid-mediated KPC-2 brane protein loss. Int. J. Antimicrob. Chemother. 56, 1693–1697. Eleven novel OXA-51-like enzymes carbapenem-hydrolysing enzyme in Agents 31, 523–526. El-Gamal, M. I., and Oh, C. H. (2010). from clinical isolates of Acinetobac- three unusual species of the Enter- Gupta, N., Limbago, B. M., Patel, Current status of carbapenem antibi- ter baumannii. Clin. Microbiol. Infect. obacteriaceae family in Israel. J. J. B., and Kallen, A. J. (2011). otics. Curr. Top. Med. Chem. 10, 13, 1137–1138. Antimicrob. Chemother. 68, 719–720. Carbapenem-resistant Enterobacte- 1882–1897. Fairfax, M. R., Queenan, A. M., Lep- Ghazawi, A., Sonnevend, A., Bonnin, riaceae: epidemiology and preven- El Salabi, A., Borra, P. S., Tole- hart, P. R., Kaye, K. S., Dror, M., R. A., Poirel, L., Nordmann, P., tion. Clin. Infect. Dis. 53, 60–67. man, M. A., Samuelsen, O., and Arnous, N., et al. (2011). Detection of Hashmey, R., et al. (2012). NDM- Halaby, T., Reuland, A. E., Al Walsh, T. R. (2012). Genetic and 2 SME-1 carbapenemase-producing 2 carbapenemase-producing Acineto- Naiemi, N., Potron, A., Savelkoul, biochemical characterization of a Serratia marcescens in Detroit. Diagn. bacter baumannii in the United Arab P. H., Vandenbroucke-Grauls, C. M., novel metallo-beta-lactamase, TMB- Microbiol. Infect. Dis. 71, 325–326. Emirates. Clin. Microbiol. Infect. 18, et al. (2012). A case of New Delhi 1, from an Achromobacter xylosoxi- Falagas, M. E., Kanellopoulou, M. D., E34–E36. metallo-beta-lactamase 1 (NDM- dans strain isolated in Tripoli, Libya. Karageorgopoulos, D. E., Dimopou- Giakkoupi, P., Papagiannitsis, C. C., 1)-producing Klebsiella pneumoniae Antimicrob. Agents Chemother. 56, los, G., Rafailidis, P. I., Skarmoutsou, Miriagou, V., Pappa, O., Polemis, M., with putative secondary transmission 2241–2245. N. D., et al. (2008). Antimicrobial Tryﬁnopoulou, K., et al. (2011). An from the Balkan region in the Nether- Endimiani, A., Bethel, C., Choudhary, susceptibility of multidrug-resistant update of the evolving epidemic of lands. Antimicrob. Agents Chemother. Y., and Bonomo, R. A. (2010a). In Gram negative bacteria to fos- blaKPC-2-carrying Klebsiella pneu- 56, 2790–2791. vitro activity of penem-1 in com- fomycin. Eur. J. Clin. Microbiol. moniae in Greece (2009–10). J. Hays, C., Benouda, A., Poirel, L., bination with beta-lactams against Infect. Dis. 27, 439–443. Antimicrob. Chemother. 66, 1510– Elouennass, M., and Nordmann, blaKPC-possessing Klebsiella pneu- Falagas, M. E., Karageorgopoulos, 1513. P. (2012). Nosocomial occurrence moniae isolates. Antimicrob. Agents D. E., and Nordmann, P. (2011). Giakkoupi, P., Xanthaki, A., of OXA-48-producing enterobacte- Chemother. 54, 1650–1651. Therapeutic options for infections Kanelopoulou, M., Vlahaki, A., rial isolates in a Moroccan hospital. Endimiani, A., Patel, G., Hujer, K. M., with Enterobacteriaceae producing Miriagou,V.,Kontou, S., et al. (2003). Int. J. Antimicrob. Agents 39, 545–547. Swaminathan, M., Perez, F., Rice, L. carbapenem-hydrolyzing enzymes. VIM-1 metallo-beta-lactamase- Henriques, I., Moura, A., Alves, A., B., et al. (2010b). In vitro activity Future Microbiol. 6, 653–666. producing Klebsiella pneumoniae Saavedra, M. J., and Correia, A. of fosfomycin against blaKPC- Falagas, M. E., and Kasiakou, S. strains in Greek hospitals. J. Clin. (2004). Molecular characterization containing Klebsiella pneumoniae K. (2005). Colistin: the revival Microbiol. 41, 3893–3896. of a carbapenem-hydrolyzing class isolates, including those nonsuscep- of polymyxins for the manage- Giani, T., Conte, V., Di Pilato, V., A beta-lactamase, SFC-1, from Ser- tible to tigecycline and/or colistin. ment of multidrug-resistant Gram- Aschbacher, R., Weber, C., Larcher, ratia fonticola UTAD54. Antimicrob. Antimicrob. Agents Chemother. 54, negative bacterial infections. Clin. C., et al. (2012). Escherichia coli from Agents Chemother. 48, 2321–2324. 526–529. Infect. Dis. 40, 1333–1341. Italy producing OXA-48 carbapen- Héritier, C., Dubouix, A., Poirel, Endimiani, A., Carias, L. L., Hujer, Falagas, M. E., Kastoris, A. C., Kara- emase encoded by a novel Tn1999 L., Marty, N., and Nordmann, A. M., Bethel, C. R., Hujer, K. M., georgopoulos, D. E., and Rafailidis, transposon derivative. Antimicrob. P. (2005a). A nosocomial out- Perez, F., et al. (2008). Presence of P. I. (2009). Fosfomycin for the Agents Chemother. 56, 2211–2213. break of Acinetobacter baumannii

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 77

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 12 — #12 Patel and Bonomo The global emergence of carbapenemases

isolates expressing the carbapenem- with ME1071, a novel metallo- Genetic factors associated with ele- enzymes in North America. Antimi- hydrolysing oxacillinase OXA-58. J. beta-lactamase inhibitor, against vated carbapenem resistance in KPC- crob Agents Chemother. 57, 130–136. Antimicrob. Chemother. 55, 115–118. metallo-beta-lactamase-producing producing Klebsiella pneumoniae. Lauretti, L., Riccio, M. L., Mazzariol, Héritier, C., Poirel, L., Fournier, P.- Pseudomonas aeruginosa clinical iso- Antimicrob. Agents Chemother. 54, A., Cornaglia, G., Amicosante, G., E., Claverie, J.-M., Raoult, D., and lates. Antimicrob. Agents Chemother. 4201–4207. Fontana, R., et al. (1999). Cloning Nordmann, P. (2005b). Character- 54, 3625–3629. Kitchel, B., Rasheed, J. K., Patel, J. and characterization of blaVIM, a ization of the naturally occurring Ito, H., Arakawa, Y., Ohsuka, S., B., Srinivasan, A., Navon-Venezia, S., new integron-borne metallo-beta- oxacillinase of Acinetobacter bauman- Wacharotayankun, R., Kato, N., and Carmeli, Y., et al. (2009a). Molecu- lactamase gene from a Pseudomonas nii. Antimicrob. Agents Chemother. Ohta, M. (1995). Plasmid-mediated lar epidemiology of KPC-producing aeruginosa clinical isolate. Antimi- 49, 4174–4179. dissemination of the metallo-beta- Klebsiella pneumoniae isolates in crob. Agents Chemother. 43, 1584– Héritier, C., Poirel, L., and Nord- lactamase gene blaIMP among the United States: clonal expansion 1590. mann, P. (2004). Genetic and clinically isolated strains of Serra- of multilocus sequence type 258. Lautenbach, E., Synnestvedt, M., biochemical characterization of a tia marcescens. Antimicrob. Agents Antimicrob. Agents Chemother. 53, Weiner, M. G., Bilker, W. B., Vo, chromosome-encoded carbapenem- Chemother. 39, 824–829. 3365–3370. L., Schein, J., et al. (2009). Epi- hydrolyzing ambler class D β- Jeong, S. H., Bae, I. K., Kim, D., Hong, Kitchel, B., Sundin, D. R., and Patel, J. B. demiology and impact of imipenem lactamase from Shewanella algae. S. G., Song, J. S., Lee, J. H., et al. (2009b). Regional dissemination of resistance in Acinetobacter bauman- Antimicrob. Agents Chemother. 48, (2005). First outbreak of Klebsiella KPC-producing Klebsiella pneumo- nii. Infect. Control Hosp. Epidemiol. 1670–1675. pneumoniae clinical isolates produc- niae. Antimicrob. Agents Chemother. 30, 1186–1192. Higgins, P. G., Janssen, K., Fresen, M. ing GES-5 and SHV-12 extended- 53, 4511–4513. Lautenbach, E., Synnestvedt, M., M., Wisplinghoff, H., and Seifert, spectrum beta-lactamases in Korea. Kotsakis, S. D., Miriagou, V., Tzelepi, Weiner, M. G., Bilker, W. B., Vo, L., H. (2012a). Molecular epidemiology Antimicrob. Agents Chemother. 49, E., and Tzouvelekis, L. S. (2010). Schein, J., et al. (2010). Imipenem of Acinetobacter baumannii blood- 4809–4810. Comparative biochemical and com- resistance in Pseudomonas aerugi- stream isolates obtained in the United Jernigan, M. G., Press, E. G., Nguyen, putational study of the role of natu- nosa: emergence, epidemiology, and States from 1995 to 2004 using rep- M. H., Clancy, C. J., and Shields, rally occurring mutations at Ambler impact on clinical and economic out- PCR and multilocus sequence typing. R. K. (2012). The combination of positions 104 and 170 in GES comes. Infect. Control Hosp. Epi- J. Clin. Microbiol. 50, 3493–3500. doripenem and colistin is bactericidal beta-lactamases. Antimicrob. Agents demiol. 31, 47–53. Higgins, P. G., Stefanik, D., Page, and synergistic against colistin- Chemother. 54, 4864–4871. Lee, J., Patel, G., Huprikar, S., M. G. P., Hackel, M., and resistant, carbapenemase-producing Kumarasamy, K. K., Toleman, M. A., Calfee, D. P., and Jenkins, S. Seifert, H. (2012b). In vitro activ- Klebsiella pneumoniae. Antimi- Walsh, T. R., Bagaria, J., Butt, F., G. (2009). Decreased susceptibility ity of the siderophore monosulfactam crob. Agents Chemother. 56, 3395– Balakrishnan, R., et al. (2010). Emer- to polymyxin B during treatment BAL30072 against meropenem-non- 3398. gence of a new antibiotic resistance for carbapenem-resistant Klebsiella susceptible Acinetobacter baumannii. Jovcic, B., Lepsanovic, Z., Suljagic, mechanism in India, Pakistan, and pneumoniae infection. J. Clin. Micro- J. Antimicrob. Chemother. 67, 1167– V., Rackov, G., Begovic, J., Top- the UK: a molecular, biological, and biol. 47, 1611–1612. 1169. isirovic, L., et al. (2011). Emergence epidemiological study. Lancet Infect. Lee, K., Yum, J. H., Yong, D., Lee, H. Higgins, P. G., Poirel, L., Lehmann, M., of NDM-1 metallo-beta-lactamase in Dis. 10, 597–602. M., Kim, H. D., Docquier, J.-D., et al. Nordmann, P.,and Seifert, H. (2009). Pseudomonas aeruginosa clinical iso- Kus, J. V., Tadros, M., Simor, A., Low, (2005). Novel acquired metallo-beta- OXA-143, a novel carbapenem- lates from Serbia. Antimicrob. Agents D. E., Mcgeer, A. J., Willey, B. M., lactamase gene, blaSIM-1, in a class hydrolyzing class D beta-lactamase in Chemother. 55, 3929–3931. et al. (2011). New Delhi metallo- 1 integron from Acinetobacter bau- Acinetobacter baumannii. Antimicrob. Kaase, M., Nordmann, P., Wichelhaus, beta-lactamase-1: local acquisition mannii clinical isolates from Korea. Agents Chemother. 53, 5035–5038. T. A., Gatermann, S. G., Bonnin, R. in Ontario, Canada, and challenges Antimicrob. Agents Chemother. 49, Hirsch, E. B., Ledesma, K. R., Chang, A., and Poirel, L. (2011). NDM-2 in detection. CMAJ 183, 1257– 4485–4491. K. T., Schwartz, M. S., Motyl, M. carbapenemase in Acinetobacter bau- 1261. Levasseur, P., Girard, A. M., Claudon, R., and Tam, V. H. (2012). In vitro mannii from Egypt. J. Antimicrob. Landman, D., Georgescu, C., Martin, M., Goossens, H., Black, M. T., activity of MK-7655, a novel beta- Chemother. 66, 1260–1262. D.A., and Quale, J. (2008). Polymyx- Coleman, K., et al. (2012). In lactamase inhibitor, in combination Karageorgopoulos, D. E., Wang, R., Yu, ins revisited. Clin. Microbiol. Rev. 21, vitro antibacterial activity of the with imipenem against carbapenem- X.-H., and Falagas, M. E. (2012). Fos- 449–465. ceftazidime–avibactam (NXL104) resistant Gram-negative bacteria. fomycin: evaluation of the published Landman, D., Kelly, P., Bäcker, M., combination against Pseudomonas Antimicrob. Agents Chemother. 56, evidence on the emergence of antimi- Babu, E., Shah, N., Bratu, S., et al. aeruginosa clinical isolates. Antimi- 3753–3757. crobial resistance in Gram-negative (2011). Antimicrobial activity of a crob. Agents Chemother. 56, Hirsch, E. B., and Tam, V. H. (2010). pathogens. J. Antimicrob. Chemother. novel aminoglycoside, ACHN-490, 1606–1608. Detection and treatment options 67, 255–268. against Acinetobacter baumannii and Li, J., Nation, R. L., Turnidge, for Klebsiella pneumoniae carbapen- Kassis-Chikhani, N., Decre, D., Gau- Pseudomonas aeruginosa from New J. D., Milne, R. W., Coulthard, emases (KPCs): an emerging cause tier, V., Burghoffer, B., Saliba, F., York Cit y. J. Antimicrob. Chemother. K., Rayner, C. R., et al. (2006a). of multidrug-resistant infection. J. Mathieu, D., et al. (2006). First out- 66, 332–334. Colistin: the re-emerging antibi- Antimicrob. Chemother. 65, 1119– break of multidrug-resistant Kleb- Lascols, C., Hackel, M., Marshall, S. H., otic for multidrug-resistant Gram- 1125. siella pneumoniae carrying blaVIM-1 Hujer, A. M., Bouchillon, S., Badal, negative bacterial infections. Lancet Hujer, K. M., Hujer, A. M., Hulten, E. and blaSHV-5 in a French university R., et al. (2011). Increasing preva- Infect. Dis. 6, 589–601. A., Bajaksouzian, S., Adams, J. M., hospital. J. Antimicrob. Chemother. lence and dissemination of NDM-1 Li, J., Rayner, C. R., Nation, R. L., Owen, Donskey, C. J., et al. (2006). Anal- 57, 142–145. metallo-β-lactamase in India: data R. J., Spelman, D., Tan, K. E., et al. ysis of antibiotic resistance genes in King, D. T., Worrall, L. J., Gruninger, from the SMART study (2009). J. (2006b). Heteroresistance to col- multidrug-resistant Acinetobacter sp. R., and Strynadka, N. C. (2012). New Antimicrob. Chemother. 66, 1992– istin in multidrug-resistant Acineto- isolates from military and civilian Delhi metallo-beta-lactamase: struc- 1997. bacter baumannii. Antimicrob. Agents patients treated at the Walter Reed tural insights into beta-lactam recog- Lascols, C., Peirano, G., Hackel, M., Chemother. 50, 2946–2950. Army Medical Center. Antimicrob. nition and inhibition. J. Am. Chem. Laupland, K. B., and Pitout, J. D. Livermore, D. M., Mushtaq, S., Mori- Agents Chemother. 50, 4114–4123. Soc. 134, 11362–11365. (2012). Surveillance and molecular naka, A., Ida, T., Maebashi, K., Ishii, Y., Eto, M., Mano, Y., Tateda, Kitchel, B., Rasheed, J. K., Endimi- epidemiology of Klebsiella pneumo- and Hope, R. (2013). Activity of K., and Yamaguchi, K. (2010). In ani, A., Hujer, A. M., Anderson, niae that produce carbapenemases; carbapenems with ME1071 (dis- vitro potentiation of carbapenems K. F., Bonomo, R. A., et al. (2010). the ﬁrst report of OXA-48-like odium 2,3-diethylmaleate) against

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 78

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 13 — #13 Patel and Bonomo The global emergence of carbapenemases

Enterobacteriaceae and Acinetobacter Infect. Control Hosp. Epidemiol. 33, Morfin-Otero, R., Rodriguez-Noriega, Antimicrob. Agents Chemother. 38, spp. with carbapenemases, includ- 817–830. E., Deshpande, L. M., Sader, H. S., 1262–1270. ing NDM enzymes. J. Antimicrob. Marchaim, D., Chopra, T., Perez, and Castanheira, M. (2009). Dis- Navon-Venezia, S., Leavitt, A., and Chemother. 68, 153–158. F., Hayakawa, K., Lephart, P. R., semination of a bla(VIM-2)-carrying Carmeli, Y. (2007). High tige- Livermore, D. M., Mushtaq, S., Bheemreddy, S., et al. (2011). Out- integron among Enterobacteriaceae cycline resistance in multidrug- and Warner, M. (2010). Activ- comes and genetic relatedness of species in Mexico: report from the resistant Acinetobacter baumannii. J. ity of BAL30376 (monobactam carbapenem-resistant enterobacteri- SENTRY Antimicrobial Surveillance Antimicrob. Chemother. 59, 772–774. BAL19764 + BAL29880 + clavu- aceae at Detroit medical center. Program. Microb. Drug Resist. 15, Navon-Venezia, S., Leavitt, A., lanate) versus Gram-negative Infect. Control Hosp. Epidemiol. 32, 33–35. Schwaber, M. J., Rasheed, J. K., Srini- bacteria with characterized resis- 861–871. Mostachio, A. K., Levin, A. S., Rizek, vasan, A., Patel, J. B., et al. (2009). tance mechanisms. J. Antimicrob. Mataseje, L. F., Bryce, E., Roscoe, D., C., Rossi, F., Zerbini, J., and Costa, First report on a hyperepidemic clone Chemother. 65, 2382–2395. Boyd, D. A., Embree, J., Gravel, S. F. (2012). High prevalence of of KPC-3-producing Klebsiella pneu- Livermore, D. M., Mushtaq, S., Warner, D., et al. (2012). Carbapenem- OXA-143 and alteration of outer moniae in Israel genetically related M., Miossec, C., and Woodford, N. resistant Gram-negative bacilli in membrane proteins in carbapenem- to a strain causing outbreaks in the (2008). NXL104 combinations ver- Canada 2009–10: results from the resistant Acinetobacter spp. isolates in United States. Antimicrob. Agents sus Enterobacteriaceae with CTX-M Canadian Nosocomial Infection Brazil. Int. J. Antimicrob. Agents 39, Chemother. 53, 818–820. extended-spectrum beta-lactamases Surveillance Program (CNISP). J. 396–401. Nordmann, P., Boulanger, A. E., and and carbapenemases. J. Antimicrob. Antimicrob. Chemother. 67, 1359– Moubareck, C., Bremont, S., Conroy, Poirel, L. (2012a). NDM-4 metallo- Chemother. 62, 1053–1056. 1367. M. C., Courvalin, P., and Lambert, beta-lactamase with increased car- Livermore, D. M., Mushtaq, S., Warner, Mathers, A. J., Cox, H. L., Kitchel, T. (2009). GES-11, a novel integron- bapenemase activity from Escherichia M., Zhang, J.-C., Maharjan, S., B., Bonatti, H., Brassinga, A. K., associated GES variant in Acineto- coli. Antimicrob. Agents Chemother. Doumith, M., et al. (2011a). Activ- Carroll, J., et al. (2011). Molec- bacter baumannii. Antimicrob. Agents 56, 2184–2186. ity of aminoglycosides, including ular dissection of an outbreak of Chemother. 53, 3579–3581. Nordmann, P., Couard, J.-P., Sansot, D., ACHN-490, against carbapenem- carbapenem-resistant enterobacteri- Mugnier, P. D., Poirel, L., Naas, T., and Poirel, L. (2012b). Emergence of resistant Enterobacteriaceae isolates. aceae reveals intergenus KPC car- and Nordmann, P. (2010). World- an autochthonous and community- J. Antimicrob. Chemother. 66, 48–53. bapenemase transmission through wide dissemination of the blaOXA-23 acquired NDM-1–producing Kleb- Livermore, D. M., Mushtaq, S., Warner, a promiscuous plasmid. MBio 2, carbapenemase gene of Acinetobac- siella pneumoniae in Europe. Clin. M., Zhang, J., Maharjan, S., Doumith, e00204–e00211. ter baumannii. Emerg. Infect. Dis. 16, Infect. Dis. 54, 150–151. M., et al. (2011b). Activities of Mathers, A. J., Hazen, K. C., Carroll, J., 35–40. Nordmann, P., Gniadkowski, M., Giske, NXL104 combinations with cef- Yeh, A. J., Cox, H. L., Bonomo, R. Mushtaq, S., Irfan, S., Sarma, J. B., C. G., Poirel, L., Woodford, N., and tazidime and aztreonam against A., et al. (2012). First clinical cases Doumith, M., Pike, R., Pitout, J., Miriagou, V. (2012c). Identification carbapenemase-producing Enter- of OXA-48 producing carbapenem et al. (2011). Phylogenetic diversity and screening of carbapenemase- obacteriaceae. Antimicrob. Agents resistant Klebsiella pneumoniae in the of Escherichia coli strains produc- producing Enterobacteriaceae. Clin. Chemother. 55, 390–394. United States: the “menace” arrives in ing NDM-type carbapenemases. J. Microbiol. Infect. 18, 432–438. Livermore, D. M., Walsh, T. R., Tole- the New World. J. Clin. Microbiol. 51, Antimicrob. Chemother. 66, 2002– Nordmann, P., Cuzon, G., and Naas, man, M., and Woodford, N. (2011c). 680–683. 2005. T. (2009). The real threat of Kleb- Balkan NDM-1: escape or transplant? Meletis, G., Tzampaz, E., Sianou, Mushtaq, S., Warner, M., and Liv- siella pneumoniae carbapenemase- Lancet Infect. Dis. 11, 164. E., Tzavaras, I., and Sofianou, ermore, D. (2010a). Activity of producing bacteria. Lancet Infect. Dis. Livermore, D. M., Warner, M., Mush- D. (2011). Colistin heteroresistance the siderophore monobactam 9, 228–236. taq, S., Doumith, M., Zhang, J., and in carbapenemase-producing Kleb- BAL30072 against multiresistant Nordmann, P., Mariotte, S., Naas, Woodford, N. (2011d). What remains siella pneumoniae. J. Antimicrob. non-fermenters. J. Antimicrob. T., Labia, R., and Nicolas, M. against carbapenem-resistant Enter- Chemother. 66, 946–947. Chemother. 65, 266–270. H. (1993). Biochemical properties obacteriaceae? Evaluation of chlo- Mendes, R. E., Bell, J. M., Turnidge, J. Mushtaq, S., Warner, M., and Liv- of a carbapenem-hydrolyzing beta- ramphenicol, ciprofloxacin, colistin, D., Castanheira, M., and Jones, R. N. ermore, D. M. (2010b). In vitro lactamase from Enterobacter cloa- fosfomycin, minocycline, nitrofuran- (2009). Emergence and widespread activity of ceftazidime + NXL104 cae and cloning of the gene into toin, temocillin and tigecycline. Int. J. dissemination of OXA-23, -24/40 against Pseudomonas aeruginosa and Escherichia coli. Antimicrob. Agents Antimicrob. Agents 37, 415–419. and -58 carbapenemases among other non-fermenters. J. Antimicrob. Chemother. 37, 939–946. Lolans, K., Rice, T. W., Munoz-Price, L. Acinetobacter spp. in Asia-Pacific Chemother. 65, 2376–2381. Nordmann, P., Poirel, L., Walsh, T. S., and Quinn, J. P. (2006). Multic- nations: report from the SENTRY Mushtaq, S., Warner, M., Williams, R., and Livermore, D. M. (2011). ity outbreak of carbapenem-resistant Surveillance Program. J. Antimicrob. G., Critchley, I., and Liver- The emerging NDM carbapene- Acinetobacter baumannii isolates pro- Chemother. 63, 55–59. more, D. M. (2010c). Activity mases. Trends Microbiol. 19, 588–595. ducing the carbapenemase OXA-40. Michalopoulos, A., Virtzili, S., Rafai- of chequerboard combinations of Noskin, G. A. (2005). Tigecycline: a new Antimicrob. Agents Chemother. 50, lidis, P., Chalevelakis, G., Damala, ceftaroline and NXL104 versus β- glycylcycline for treatment of seri- 2941–2945. M., and Falagas, M. E. (2010). Intra- lactamase-producing Enterobacteri- ous infections. Clin. Infect. Dis. 41, Lopez-Otsoa, F., Gallego, L., Towner, venous fosfomycin for the treatment aceae. J. Antimicrob. Chemother. 65, S303–S314. K. J., Tysall, L., Woodford, N., and of nosocomial infections caused 1428–1432. Osano, E., Arakawa, Y., Wacharo- Livermore, D. M. (2002). Endemic by carbapenem-resistant Klebsiella Naas, T., Cuzon, G., Villegas, M.-V.,Lar- tayankun, R., Ohta, M., Horii, T., carbapenem resistance associated pneumoniae in critically ill patients: a tigue, M.-F., Quinn, J. P., and Nord- Ito, H., et al. (1994). Molecular with OXA-40 carbapenemase among prospective evaluation. Clin. Micro- mann, P.(2008). Genetic structures at characterization of an enterobacte- Acinetobacter baumannii isolates biol. Infect. 16, 184–186. the origin of acquisition of the beta- rial metallo beta-lactamase found in a from a hospital in Northern Spain. J. Miriagou, V.,Tzouvelekis, L. S., Rossiter, lactamase blaKPC gene. Antimicrob. clinical isolate of Serratia marcescens Clin. Microbiol. 40, 4741–4743. S., Tzelepi, E., Angulo, F. J., and Agents Chemother. 52, 1257–1263. that shows imipenem resistance. Marchaim, D., Chopra, T., Bhargava, Whichard, J. M. (2003). Imipenem Naas, T., Vandel, L., Sougakoff, W., Liv- Antimicrob. Agents Chemother. 38, A., Bogan, C., Dhar, S., Hayakawa, resistance in a Salmonella clinical ermore, D. M., and Nordmann, P. 71–78. K., et al. (2012). Recent exposure strain due to plasmid-mediated class (1994). Cloning and sequence anal- Osterblad, M., Kirveskari, J., Hakanen, to antimicrobials and carbapenem- A carbapenemase KPC-2. Antimi- ysis of the gene for a carbapenem- A. J., Tissari, P.,Vaara, M., and Jalava, resistant Enterobacteriaceae: the crob. Agents Chemother. 47, 1297– hydrolyzing class A beta-lactamase, J. (2012). Carbapenemase-producing role of antimicrobial stewardship. 1300. Sme-1, from Serratia marcescens S6. Enterobacteriaceae in Finland: the

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 79

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 14 — #14 Patel and Bonomo The global emergence of carbapenemases

ﬁrst years (2008–11). J. Antimicrob. baumannii and Klebsiella pneumoniae the carbapenemase OXA-48. Antimi- Poirel, L., Naas, T., Nicolas, D., Chemother. 67, 2860–2864. across a hospital system: impact of crob. Agents Chemother. 56, 559–562. Collet, L., Bellais, S., Cavallo, Page, M. G., Dantier, C., and post-acute care facilities on dissemi- Poirel, L., Potron, A., and Nordmann, J.-D., et al. (2000b). Characteri- Desarbre, E. (2010). In vitro nation. J. Antimicrob. Chemother. 65, P. (2012c). OXA-48-like carbapen- zation of VIM-2, a carbapenem- properties of BAL30072, a novel 1807–1818. emases: the phantom menace. J. hydrolyzing metallo-beta-lactamase siderophore sulfactam with activ- Perez, F., Hujer, A. M., Hulten, E. Antimicrob. Chemother. 67, 1597– and its plasmid- and integron-borne ity against multiresistant Gram- A., Fishbain, J., Hujer, K. M., Aron, 1606. gene from a Pseudomonas aeruginosa negative bacilli. Antimicrob. Agents D., et al. (2010b). Antibiotic resis- Poirel, L., Bonnin, R. A., and Nord- clinical isolate in France. Antimicrob. Chemother. 54, 2291–2302. tance determinants in Acinetobacter mann, P. (2011a). Analysis of the Agents Chemother. 44, 891–897. Page, M. G., Dantier, C., Desar- spp and clinical outcomes in patients resistome of a multidrug-resistant Poirel, L., Marqué, S., Héritier, C., bre, E., Gaucher, B., Gebhardt, K., from a major military treatment facil- NDM-1-producing Escherichia coli Segonds, C., Chabanon, G., and and Schmitt-Hoffmann, A. (2011). ity. Am. J. Infect. Control 38, 63–65. strain by high-throughput genome Nordmann, P. (2005). OXA-58, a In vitro and in vivo properties of Perez, F., Hujer, A. M., Hujer, K. M., sequencing. Antimicrob. Agents novel class D beta-lactamase involved BAL30376, a beta-lactam and dual Decker, B. K., Rather, P. N., and Chemother. 55, 4224–4229. in resistance to carbapenems in beta-lactamase inhibitor combina- Bonomo, R. A. (2007). Global chal- Poirel, L., Castanheira, M., Carrer, Acinetobacter baumannii. Antimicrob. tion with enhanced activity against lenge of multidrug-resistant Acineto- A., Rodriguez, C. P., Jones, R. Agents Chemother. 49, 202–208. Gram-negative bacilli that express bacter baumannii. Antimicrob. Agents N., Smayevsky, J., et al. (2011b). Poirel, L., and Nordmann, P. (2006). multiple beta-lactamases. Antimi- Chemother. 51, 3471–3484. OXA-163, an OXA-48-related class Carbapenem resistance in Acineto- crob. Agents Chemother. 55, 1510– Pfeifer, Y., Schlatterer, K., Engelmann, D beta-lactamase with extended bacter baumannii: mechanisms and 1519. E., Schiller, R. A., Frangenberg, H. R., activity toward expanded-spectrum epidemiology. Clin. Microbiol. Infect. Palepou, M. F., Woodford, N., Hope, Stiewe, D., et al. (2012). Emergence cephalosporins. Antimicrob. Agents 12, 826–836. R., Colman, M., Glover, J., Kauf- of OXA-48-type carbapenemase- Chemother. 55, 2546–2551. Poirel, L., Weldhagen, G. F., De mann, M., et al. (eds). (2005). “Novel producing Enterobacteriaceae in Poirel, L., Dortet, L., Bernabeu, S., and Champs, C., and Nordmann, P. class A carbapenemase, KPC-4, in German hospitals. Antimicrob. Agents Nordmann, P. (2011c). Genetic fea- (2002). A nosocomial outbreak of an Enterobacter isolate from Scot- Chemother. 56, 2125–2128. tures of blaNDM-1-positive Enter- Pseudomonas aeruginosa isolates land, abstr. 1134_01_20,” in Program Pfeifer, Y., Wilharm, G., Zander, E., obacteriaceae. Antimicrob. Agents expressing the extended-spectrum β- and Abstracts of the 15th European Wichelhaus, T. A., Göttig, S., Hun- Chemother. 55, 5403–5407. lactamase GES-2 in South Africa. Congress of Clinical Microbiology and feld, K.-P., et al. (2011a). Molecular Poirel, L., Fortineau, N., and Nord- J. Antimicrob. Chemother. 49, Infectious Diseases, Copenhagen. characterization of blaNDM-1 in an mann, P. (2011d). International 561–565. Papp-Wallace, K. M., Bethel, C. R., Dis- Acinetobacter baumannii strain iso- transfer of NDM-1-producing Poirel, L., Wenger, A., Bille, J., Bern- tler, A. M., Kasuboski, C., Taracila, lated in Germany in 2007. J. Antimi- Klebsiella pneumoniae from Iraq abeu, S., Naas, T., and Nordmann, M., and Bonomo, R. A. (2010). crob. Chemother. 66, 1998–2001. to France. Antimicrob. Agents P. (2007). SME-2-producing Serratia Inhibitor resistance in the KPC-2 Pfeifer, Y., Witte, W., Holfelder, Chemother. 55, 1821–1822. marcescens isolate from Switzerland. beta-lactamase, a preeminent prop- M., Busch, J., Nordmann, P., Poirel, L., Ros, A., Carrer, A., Fortineau, Antimicrob. Agents Chemother. 51, erty of this class A beta-lactamase. and Poirel, L. (2011b). NDM-1- N., Carricajo, A., Berthelot, P., et al. 2282–2283. Antimicrob. Agents Chemother. 54, producing Escherichia coli in Ger- (2011e). Cross-border transmission Pollini, S., Maradei, S., Pecile, P., 890–897. many. Antimicrob. Agents Chemother. of OXA-48-producing Enterobacter Olivo, G., Luzzaro, F., Docquier, Patel, G., and Bonomo, R. A. (2011). 55, 1318–1319. cloacae from Morocco to France. J. J. D., et al. (2012). FIM-1, a Status report on carbapenemases: Poirel, L., Al Maskari, Z., Al Rashdi, Antimicrob. Chemother. 66, 1181– new acquired metallo-beta-lactamase challenges and prospects. Expert Rev. F., Bernabeu, S., and Nordmann, 1182. from a Pseudomonas aeruginosa clin- Anti Infect. Ther. 9, 555–570. P. (2010a). NDM-1-producing Kleb- Poirel, L., Héritier, C., and Nordmann, ical isolate from Italy. Antimicrob. Patel, G., Huprikar, S., Factor, S. H., siella pneumoniae isolated in the P. (2004a). Chromosome-encoded Agents Chemother. 57, 410–416. Jenkins, S. G., and Calfee, D. P. Sultanate of Oman. J. Antimicrob. ambler class D beta-lactamase of She- Potron, A., Kalpoe, J., Poirel, L., (2008). Outcomes of carbapenem- Chemother. 66, 304–306. wanella oneidensis as a progenitor and Nordmann, P. (2011a). Euro- resistant Klebsiella pneumoniae infec- Poirel, L., Naas, T., and Nord- of carbapenem-hydrolyzing oxacilli- pean dissemination of a single OXA- tion and the impact of antimicrobial mann, P. (2010b). Diversity, epi- nase. Antimicrob. Agents Chemother. 48-producing Klebsiella pneumoniae and adjunctive therapies. Infect. Con- demiology, and genetics of class D 48, 348–351. clone. Clin. Microbiol. Infect. 17, trol Hosp. Epidemiol. 29, 1099–1106. beta-lactamases. Antimicrob. Agents Poirel, L., Héritier, C., and Nordmann, E24–E26. Paterson, D. L., and Bonomo, R. Chemother. 54, 24–38. P. (2004b). Chromosome-encoded Potron, A., Poirel, L., and Nordmann, A. (2005). Extended-spectrum beta- Poirel, L., Rodriguez-Martinez, J.- ambler class D β-lactamase of She- P. (2011b). Origin of OXA-181, an lactamases: a clinical update. Clin. M., Al Naiemi, N., Debets- wanella oneidensis as a progenitor emerging carbapenem-hydrolyzing Microbiol. Rev. 18, 657–686. Ossenkopp, Y. J., and Nordmann, P. of carbapenem-hydrolyzing oxacilli- oxacillinase, as a chromosomal gene Paton, R., Miles, R. S., Hood, J., and (2010c). Characterization of DIM- nase. Antimicrob. Agents Chemother. in Shewanella xiamenensis. Antimi- Amyes, S. G. (1993). ARI 1: beta- 1, an integron-encoded metallo- 48, 348–351. crob. Agents Chemother. 55, 4405– lactamase-mediated imipenem resis- beta-lactamase from a Pseudomonas Poirel, L., Héritier, C., Tolun, V., and 4407. tance in Acinetobacter baumannii. Int. stutzeri clinical isolate in the Nether- Nordmann, P. (2004c). Emergence Potron, A., Nordmann, P., and J. Antimicrob. Agents 2, 81–87. lands. Antimicrob. Agents Chemother. of oxacillinase-mediated resistance to Poirel, L. (2013). Characterization of Peirano, G., Schreckenberger, P. C., and 54, 2420–2424. imipenem in Klebsiella pneumoniae. OXA-204, a carbapenem-hydrolyzing Pitout, J. D. (2011). Characteristics Poirel, L., Bonnin, R. A., Boulanger, A., Antimicrob. Agents Chemother. 48, class D β-lactamase from Kleb- of NDM-1-producing Escherichia coli Schrenzel, J., Kaase, M., and Nord- 15–22. siella pneumoniae. Antimicrob. Agents isolates that belong to the successful mann, P. (2012a). Tn125-related Poirel, L., Le Thomas, I., Naas, Chemother. 57, 633–636. and virulent clone ST131. Antimi- acquisition of blaNDM-like genes T., Karim, A., and Nordmann, P. Pottumarthy, S., Moland, E. S., crob. Agents Chemother. 55, 2986– in Acinetobacter baumannii. Antimi- (2000a). Biochemical sequence anal- Juretschko, S., Swanzy, S. R., 2988. crob. Agents Chemother. 56, 1087– yses of GES-1, a novel class A Thomson, K. S., and Fritsche, Perez, F., Endimiani, A., Ray, A. 1089. extended-spectrum beta-lactamase, T. R. (2003). NmcA carbapenem- J., Decker, B. K., Wallace, C. Poirel, L., Bonnin, R. A., and Nord- and the class 1 integron In52 from hydrolyzing enzyme in Enterobacter J., Hujer, K. M., et al. (2010a). mann, P. (2012b). Genetic features Klebsiella pneumoniae. Antimicrob. cloacae in North America. Emerg. Carbapenem-resistant Acinetobacter of the widespread plasmid coding for Agents Chemother. 44, 622–632. Infect. Dis. 9, 999–1002.

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 80

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 15 — #15 Patel and Bonomo The global emergence of carbapenemases

Poudyal, A., Howden, B. P., Bell, J. M., existence of bla(IMP) allelic vari- monosulfactam BAL30072 against Snitkin, E. S., Zelazny, A. M., Gao, W., Owen, R. J., Turnidge, J. D., ants carried by gene cassettes of Acinetobacter baumannii. J. Antimi- Thomas, P. J., Stock, F., Pro- et al. (2008). In vitro pharmacody- different phylogeny. Antimicrob. crob. Chemother. 66, 867–873. gram, N. C. S., Henderson, D. namics of colistin against multidrug- Agents Chemother. 44, 1229–1235. Salabi, A. E., Toleman, M. A., Weeks, J., K., et al. (2012). Tracking a hospi- resistant Klebsiella pneumoniae. J. Rice, L. B., Carias, L. L., Hutton, Bruderer, T., Frei, R., and Walsh, T. R. tal outbreak of carbapenem-resistant Antimicrob. Chemother. 62, 1311– R. A., Rudin, S. D., Endimiani, (2010). First report of the metallo- Klebsiella pneumoniae with whole- 1318. A., and Bonomo, R. A. (2008). beta-lactamase SPM-1 in Europe. genome sequencing. Sci. Transl. Med. Prabaker, K., Lin, M. Y., McNally, M., The KQ element, a complex genetic Antimicrob. Agents Chemother. 4, 148ra116. Cherabuddi, K., Ahmed, S., Nor- region conferring transferable resis- 54, 582. Souli, M., Galani, I., Boukovalas, ris, A., et al. (2012). Transfer from tance to carbapenems, aminogly- Samuelsen, O., Naseer, U., Tofteland, S., Gourgoulis, M. G., Chrys- high-acuity long-term care facilities cosides, and fluoroquinolones in S., Skutlaberg, D. H., Onken, A., souli, Z., Kanellakopoulou, K., et al. is associated with carriage of Kleb- Klebsiella pneumoniae. Antimicrob. Hjetland, R., et al. (2009). Emer- (2011). In vitro interactions of siella pneumoniae carbapenemase- Agents Chemother. 52, 3427–3429. gence of clonally related Klebsiella antimicrobial combinations with fos- producing Enterobacteriaceae: a Robledo, I. E., Aquino, E. E., Sante, pneumoniae isolates of sequence type fomycin against KPC-2-producing multihospital study. Infect. Control M. I., Santana, J. L., Otero, D. M., 258 producing plasmid-mediated Klebsiella pneumoniae and protection Hosp. Epidemiol. 33, 1193–1199. Leon, C. F., et al. (2010). Detection of KPC carbapenemase in Norway and of resistance development. Antimi- Qi, Y.,Wei, Z., Ji, S., Du, X., Shen, P.,and KPC in Acinetobacter spp. in Puerto Sweden. J. Antimicrob. Chemother. crob. Agents Chemother. 55, 2395– Yu, Y. (2011). ST11, the dominant Rico. Antimicrob. Agents Chemother. 63, 654–658. 2397. clone of KPC-producing Klebsiella 54, 1354–1357. Satlin, M. J., Kubin, C. J., Blumenthal, Struelens, M. J., Monnet, D. L., Magio- pneumoniae in China. J. Antimicrob. Robledo, I. E., Moland, E. S., Aquino, J. S., Cohen, A. B., Furuya, E. Y., rakos, A. P., Santos O’Connor, F., Chemother. 66, 307–312. E. A., Vazquez, G. J., Sante, M. Wilson, S. J., et al. (2011). Compara- and Giesecke, J. (2010). New Delhi Queenan, A. M., Shang, W., Schreck- I., Bertran, J., et al. (2007). “First tive effectiveness of aminoglycosides, metallo-beta-lactamase 1-producing enberger, P., Lolans, K., Bush, report of a KPC-4 and CTX-M polymyxin B, and tigecycline for Enterobacteriaceae: emergence and K., and Quinn, J. (2006). SME-3, producing K. pneumoniae (KP) iso- clearance of carbapenem-resistant response in Europe. Euro Surveill. 15. a novel member of the Serra- lated from Puerto Rico, abstr. C2- Klebsiella pneumoniae from urine. pii: 19716. tia marcescens SME family of 1933,” in 47th Annual Interscience Antimicrob. Agents Chemother. 55, Sun, Y., Cai, Y., Liu, X., Bai, N., Liang, carbapenem-hydrolyzing beta- Conference on Antimicrobial Agents 5893–5899. B., and Wang, R. (2012). The emer- lactamases. Antimicrob. Agents and Chemotherapy (Washington, DC: Schwaber, M. J., Klarfeld-Lidji, S., gence of clinical resistance to tigecy- Chemother. 50, 3485–3487. American Society for Microbiology). Navon-Venezia, S., Schwartz, D., cline. Int. J. Antimicrob. Agents 41, Queenan, A. M., Torres-Viera, C., Robledo, I. E., Vázquez, G. J., Aquino, Leavitt, A., and Carmeli, Y. (2008). 110–116. Gold, H. S., Carmeli, Y., Eliopou- E. A., Moland, E. S., Santé, M. I., and Predictors of carbapenem-resistant Tasina, E., Haidich, A.-B., Kokkali, S., los, G. M., Moellering, R. C. Jr., Hanson, N. D. (2008). “A novel KPC Klebsiella pneumoniae acquisition and Arvanitidou, M. (2011). Effi- et al. (2000). SME-type carbapenem- variant, KPC-6, in a Klebsiella pneu- among hospitalized adults and effect cacy and safety of tigecycline for the hydrolyzing class A beta-lactamases moniae (Kp) isolated in Puerto Rico of acquisition on mortality. Antimi- treatment of infectious diseases: a from geographically diverse Serra- (PR), abstr. C2-3738,”in 48th Annual crob. Agents Chemother. 52, 1028– meta-analysis. Lancet Infect. Dis. 11, tia marcescens strains. Antimicrob. Interscience Conference on Antimicro- 1033. 834–844. Agents Chemother. 44, 3035–3039. bial Agents and Chemotherapy and Schwaber, M. J., Lev, B., Israeli, Tibbetts, R., Frye, J. G., Marschall, J., Qureshi, Z. A., Paterson, D. L., Poto- the 46th Annual Meeting of Infectious A., Solter, E., Smollan, G., Rubi- Warren, D., and Dunne, W. (2008). ski, B. A., Kilayko, M. C., Sandovsky, Diseases Society of America (Wash- novitch, B., et al. (2011). Contain- Detection of KPC-2 in a clinical iso- G., Sordillo, E., et al. (2012). Treat- ington, DC: American Society for ment of a country-wide outbreak late of Proteus mirabilis and first ment outcome of bacteremia due Microbiology). of carbapenem-resistant Klebsiella reported description of carbapene- to KPC-producing Klebsiella pneu- Rodriguez-Rojas, A., Couce, A., and pneumoniae in Israeli hospitals via mase resistance caused by a KPC moniae: superiority of combination Blazquez, J. (2010). Frequency of a nationally implemented interven- beta-lactamase in P. mirabilis. J. Clin. antimicrobial regimens. Antimicrob. spontaneous resistance to fosfomycin tion. Clin. Infect. Dis. 52, 848–855. Microbiol. 46, 3080–3083. Agents Chemother. 56, 2108–2113. combined with different antibiotics Scott, P., Deye, G., Srinivasan, A., Mur- Tillotson, G. (2010). Stimulating antibi- Rasheed, J. K., Biddle, J. W., Ander- in Pseudomonas aeruginosa. Antimi- ray, C., Moran, K., Hulten, E., et al. otic development. Lancet Infect. Dis. son, K. F., Washer, L., Chenoweth, C., crob. Agents Chemother. 54, 4948– (2007). An outbreak of multidrug- 10, 2–3. Perrin, J., et al. (2008). Detection of 4949. resistant Acinetobacter baumannii– Toleman, M. A., Simm, A. M., Murphy, the Klebsiella pneumoniae carbapene- Rodvold, K. A., Gotfried, M. H., Cwik, calcoaceticus complex infection in T. A., Gales, A. C., Biedenbach, D. mase type 2 carbapenem-hydrolyzing M., Korth-Bradley, J. M., Dukart, G., the US military health care system J., Jones, R. N., et al. (2002). Molec- enzyme in clinical isolates of Cit- and Ellis-Grosse, E. J. (2006). Serum, associated with military operations ular characterization of SPM-1, a robacter freundii and K. oxytoca car- tissue and body fluid concentrations in Iraq. Clin. Infect. Dis. 44, 1577– novel metallo-β-lactamase isolated in rying a common plasmid. J. Clin. of tigecycline after a single 100 mg 1584. Latin America: report from the SEN- Microbiol. 46, 2066–2069. dose. J. Antimicrob. Chemother. 58, Sekiguchi, J.-I., Morita, K., Kitao, T., TRY antimicrobial surveillance pro- Rasmussen, B., Bush, K., Keeney, 1221–1229. Watanabe, N., Okazaki, M., Miyoshi- gramme. J. Antimicrob. Chemother. D., Yang, Y., Hare, R., O’Gara, Rogers, B. A., Aminzadeh, Z., Akiyama, T., et al. (2008). KHM-1, 50, 673–679. C., et al. (1996). Characterization Hayashi, Y., and Paterson, D. L. a novel plasmid-mediated metallo- Toleman, M. A., Spencer, J., Jones, L., of IMI-1 beta-lactamase, a class (2011). Country-to-country transfer beta-lactamase from a Citrobacter and Walsh, T. R. (2012). blaNDM- A carbapenem- hydrolyzing enzyme of patients and the risk of multi- freundii clinical isolate. Antimi- 1 is a chimera likely constructed in from Enterobacter cloacae. Antimi- resistant bacterial infection. Clin. crob. Agents Chemother. 52, 4194– Acinetobacter baumannii. Antimicrob. crob. Agents Chemother. 40, 2080– Infect. Dis. 53, 49–56. 4197. Agents Chemother. 56, 2773–2776. 2086. Rossi, F. (2011). The challenges of Shahid, M., Sobia, F., Singh, A., Tumbarello, M., Viale, P., Viscoli, Riccio, M. L., Franceschini, N., Boschi, antimicrobial resistance in Brazil. Malik, A., Khan, H. M., Jonas, D., C., Trecarichi, E. M., Tumietto, F., L., Caravelli, B., Cornaglia, G., Clin. Infect. Dis. 52, 1138–1143. et al. (2009). Beta-lactams and beta- Marchese, A., et al. (2012). Predictors Fontana, R., et al. (2000). Characteri- Russo, T. A., Page, M. G. P., Beanan, J. lactamase-inhibitors in current- or of mortality in bloodstream infec- zation of the metallo-beta-lactamase M., Olson, R., Hujer, A. M., Hujer, potential-clinical practice: a compre- tions caused by Klebsiella pneumoniae determinant of Acinetobacter bau- K. M., et al. (2011). In vivo and hensive update. Crit. Rev. Microbiol. carbapenemase-producing K. pneu- mannii AC-54/97 reveals the in vitro activity of the siderophore 35, 81–108. moniae: importance of combination

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 48 | 81

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 16 — #16 Patel and Bonomo The global emergence of carbapenemases

therapy. Clin. Infect. Dis. 55, Klebsiella pneumoniae in Greece Wolter, D. J., Kurpiel, P. M., Woodford, of a new metallo-beta-lactamase 943–950. involving an ST11 clone. J. Antimi- N., Palepou, M. F.,Goering, R. V.,and gene, bla(NDM-1), and a novel ery- Urban, C., Bradford, P. A., Tuckman, crob. Chemother. 68, 84–88. Hanson, N. D. (2009). Phenotypic thromycin esterase gene carried on a M., Segal-Maurer, S., Wehbeh, Vourli, S., Giakkoupi, P., Miriagou, and enzymatic comparative analy- unique genetic structure in Klebsiella W., Grenner, L., et al. (2008). V., Tzelepi, E., Vatopoulos, A. sis of the novel KPC variant KPC-5 pneumoniae sequence type 14 from Carbapenem-resistant Escherichia C., and Tzouvelekis, L. S. (2004). and its evolutionary variants, KPC- India. Antimicrob. Agents Chemother. coli harboring Klebsiella pneumoniae Novel GES/IBC extended-spectrum 2 and KPC-4. Antimicrob. Agents 53, 5046–5054. carbapenemase β-lactamases associ- β-lactamase variants with carbapen- Chemother. 53, 557–562. Yong, D., Walsh, T. R., Bell, J., Ritchie, ated with long-term care facilities. emase activity in clinical enterobac- Woodford, N., Tierno, P. M. Jr., B., Pratt, R., and Toleman, M. A. Clin. Infect. Dis. 46, e127–e130. teria. FEMS Microbiol. Lett. 234, Young, K., Tysall, L., Palepou, M. (2007). “A novel subgroup metallo Urban, C., Mariano, N., and 209–213. F., Ward, E., et al. (2004). Outbreak β-lactamase,AIM-1, emerges in Pseu- Rahal, J. J. (2010). In vitro dou- Wachino, J., Doi, Y., Yamane, K., of Klebsiella pneumoniae producing a domonas aeruginosa from Australia ble and triple bactericidal activi- Shibata, N., Yagi, T., Kubota, T., new carbapenem-hydrolyzing class A abstr. C1-593,” in 47th Annual Inter- ties of doripenem, polymyxin B, et al. (2004). Molecular characteri- beta-lactamase, KPC-3, in a NewYork science Conference on Antimicrobial and rifampin against multidrug- zation of a cephamycin-hydrolyzing Medical Center. Antimicrob. Agents Agents and Chemotherapy (Washing- resistant Acinetobacter baumannii, and inhibitor-resistant class A beta- Chemother. 48, 4793–4799. ton, DC:American Society for Micro- Pseudomonas aeruginosa, Klebsiella lactamase, GES-4, possessing a single Woodford, N., Turton, J. F., and Liv- biology). pneumoniae, and Escherichia coli. G170S substitution in the omega- ermore, D. M. (2011). Multiresistant Yu, Y.-S., Du, X.-X., Zhou, Z.-H., Chen, Antimicrob. Agents Chemother. 54, loop. Antimicrob. Agents Chemother. Gram-negative bacteria: the role of Y.-G., and Li, L.-J. (2006). First isola- 2732–2734. 48, 2905–2910. high-risk clones in the dissemination of blaIMI-2 in an Enterobacter Vaara, M., Sader, H. S., Rhomberg, P. Wachino, J., Yoshida, H., Yamane, K., tion of antibiotic resistance. FEMS cloacae clinical isolate from China. R., Jones, R. N., and Vaara, T. (2012). Suzuki, S., Matsui, M., Yamagishi, T., Microbiol. Rev. 35, 736–755. Antimicrob. Agents Chemother. 50, Antimicrobial activity of the novel et al. (2011). SMB-1, a novel subclass Woodford, N., Zhang, J., Warner, M., 1610–1611. polymyxin derivative NAB739 tested B3 metallo-beta-lactamase, associ- Kaufmann, M. E., Matos, J., Mac- Zarrilli, R., Pournaras, S., Gian- against Gram-negative pathogens. J. ated with ISCR1 and a class 1 inte- donald, A., et al. (2008). Arrival nouli, M., and Tsakris, A. (2013). Antimicrob. Chemother. 68, 636–639. gron, from a carbapenem-resistant of Klebsiella pneumoniae producing Global evolution of multidrug- Vaara, M., Siikanen, O., Apa- Serratia marcescens clinical isolate. KPC carbapenemase in the United resistant Acinetobacter baumannii jalahti, J., Frimodt-Moller, N., Antimicrob. Agents Chemother. 55, Kingdom. J. Antimicrob. Chemother. clonal lineages. Int. J. Antimicrob. and Vaara, T. (2010). Suscepti- 5143–5149. 62, 1261–1264. Agents 41, 11–19. bility of carbapenemase-producing Walkty, A., Decorby, M., Lagace-Wiens, Yahav, D., Lador, A., Paul, M., and Zhanel, G. G., Lawson, C. D., Zelenitsky, strains of Klebsiella pneumoniae and P. R., Karlowsky, J. A., Hoban, D. Leibovici, L. (2011). Efficacy and S., Findlay, B., Schweizer, F., Adam, Escherichia coli to the direct antibac- J., and Zhanel, G. G. (2011). In safety of tigecycline: a systematic H., et al. (2012). Comparison of the terial activity of NAB739 and to vitro activity of ceftazidime com- review and meta-analysis. J. Antimi- next-generation aminoglycoside pla- the synergistic activity of NAB7061 bined with NXL104 versus Pseu- crob. Chemother. 66, 1963–1971. zomicin to gentamicin, tobramycin with rifampicin and clarithromycin. domonas aeruginosa isolates obtained Yang, Y. J., Wu, P. J., and Livermore, and amikacin. Expert Rev. Anti Infect. J. Antimicrob. Chemother. 65, 942– from patients in Canadian hospitals D. M. (1990). Biochemical charac- Ther. 10, 459–473. 945. (CANWARD 2009 study). Antimi- terization of a beta-lactamase that Zhang, H., and Hao, Q. (2011). Crystal van der Bij, A. K., and Pitout, J. crob. Agents Chemother. 55, 2992– hydrolyzes penems and carbapenems structure of NDM-1 reveals a com- D. (2012). The role of international 2994. from two Serratia marcescens isolates. mon β-lactam hydrolysis mechanism. travel in the worldwide spread of Walsh, T. R., Toleman, M. A., Poirel, L., Antimicrob. Agents Chemother. 34, FASEB J. 25, 2574–2582. multiresistant Enterobacteriaceae. J. and Nordmann, P. (2005). Metallo- 755–758. Antimicrob. Chemother. 67, 2090– beta-lactamases: the quiet before the Yau,W., Owen, R. J., Poudyal, A., Bell, J. Conflict of Interest Statement: The 2100. storm? Clin. Microbiol. Rev. 18, 306– M., Turnidge, J. D., Yu, H. H., et al. authors declare that the research was Verde, P. E., and Curcio, D. (2012). 325. (2009). Colistin hetero-resistance conducted in the absence of any com- Imbalanced mortality evidence for Walsh,T. R., Weeks, J., Livermore, D. M., in multidrug-resistant Acinetobacter mercial or financial relationships that tigecycline: 2011, the year of the and Toleman, M. A. (2011). Dissem- baumannii clinical isolates from the could be construed as a potential con- meta-analysis. Clin. Infect. Dis. 55, ination of NDM-1 positive bacteria Western Pacific region in the SEN- flict of interest. 471–472. in the New Delhi environment and TRY antimicrobial surveillance pro- Viau, R. A., Hujer, A. M., Marshall, S. its implications for human health: gramme. J. Infect. 58, 138–144. Received: 19 December 2012; paper pend- H., Perez, F., Hujer, K. M., Briceño, D. an environmental point prevalence Yigit, H., Queenan, A. M., Ander- ing published: 07 January 2013; accepted: F., et al. (2012). “Silent” dissemina- study. Lancet Infect. Dis. 11, 355–362. son, G. J., Domenech-Sanchez, A., 20 February 2013; published online: 14 tion of Klebsiella pneumoniae isolates Walther-Rasmussen, J., and Høiby, N. Biddle, J. W., Steward, C. D., March 2013. bearing K. pneumoniae carbapene- (2007). Class A carbapenemases. J. et al. (2001). Novel carbapenem- Citation: Patel G and Bonomo RA (2013) mase in a long-term care facility for Antimicrob. Chemother. 60, 470–482. hydrolyzing beta-lactamase, KPC-1, “Stormy waters ahead”: global emergence children and young adults in North- Wang, Y. F., and Dowzicky, M. J. from a carbapenem-resistant strain of carbapenemases. Front. Microbiol. east Ohio. Clin. Infect. Dis. 54, (2010). In vitro activity of tigecy- of Klebsiella pneumoniae. Antimi- 4:48. doi: 10.3389/fmicb.2013.00048 1314–1321. cline and comparators on Acineto- crob. Agents Chemother. 45, 1151– This article was submitted to Fron- Villegas, M. V., Lolans, K., Cor- bacter spp. isolates collected from 1161. tiers in Antimicrobials, Resistance and rea, A., Kattan, J. N., Lopez, patients with bacteremia and MIC Yigit, H., Queenan, A. M., Rasheed, Chemotherapy, a specialty of Frontiers in J. A., and Quinn, J. P. (2007). change during the Tigecycline Eval- J. K., Biddle, J. W., Domenech- Microbiology. First identification of Pseudomonas uation and Surveillance Trial, 2004 to Sanchez, A., Alberti, S., et al. (2003). Copyright © 2013 Patel and Bonomo. aeruginosa isolates producing a 2008. Diagn. Microbiol. Infect. Dis. 68, Carbapenem-resistant strain of Kleb- This is an open-access article distributed KPC-type carbapenem-hydrolyzing 73–79. siella oxytoca harboring carbapenem- under the terms of the Creative Commons beta-lactamase. Antimicrob. Agents Werneck, J. S., Picao, R. C., Girardello, hydrolyzing beta-lactamase KPC-2. Attribution License, which permits use, Chemother. 51, 1553–1555. R., Cayo, R., Marguti, V.,Dalla-Costa, Antimicrob. Agents Chemother. 47, distribution and reproduction in other Voulgari, E., Zarkotou, O., Ranellou, K., L., et al. (2011). Low prevalence of 3881–3889. forums, provided the original authors and Karageorgopoulos, D. E., Vrioni, G., blaOXA-143 in private hospitals in Yong, D., Toleman, M. A., Giske, C. source are credited and subject to any Mamali, V., et al. (2012). Outbreak of Brazil. Antimicrob. Agents Chemother. G., Cho, H. S., Sundman, K., Lee, copyright notices concerning any third- OXA-48 carbapenemase-producing 55, 4494–4495; author reply 4495. K., et al. (2009). Characterization party graphics etc.

www.frontiersin.org March 2013 | Volume 4 | Article 48 | 82

“fmicb-04-00048” — 2013/3/13 — 9:48 — page 17 — #17 REVIEW ARTICLE published: 12 March 2013 doi: 10.3389/fmicb.2013.00047 The multifaceted roles of antibiotics and antibiotic resistance in nature

Saswati Sengupta1, Madhab K. Chattopadhyay 2 and Hans-Peter Grossart 3,4* 1 Independent, Hyderabad, India 2 Centre for Cellular and Molecular Biology (Council of Scientiﬁc and Industrial Research), Hyderabad, India 3 Limnology of Stratiﬁed Lakes, Leibniz Institute of Freshwater Ecology and Inland Fisheries, Stechlin, Germany 4 Institute for Biochemistry and Biology, Potsdam University, Potsdam, Germany

Edited by: Antibiotics are chemotherapeutic agents, which have been a very powerful tool in the Fiona Walsh, Agroscope clinical management of bacterial diseases since the 1940s. However, benefits offered Changins-Wädenswil, Switzerland by these magic bullets have been substantially lost in subsequent days following the Reviewed by: widespread emergence and dissemination of antibiotic-resistant strains. While it is obvious Jun Lin, The University of Tennessee, USA that excessive and imprudent use of antibiotics significantly contributes to the emergence Eddie Cytryn, Agricultural Research of resistant strains, antibiotic resistance is also observed in natural bacteria of remote Organization, Volcani Agriculture places unlikely to be impacted by human intervention. Both antibiotic biosynthetic genes Research Center, Israel and resistance-conferring genes have been known to evolve billions of years ago, long *Correspondence: before clinical use of antibiotics. Hence it appears that antibiotics and antibiotics resistance Hans-Peter Grossart, Limnology of Stratified Lakes, Leibniz Institute of determinants have some other roles in nature, which often elude our attention because Freshwater Ecology and Inland of overemphasis on the therapeutic importance of antibiotics and the crisis imposed by Fisheries, Alte Fischerhuette 2, the antibiotic resistance in pathogens. In the natural milieu, antibiotics are often found to 16775 Stechlin, Germany. e-mail: [email protected] be present in sub-inhibitory concentrations acting as signaling molecules supporting the process of quorum sensing and biofilm formation. They also play an important role in the production of virulence factors and influence host–parasite interactions (e.g., phagocytosis, adherence to the target cell, and so on). The evolutionary and ecological aspects of antibiotics and antibiotic resistance in the naturally occurring microbial community are little understood. Therefore, the actual role of antibiotics in nature warrants in-depth investigations. Studies on such an intriguing behavior of the microorganisms promise insight into the intricacies of the microbial physiology and are likely to provide some lead in controlling the emergence and subsequent dissemination of antibiotic resistance. This article highlights some of the recent findings on the role of antibiotics and the genes that confer resistance to antibiotics in nature.

Keywords: antibiotics, sub-inhibitory concentration, quorum sensing, virulence, stress response, antibiotic resistance, antibiotic paradox

INTRODUCTION groups. Antibiotics target bacterial physiology and biochemistry, The term “antibiotics” was first coined by the American microbi- causing microbial cell death or the cessation of growth. A signifi- ologist Selman Waksman and his colleagues to describe chemical cant number of these antibiotics affect cell walls or membranes substances produced by microorganisms and having antagonis- (e.g., β-lactam and glycopeptides), while several others exert tic effects on the growth of other microorganisms. It excluded their antibacterial activity by targeting protein synthetic machin- synthetic antimicrobials (sulfur drugs) and biological products ery via interaction with ribosomal subunits and these include of non-microbial origin having antagonistic effects on bacteria. antibiotics such as macrolides, chloramphenicol, tetracycline, line- Though antibiotics were introduced into the clinical practice only zolid, and aminoglycosides. Other “mechanistic” groups include in the middle of the last century, the use of microorganisms for molecules which interfere with the nucleic acid synthesis [e.g., the management of microbial infections in ancient Egypt, Greece, fluoroquinolones (FQ) and rifampin], while some others exert China, and some other places of the world is well-documented. their effects by interfering with the metabolic pathways (e.g., The modern era of antibiotics started with the serendipitous dis- sulphonamides and folic acid analog) or by disruption of the bac- covery of penicillin from the culture filtrate of a fungus, Penicillium terial membrane structure (e.g., polymyxins, daptomycin, and notatum by Alexander Fleming in 1928 (Fleming, 1929). others). In the present scenario, antibiotics available in the market A surge of discovery of several such antibacterial and antifungal are either produced by microbial fermentation or are derived antibiotics accompanied with a new generation of semi-synthetic via semi-synthetic route using the existing antibiotic backbone drugs initially led to euphoria that any infectious disease could be structure. They are classified into different chemically defined successfully controlled using antibiotics. However, emergence and

www.frontiersin.org March 2013 | Volume 4 | Article 47 | 83

“fmicb-04-00047” — 2013/3/12 — 11:18 — page1—#1 Sengupta et al. Ecological role of antibiotics in nature

propagation of bacterial strains, resistant to almost all the thera- of the producer microorganisms that they use against other nat- peutically useful antibiotics during the past few decades revealed urally occurring cohabiting microorganisms and eliminate these the limitation of the wonder drugs. Though imprudent and exces- competing bacteria for the purpose of “empire building” in the sive use of antibiotics is highlighted as a major causative factor microbial community (Davies, 1990). The concept of the empire behind the setback, it is evident by this time that antibiotic resis- building role of antibiotics stemmed from the soil borne nature tance does not call for exposure of the organisms to antibiotics. of the antibiotic-producing microorganisms. In certain instances It is also found that genes involved in the biosynthesis of antibi- like plant–microbe interactions, the antibiotic-producing organ- otics and antibiotic resistance evolved thousands of years before isms have indeed been found to secrete antibiotics to eliminate the antibiotics were introduced into the clinical practice. Hence both competing bacteria in the vicinity. Pseudomonas fluorescens 2-79 antibiotic and its resistance determinants have some other role in (NRRL B-15132) colonizing the rhizosphere of wheat, secrete the bacterial physiology. antibiotic, phenazine which inhibits the growth of Gaeumanno- myces graminis var. tritici thus suppressing a major root and crown ROLE OF ANTIBIOTICS IN NATURE disease of wheat and barley (Thomashow and Weller, 1988). Few Antibiotics in the biosphere are produced by microorganisms as other ecological examples of probable antibiotic function of these secondary metabolites at a concentration much lower than the secondary metabolites in nature include the fungus-growing ants, therapeutic dose. Waksman was convinced that antibiotics play which carry an antibiotic-producing actinomycete (Pseudonocar- “no real part in modifying or influencing living processes that dia sp.) on their cuticle specifically for biocontrol of the fungal occur in nature” (Waksman, 1961) though there is evidence to garden parasite, Escovopsis sp. (Currie et al., 1999; Cafaro and the contrary (Gullberg et al., 2011). Antibiotics are produced in Currie, 2005). Another example is the antibiotic-producing strain the late stages of microbial stationary growth phase, decoupled Streptomyces diastatochromogenes strain PonSSII controlling the from the doubling time, implying that they are not indispens- growth of Streptomyces scabies strain RB4, the causative agent of able for sustenance of life of the producer organism. However, potato scab (Neeno-Eckwall et al., 2001). it is also a fact that the production of an antibiotic is a multi- step process that involves a number of genes. Hence it does not ROLE OF ANTIBIOTICS AS SIGNALING MOLECULES seem tenable that such a complex anabolic process has been sus- During primordial development, the cells instead of antagonizing tained through evolution without having any obvious purpose to each other remained in a communal harmony to share an evo- serve. Recent studies reveal that antibiotics do have some specific lutionary advantage and the symbiotic coexistence of microbes effects on the natural milieu of the microbes while they assume in biofilms, in lichens and in a metabolically quiescent state is an entirely different role as antibacterial agents in the dose used well-documented (Davies, 2006). Chemical communication, an in therapeutics. This dual role of antibiotics remains a riddle. essential feature in such mixed populations, helps the microor- In the natural habitat, the diverse microbial communities exist ganisms coordinate with each other in an orchestrated fashion. as a multi-cellular network. They constitute a huge reservoir of Probably the small molecule antibiotics act as a liaison in the com- different metabolic activities and have to evolve continuously to munication between the microbes. It has been postulated that encounter the constant environmental threat and different selec- antibiotics in the early stages of biochemical evolution had some tion pressure. Survival is most often a major challenge because of functions mediated by their interaction with primitive macro- the limiting nutrients. Under nutrient starvation, the organisms molecule receptors. Later on, many of these low molecular weight start secreting secondary metabolites, which are a diverse array interacting partners were employed to antagonize the original of low molecular weight organic molecules, known as the par- receptor sites in macromolecular structure thus imparting the vome (Davies, 2009). The total number of parvome detected so antibiosis property. Such a switchover in the role of the pri- far is at least an order of magnitude larger than the number of mordial effector molecule has been hypothesized in a schematic bacteria in the biosphere. Bacteria of the phylum Actinobacteria biochemical pathway by J. Davies (Davies, 1990). During the past (Ventura et al., 2007), a huge taxonomic group of diverse gen- seven decades, the scientists were mostly preoccupied with study- era with a high genomic guanine–cytosine (GC) content, produce ing the antibiotic property of these small molecules because of millions of complex bioactive small molecules. The ecological role its overwhelming success in pharmacotherapy. Only during the of the majority of these small molecules in nature remains largely recent past antibiotics were implicated in several other physi- unknown. Amongst these molecules, only a fraction has been iden- ological phenomena ranging from control of transcription and tified to have antibiotic activity and has been extensively studied translation to growth of the producer organism in the microbial only from the therapeutic point of view whereas the significance communities. From a broader perspective of evolutionary and of antibiotics in nature still remains a mystery. The prevail- ecological point of view, antibiotics, apart from having a growth ing confusion about its role in nature has been dealt by Julian inhibitory activity, appear to have a role in intra- and inter-domain Davis in an article titled: “are antibiotics naturally antibiotic?” communication in various ecosystems. Under normal physiologi- (Davies, 2006). cal condition, microorganisms in their marine and soil habitats produce antibiotics at sub-minimum inhibitory concentration ROLE OF ANTIBIOTICS IN INTERSPECIES COMPETITION (sub-MIC). Despite having evidence that resistant bacteria repre- Antibiotics are critical to the producer organisms in the natural senting a low fraction in a sample were enriched at sub-inhibitory environment as they are needed both for survival and competi- concentrations of three clinically useful antibiotics (Gullberg et al., tive advantage. Thus antibiotics are widely perceived as an arsenal 2011), antibiotics are believed not to display any antibiotic activity

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 47 | 84

“fmicb-04-00047” — 2013/3/12 — 11:18 — page2—#2 Sengupta et al. Ecological role of antibiotics in nature

at concentrations lower than 1/100 of the minimum inhibitory a broad range of antimicrobial activities against most Gram- concentrations (Davies, 2006). Instead, many antibiotics are negative and Gram-positive bacteria, fungi, enveloped viruses, known to possess biological activities other than the inhibitory role and eukaryotic parasites, also have a broad spectrum of pro- at such a low concentration and have major effects on global tran- inflammatory and anti-inflammatory effects on the host immune scription pattern. Antibiotics such as erythromycin (an inhibitor system. Apart from their antimicrobial activity, these molecules of translation) and rifampicin (an inhibitor of transcription), at also act as transcription modulators. Cecropin, an insect-derived low concentrations, could modulate global bacterial transcription cationic peptide, was shown to alter the transcription pattern of pattern in a random promoter library construct of Salmonella 26 genes when added to Escherichia coli (Hong et al., 2003). Some typhimurium having lux as the reporter gene. In response to the low cationic peptides of microbial- and mammalian origin (polymyx- concentration of different antibiotics, a variety of promoters were ins B and E, cattle indolicidin and human LL-37) were found to activated, including those involved in virulence, metabolic, and activate the two component pmrA–pmrB regulatory operon, which adaptive functions. However, transcription was markedly reduced regulates the resistance to polymyxin B and cationic antimicro- in antibiotic-resistant hosts but the mutants defective in stress bial peptides in Pseudomonas aeruginosa (McPhee et al., 2003). responses such as rec and lex (SOS), dnaJ and ΔdnaKJ (heat shock Inhibitory effect of several antibiotics on sporulation of bacteria response), and ΔrelA ΔspoT (universal stress response) could not is well-known (Ochi and Freese, 1983; Babakhani et al., 2012). prevent antibiotic-induced modulation of transcription pattern at sub-inhibitory concentration (Goh et al.,2002). Antibiotics having ROLE OF ANTIBIOTICS IN VIRULENCE different binding sites on the ribosome (chloramphenicol, amino- Some antibiotics, at concentrations below the MIC, are able to glycosides, macrolides, and tetracycline), or intruding into the modulate the expression of some genes associated with virulence. process of cell-wall synthesis (some β-lactams and fosfomycin) Up- and downregulation of the transcription of virulence and were reported to alter the transcription pattern under different motility genes were induced by the RNA polymerase inhibitor, circumstances. So it has been postulated that the transcription rifampicin (Yim et al., 2006b). Sub-inhibitory concentration of machinery must be capable of sensing these subtle variations in several other antibiotics such as metronidazole, vancomycin, clin- the conformation or stoichiometry of small molecule antibiotics damycin, and linezolid could induce the early transcription of to respond to specific up- or downregulation. tcdA (toxin A) and tcdB (toxin B), the major virulence factors- These small molecules have a significant role in the dynam- encoding genes of Clostridium difficile, a nosocomial pathogen ics of bacterial communities in nature, thus contributing to both responsible for diarrhea, during exponential phase growth of the competitive and interactive responses. Inhibition occurs when organism (Gerber et al., 2008). Imipenems, at sub-inhibitory con- high concentrations are attained, transcriptional changes occur centration, were shown to induce β-lactamase and production at low concentrations (Davies et al., 2006). This dose-dependent of alginate during biofilm formation in Pseudomonas aeruginosa. dual role, defined as hormesis, is not exclusively associated with Sub-inhibitory concentration of an aminoglycoside antibiotic antibiotics but also found in different other biomolecules (Davies tobramycin induced biofilm formation in Pseudomonas aerugi- et al., 2006; Calabrese, 2009). The dual role of antibiotics has been nosa and Escherichia coli (Hoffman et al.,2005). On the other hand, confirmed by the detection of a variety of bioactivities of several sub-inhibitory level of the macrolide antibiotic azithromycin was antibiotics. Some β-lactam antibiotics were shown to stimulate the shown to elicit the exactly opposite phenomenon like suppression glutamate transporter-1 (GLT1) both in cell culture and in animal of the alginate overproduction and biofilm formation in Pseu- models (Rothstein et al., 2005). Thiostrepton, a potent protein domonas aeruginosa (Ichimiya et al., 1996). These contradictory synthesis inhibitor which binds to 23S RNA, was found to act as a observations suggest that the molecular targets of different antibi- transcription inducer at a concentration lower than the inhibitory otics for the execution of their inhibitory effects could be similar concentration (Murakami et al., 1989). Lincomycin, which termi- but they might differ extensively while functioning as signaling nates the peptide bond elongation, could stabilize certain mRNAs molecules (Aminov, 2009). This postulation is substantiated by like the bla mRNA (Matsushita et al., 1989). Puromycin, in spite the fact that tobramycin at sub-inhibitory concentrations does of having a chain termination activity in polypeptide synthesis, not bind the ribosome (the usual target for its role as an antibi- can affect nucleic acid synthesis, mediated by its aminonucleo- otic), rather it targets the aminoglycoside response regulator gene sidemoiety(Yarmolinsky and Haba, 1959). Edeine, apart from its (arr) which is responsible for the induction of biofilm-specific inhibitory role in protein synthesis, has specific effects on nucleic aminoglycoside resistance. The gene is also predicted to encode acid synthesis (Kurylo-Borowska and Szer, 1972). Many of the so- an inner-membrane phosphodiesterase acting on the substrate, called antibiotics have been shown to have sex pheromone activity cyclic di-guanosine monophosphate (c-di-GMP), a bacterial sec- and found to stimulate bacterial conjugation by several orders ond messenger that regulates cell surface adhesiveness (Hoffman of magnitude (Evans and Dyke, 1988). Sub-MICs of antibiotics et al., 2005). are thus known to induce extensive transcriptional changes in The influence of sub-inhibitory doses of several antibiotics on bacteria (Tsui et al., 2004; Yim et al., 2006a). In the majority of the expression of the gene encoding the Staphylococcus aureus cases the underlying mechanisms of the different ecological role alpha-toxin (hla), a major virulence factor of the pathogen, of the antibiotics at sub-inhibitory concentration still remain to revealed that glycopeptide antibiotics had no effect whereas β- be elucidated. lactams induced a strong expression of hla. On the other hand, A second level of signaling via antibiotics has also been doc- the macrolide erythromycin and several aminoglycosides reduced, umented in different organisms. Cationic peptides, which have while FQs slightly stimulated hla expression and an almost

www.frontiersin.org March 2013 | Volume 4 | Article 47 | 85

“fmicb-04-00047” — 2013/3/12 — 11:18 — page3—#3 Sengupta et al. Ecological role of antibiotics in nature

complete inhibition of alpha-toxin expression was obtained using was demonstrated in Escherichia coli by quinolones (DNA- clindamycin. Furthermore, methicillin enhanced Hla produc- damaging antibiotics) induced emergence of drug resistance tion of both MSSA (methicillin-sensitive Staphylococcus aureus) (López et al., 2007). and MRSA (methicillin-resistant Staphylococcus aureus; Ohlsen Fluoroquinolones at sub-MIC have been shown to upregulate et al., 1998). The sub-inhibitory concentration of ceftazidime (β- both the SOS and the methyl mismatch repair (MMR) path- lactam) and ciprofloxacin (FQ) appeared to be effective in decreas- way genes in Staphylococcus aureus with different SOS response ing the expression of a range of quorum sensing (QS)-regulated and DNA repair-associated promoter-lux gene fusion construct virulence factors (Skindersoe et al., 2008). (Mesak et al., 2008). Upregulation of several genes involved in Genomic rearrangements in pathogenic strains of Escherichia the SOS response such as umuD, lexA, sbmC, and dinP was coli and Shigella following exposure to sub-inhibitory concentra- demonstrated in Salmonella using sub-inhibitory concentrations tions of kanamycin and ampicillin was demonstrated (Pedro et al., of quinolones. In addition, transcriptional regulators, genes puta- 2011). Some of the rearrangements may engender genotypes with tively associated with membrane integrity (spr), virulence (sicA), increased virulence. SOS response, induced by ciprofloxacin, was and metabolism (plsB) were also affected (Yim et al., 2011). On found to stimulate transfer of pathogenicity Island in Staphylo- the contrary, it has also been shown that exposure of Streptococ- coccus aureus (Ubeda et al., 2005). Thus antibiotic used for the cus pneumoniae to quinolones, aminoglycosides, and penicillin at treatment of clinical infections may lead to the dissemination of sub-inhibitory concentrations may result in increased mutability, virulence in some cases. without the apparent involvement of SOS-response components (Henderson-Begg et al., 2006; Cortes et al., 2008). ROLE OF ANTIBIOTICS IN HOST–PARASITE INTERACTION SOS response also enhances frequency of mutations. Hence Antibiotics have also been found to modulate host immune cell antibiotics which induce SOS response, contribute to the increase response, mostly mediated by cytokines. There are ample evi- in the frequency of resistant strains. Exposure to ciprofloxacin was dences of sub-inhibitory concentrations of antibiotics interference found to increase in the frequency of rifampicin-resistant mutants with processes of host–parasite interactions such as phagocytosis, of Streptococcus pneumoniae almost by fivefold (Henderson-Begg adherence, and virulence. Antibiotics promote phagocytosis by et al., 2006). Carbapenem resistance in Pseudomonas aeruginosa altering the surface properties of pathogens. Many clinical iso- was also found to be enhanced in presence of the same antibiotic lates of Enterococcus faecium are resistant to neutrophil-mediated almost in the same order (Tanimoto et al., 2008). phagocytosis but exposure of vancomycin-susceptible Enterococ- Thus low antibiotic concentration regulates the transcription cus faecium to quinupristin/dalfopristin at concentrations both of different genes in target bacteria while increasing higher con- at either sub-inhibitory or suprainhibitory concentrations pro- centration elicits a stress response and even higher concentration moted susceptibility to subsequent PMN (polymorphonuclear is lethal. Therefore, it is apparent that these small molecule at a leucocytes)-mediated phagocytosis. Vancomycin-resistant strains sub-inhibitory concentration participate in a new form of signal- on the contrary exhibited very little change in their binding toward ing network by binding to different cytoplasmic macromolecular PMNs after antibiotic pretreatment (Herrera-Insúa et al., 1997). receptors such as ribosomes, and those involved in DNA repli- Use of amoxicillin for the treatment of Haemophilus influenzae cation, RNA transcription machinery, and cell-wall biosynthesis in acute otitis media caused upregulation in the expression of (Ryan and Dow, 2008). These receptors were also identified earlier cytokines, interleukin (IL)-6, tumor necrosis factor-α, and IL-10 as the inhibitory targets for the bioactive molecules at higher con- (Melhus, 2001). centration. The cellular targets for the lethal and sub-inhibitory concentration sometimes also differ thus implying that antibi- ROLE OF ANTIBIOTICS IN SOS AND DNA REPAIR GENE EXPRESSION otics are multi-ligand-specific sensor molecules targeting dif- The SOS response, a regulatory network present in most bacte- ferent ligands in a concentration-dependent manner (Hoffman ria is induced in response to DNA damage and has been shown et al., 2005). to promote stress-induced mutagenesis, virulence, and dissemination of antibiotic resistance specifically by horizontal gene transfer ANTIBIOTICS AND QUORUM SENSING (HGT). Beaber et al. (2004) showed that ciprofloxacin induced Quorum sensing is a mode of bacterial communication in transfer of the Vibrio cholerae SXT integrative conjugative element response to release of chemical messenger which regulate bac- (ICE) was controlled by SOS through RecA-dependent cleavage terial gene expression (Bassler, 1999). Secondary metabolites are of the SXT encoded repressor SetR. SXT is a 100-kb ICE derived activated during slow growth in the stationary phase and trig- from V. cholerae that encodes genes for the resistance toward chlo- ger the transition from primary to secondary metabolism (Bibb, ramphenicol, sulfamethoxazole, trimethoprim, and streptomycin 2005). This transition is a complex process and involves many (Beaber et al., 2004). Even the induction of the CTX prophage signals, including those by small signaling molecules called γ- which encodes the cholera toxin is regulated by the SOS repres- butyrolactones (also known as autoregulatory factor or A-factor). sor LexA in conjunction with the phage repressor rstR which is These signaling molecules, found mainly in Streptomyces species, also regulated by LexA (Quinones et al., 2005). In V. cholerae, the are considered “bacterial hormones” or the QS factor. The QS recombination-mediated excision of integron cassette was induced signaling network initiates a set of metabolic changes leading to by SOS in response to different antibiotics (Guerin et al., 2009). a number of events including morphological differentiation and On the other hand, SOS-independent recombination between synthesis of secondary metabolites such as antibiotics. The γ- divergent sequences either by RecBCD or RecFOR pathways butyrolactones constitute the major group of QS molecules in

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 47 | 86

“fmicb-04-00047” — 2013/3/12 — 11:18 — page4—#4 Sengupta et al. Ecological role of antibiotics in nature

bacteria. The first γ-butyrolactone, A-factor, was identified in and its tetrameric acid degradation product in Pseudomonas aerug- Streptomyces griseus in 1967 (Hsiao et al., 2009). inosa have antibacterial properties against Gram-positive bacteria Biosynthesis of γ-butyrolactones is not well understood, but (Kaufmann et al., 2005). N-(3-oxododecanoyl) homoserine lac- seems to be mediated by γ-butyrolactone synthase AfsA. The A- tone and other 3-oxo series homoserine lactones with 8, 10, and factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactones), at a 14 length carbon chains were shown to inhibit growth of the certain critical concentration, binds to cytoplasmic receptor pro- Gram-positive Staphylococcus aureus (Qazi et al., 2006). tein, a dimer of ArpA,which primarily acts as repressors by binding to the promoter region of adpA (A-factor-responsive transcrip- ANTIBIOTIC RESISTANCE, THE TIP OF THE ICEBERG tional activator, a key pleiotropic regulator). The binding of the In the late 1960s, the unprecedented successes of early antibiotic A factor dissociates ArpA from the promoter, thus facilitating therapies led US Surgeon General William H. Stewart to make the the transcription and translation of adpA. Subsequently, AdpA famous declaration: “it is time to close the book on infectious dis- binds an upstream activation sequence to initiate the transcrip- eases and declare the war against pestilence won” (Spellberg et al., tion of strR (a pathway-specific regulatory gene responsible for 2008). The euphoria did not last long and the magic bullets started transcription of other streptomycin biosynthetic genes cluster). losing the efficacy because of the steady emergence of antibiotic- strR is a pathway-specific regulator which acts as a transcriptional resistant pathogens simultaneously with their widespread use. activator and induces transcription of most of the streptomycin Following the success of penicillin in controlling bacterial infec- biosynthetic genes by binding multiple sites in the gene cluster tion among the soldiers during the Second World War, emergence (Retzlaff and Distler, 1995). Thus onset of streptomycin biosyn- of penicillin-resistant strain was evidenced in the 1940s. By 1960, thesis is initiated by the QS factor, γ-butyrolactones. The gene it assumed the shape of a pandemic problem. New β-lactam aphD, encoding a major streptomycin resistance determinant antibiotics were introduced into the clinical practice to restrain streptomycin-6-phosphotransferase, is also under the control of the problem. Simultaneously bacterial strains resistant to them the adpA-dependent promoter. The binding to γ-butyrolactones came into being, a phenomenon dubbed as β-lactamase cycle. The thus induces expression of the target genes (transcription fac- first case of MRSA was identified in UK in 1961 (Johnson, 2011) tors) many of which are involved in regulation of specific and the first report on MRSA in the United States came in 1968. antibiotic biosynthesis clusters. Onset of streptomycin biosyn- Now it is prevalent all over the world. MRSA is actually resistant thesis is initiated by the QS factor, γ-butyrolactones (Retzlaff to the entire class of penicillin-like antibiotics called β-lactams. and Distler, 1995). The expression of the gene aphD, encoding Presence of an enzyme called New Delhi metallo-β-lactamase a major streptomycin resistance determinant streptomycin-6- (NDM-1) in some Gram-negative bacteria (notably Escherichia phosphotransferase, is also a QS-mediated event. Each receptor coli and Klebsiella pneumoniae) makes them resistant to virtually protein is highly specific for its cognate γ-butyrolactone. In all β-lactams including carbapenems, which are most often consid- Erwinia carotovora carbapenem biosynthesis is regulated by a clas- ered the last line of defense against multidrug-resistant pathogens sical autoinducer N-(3-oxohexanoyl)-L-homoserine. In another (Kumarasamy et al., 2010). Isolation of bacterial strains producing cephamycin C-producing bacterium, Streptomyces clavuligerus, extended spectrum β-lactamase (ESBL) resistant to third genera- antibiotic synthesis is regulated at the primary regulatory level tion of cephalosporins and monobactams is also reported from by γ-butyrolactone (Liras et al., 2008). time to time (Kurihara et al., 2013). Some 10–11 probable γ-butyrolactone synthates, all from The glycopeptide antibiotic vancomycin was introduced in clin- the Streptomyces genus were found while 37–42 putative γ-but- ical practice in 1958 for the treatment of methicillin resistance yrolactone receptors were found in the genome of different other in both Staphylococcus aureus and coagulase-negative staphylo- bacteria apart from Streptomyces. Thus, it is believed that many of cocci. Vancomycin resistance was so difficult to induce that it was the cellular activities including the antibiotic production is under believed to be very unlikely to occur in a clinical setting. Until the the control of a well-orchestrated QS signaling network mediated late 1980s, the glycopeptide antibiotic vancomycin was considered by a small population of γ-butyrolactone producers acting as a the drug of last resort for treatment of diseases caused by Gram- signaling factor for a diverse population of signal recipient positive bacteria such as Enterococcus faecalis, MRSA, Streptococcus (Aminov, 2009). pneumoniae, and Clostridium difficile (Cunha, 1995). However, Structural similarity between antibiotic and intracellular sig- vancomycin resistance was first reported in coagulase-negative naling molecule has also been evidenced in several cases. The staphylococci in 1979 and 1983 (Srinivasan et al., 2002). At present quinolones produced by Pseudomonas aeruginosa have a wide vancomycin-resistant enterococci (VRE) pose a major chal- range of activities starting from antibiotics to autoinducers. 2- lenge in therapeutics. Multidrug-resistant tuberculosis (caused Heptyl-3-hydroxy-4-quinolone (pseudomonas quinolone signal; by Mycobacterium tuberculosis) has assumed an epidemic pro- PQS), belonging to the family of 2-alkyl-4-quinolones (AQs), was portion in some parts of the world. Multidrug-resistant strains previously described for their antimicrobial activities. Later on, it of M. tuberculosis, now known to be present in 50 countries, was found that PQS is integrated within an intricate QS circuit and heighten the threat posed by untreatable and fatal human tuber- plays an important role in Pseudomonas aeruginosa pathogenesis culosis. by regulating the production of diverse virulence factors including Because of the rapid dissemination of antibiotic resistance in elastase, pyocyanin, and LecA lectin in addition to affecting biofilm pathogens, many of the antibiotics, which were highly effective ear- formation (Dubern and Diggle, 2008; Heeb et al., 2011). Several lier, became obsolete during the past few decades. The efficacy of other autoinducers like N-(3-oxododecanoyl) homoserine lactone antibiotic treatment is on the wane as a result of the emergence and

www.frontiersin.org March 2013 | Volume 4 | Article 47 | 87

“fmicb-04-00047” — 2013/3/12 — 11:18 — page5—#5 Sengupta et al. Ecological role of antibiotics in nature

dissemination of antibiotic resistance amongst the pathogens. This The World Health Organization (WHO) has long recognized warrants the discovery of newer and more promising antibiotics anti microbial resistance (AMR) as a growing global health threat, with long-term efficacy against various life threatening infections. and the World Health Assembly, through several resolutions over However, the steady discovery of antibiotics witnessed during two decades, has called upon Member States and the international 1940–1980 could not be sustained. In the last two decades, only community to take measures to curtail the emergence and spread one new antibiotic class viz the oxazolidinone was introduced in of AMR. the year 1990 and all other entrants were just the variation of the existing one (Raghunath, 2008). Eventually, clinical manage- EMERGENCE OF RESISTANCE PHENOTYPE ment of antibiotic-resistant superbugs is assuming the shape of Antibiotic resistance is known to occur both in the pre-antibiotic a gruesome problem because of the depleting antibiotic reserve and antibiotic-era. The pre-antibiotic era constitutes the time (Norrby et al., 2005). The widespread apprehension that we might before the introduction of sulphonamides in 1930. It is well be heading to a situation similar to the pre-antibiotic era cannot established that resistance phenotype and the antibiotics in the be dismissed as a far-stretched imagination. pre-antibiotic era coexisted in the natural environment with- Imprudent and excessive use of antibiotics is highlighted as a out facilitating the process of selection of the deadly resistant major causative factor behind the setback. The idea dubbed as pathogens. Antibiotic Paradox by Levy (1992) is corroborated by numerous The antibiotic era started following its discovery of antibiotics evidences. In 1967, the first penicillin-resistant Streptococcus pneu- and their use in different spheres of life. The use of high concen- monia was observed in Australia, and after 7 years another case tration of lethal dose of antibiotics as a consequence of human of penicillin-resistant Streptococcus pneumonia was reported from activity led to a major change in innate functional role to give the U.S. in a patient with pneumococcal meningitis (Doern et al., rise to the emergence of antibiotic-resistant pathogens within a 2001). In 1980, 3–5% of Streptococcus pneumonia was estimated short span of time. Antibiotic resistance genes, which were once to be penicillin-resistant and by 1998, 34% of the Streptococcus involved in other cellular functions before human intervention, pneumonia sampled was resistant to penicillin (Doern et al., 2001). have been subsequently selected for the resistance phenotype with Antibiotic resistance in other organisms reflects the same trend as increased use of antibiotics. They have been mobilized from the observed in Streptococcus pneumonia against penicillin. Tetracy- environmental genomic reservoirs, with the rapid dissemination cline resistance by normal human intestinal flora exploded from into taxonomically divergent commensal and pathogenic bacte- 2% in the 1950s to 80% in the 1990s (Shoemaker et al., 2001). ria. Metagenomic approach in conjunction with a pipeline for Kanamycin, an antibiotic used in the 1950s, has become clini- the de novo assembly of short-read sequence data from func- cally obsolete as a result of the prevalence of kanamycin-resistant tional selections (termed PARFuMS) has shown the evidence of bacteria (e.g., M. tuberculosis, Campylobacter jejuni). lateral transmission of five different antibiotic resistance genes On the other hand, it is also a fact that antibiotic resistance is along with different non-coding region as well as multiple mobi- observed even in bacteria isolated from totally uninhabited and lization sequences from environmental reservoir of soil bacteria thinly populated places, where they are unlikely or least likely to to the clinical pathogens (Forsberg et al., 2012). Thus HGT or come in contact with the antibiotics (Chattopadhyay and Grossart, lateral gene transfer, which contributes significantly to the evolu- 2010). It is evident that antibiotic resistance is an outcome of evo- tion, maintenance, and transmission of virulence in pathogenic lution and pre-exposure of microorganisms to antibiotics is not a bacteria, also plays a pivotal role in dissemination of antibi- pre-requisite for emergence of resistance. In a systematic analysis, otic resistance conferring genes from the environment in clinical the conjugative plasmids occurring in several bacteria belonging to settings (Colomer-Lluch et al., 2011). Enterobacteriaceae obtained from Murray collection were found Epigenetic inheritance-based evolution of antibiotic resistance to belong to the same incompatibility group as those present in genes has been reported in an isogenic population of Escherichia contemporary Enterobacteriaceae (Datta and Hughes, 1983). It coli, exposed to gradually increasing concentration of different was shown that emergence and spread of conjugative plasmids, antibiotics such as ampicillin, nalidixic acid, and tetracycline. The which are known to be a major vehicle for transmission of resis- high frequency of survival on low antibiotic concentration could tance, were not fostered by the widespread use of antibiotics, rather not be accounted for by the occurrence of random spontaneous these plasmids evolved by inclusion of resistance-conferring genes mutation. Instead it suggested that the antibiotic resistance genes in pre-existing plasmids (Datta and Hughes, 1983; Hughes and were acquired by epigenetic inheritance. High reversion of this Datta, 1983). resistance phenotype further proved that it was indeed a case of Despite intensive investigations continuing for the last few epigenetic inheritance which does not impart a stable phenotype decades in a global scale, no significant achievement has been due to maintenance of certain chromatin configuration or DNA made so far in the prevention and control of resistance develop- methylation state (Adam et al., 2008). ment. A recent database reveals the existence of more than 20,000 There are ample evidences to suggest that anthropogenic fac- potential resistance genes of nearly 400 different types in the avail- tors (use of antibiotics by human both for therapeutic and able bacterial genome sequences. The fact that the number of non-therapeutic purposes and disposal of the unused antibiotic functional resistance determinants in pathogens is much smaller formulations) inflict significant changes on the natural flora of (Liu and Pop, 2009) provides no relief since the new generation of bacteria. But the actual concentrations of antibiotics (and other therapeutically useful antibiotics are reaching the market only in pharmaceuticals) that bacteria in the natural population is exposed trickle. to, remain unknown in most of the cases (Kümmerer, 2003, 2004).

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 47 | 88

“fmicb-04-00047” — 2013/3/12 — 11:18 — page6—#6 Sengupta et al. Ecological role of antibiotics in nature

Its role in the emergence of antibiotic resistance still remains highly vitro and in vivo. Hence antibiotic-induction of phage may also controversial (Bhullar et al., 2012). contribute to increased virulence (Zhang et al., 2000). In therapeutics, the pathogens are challenged with an over- In many cases, exposure to a low-dose antibiotic is associ- whelmingly high concentration of a single or few antibiotics ated with mobile element-mediated dissemination of antibiotic whereas the soil borne microbes exist in a complex microenvi- resistance genes through HGT which is known to be enhanced ronment and encounter a number of simultaneously occurring by sub-MIC concentrations of tetracycline (Celli and Trieu-Cuot, stress factors in a network of multiple interactions. Probably, 1998). Tetracycline at a sub-inhibitory concentration in the mating these interactions have a nullifying effect on each other thus medium substantially enhanced Tn916 mediated conjugal trans- favoring the evolutionary selection of antibiotic sensitivity over fer to the recipient Bacillus thuringiensis subsp. israelensis (Showsh resistance (Chait et al., 2011). Therefore, the dynamics of antibi- and Andrews, 1992). Spread of antibiotic resistance genes between otic resistance in clinical settings is believed to be inflicted by human colonic Bacteroides spp. is mediated by self-transmissible anthropocentric factors and is quite different from naturally occur- elements known as conjugative transposons (CTns). The expo- ring resistance. Simulation of these natural niches may reveal the sure of donor Bacteroides cells to low concentration of tetracycline dynamics by which antibiotic resistance is disseminated across the appeared to be a pre-requisite for the excision of the CTnDOT microbial population. family of CTns from the chromosome and conjugal transfer of the excised elements. Virtually no transfer occurs without the tetracy- IMPLICATION OF SUBLETHAL CONCENTRATIONS OF cline induction of donor cells (Stevens et al., 1993; Whittle et al., ANTIBIOTICS IN DISSEMINATION OF RESISTANCE 2002). Low concentrations of tetracycline might increase the likeli- In nature, sub-inhibitory concentrations of antibiotics are prob- hood of HGT of integrated mobile elements, such as CTnDOT and ably encountered by bacteria more frequently than inhibitory NBU1. At sub-inhibitory tetracycline concentration, the excision concentrations. Most often bacteria are exposed to antibiotics is not associated with growth phase (Song et al., 2005). Mobi- at a sublethal concentration in certain clinical situations such as lization of coresident non-conjugative plasmids (from Bacteroides incomplete treatment of an infection, patient non-compliance, strain to Bacteroides strain or Escherichia coli) by chromosoma- and reduced or limited drug accessibility to certain tissues (e.g., lly encoded tetracycline conjugal elements (Valentine et al., 1988) bone or cerebrospinal fluid; Bryskier, 2005). In the colon of a was enhanced by 20–10,000-folds when the donor was pre-grown person taking antibiotics, sub-inhibitory concentrations would be in a sub-inhibitory concentration of tetracycline (1 μg/ml). The experienced by colonic bacteria during the initial phase of treat- similar stimulatory effect of tetracycline on conjugation trans- ment and at the end of treatment. Thus host tissues are exposed fer was also demonstrated for the conjugative transposon Tn925 to a range of drug concentration starting from higher to a sub- (Torres et al., 1991). inhibitory concentration. Therefore, micro-niches within the host, These in vitro observations were validated in vivo in gnoto- such as epidermis, lungs, and joints, may attain significantly lower biotic mice where tetracycline at a sublethal concentration in drug concentrations than the plasma (Rybak, 2006). Outside clin- drinking water could induce the frequency of conjugative trans- ical settings, sub-inhibitory condition is established because of the fer of the transposon Tn1545 from Enterococcus faecalis to Listeria use of manure from livestock whose feed is supplemented with monocytogenes in the digestive tract and there was an approxi- antibiotics and in consequence multiple drugs in a very low level mately 10-fold increase in the transposition (Doucet-Populaire find their way both to the soil and aqueous environment. et al., 1991). In gnotobiotic rats, selection for the resistant phe- Due to such imprudent use of antibiotics, bacteria are quite notype was the major factor causing higher numbers of Tn916 likely to experience sublethal levels of antibiotics which have high transconjugants in the presence of tetracycline (Bahl et al., 2004). implications in the spread of multidrug resistance. There is evi- Therefore, the enhancement of conjugal transfer of antibiotic dence that low level of antibiotics gives rise to mutagenesis in resistance-carrying transposons in the presence of sub-inhibitory a wider range of antibacterial resistance genes and drug efflux concentration of antibiotics is not only an in vitro phenomenon systems resulting in multidrug resistance (Girgis et al., 2009). Sub- but also takes place in the human intestinal microbiome of animal inhibitory concentrations of β-lactams were found to enhance the models. transfer of tetracycline resistance plasmids in Staphylococcus aureus Adverse effect of some antibiotics on bacterial motility was by up to 1,000-fold (Barr et al., 1986). Ampicillin treatment of reported earlier (Molinari et al., 1992; Kawamura-Sato et al., Escherichia coli was associated with the formation of norfloxacin- 2000). However, sub-inhibitory concentration of tobramycin was resistant isolates with mutations in gyrA, gyrB, or the acrAB observed to increase motility of Pseudomonas aeruginosa and promoter (PacrAB) and kanamycin-resistant isolates with muta- enhance the expression of virulence determinants of the organism tions in rpsL or arcA (A and B). It has been hypothesized that low (Linares et al., 2006). level of bactericidal antibiotics give rise to reactive oxygen species (ROS) which leads to DNA damage-induced mutations thus facil- ANTIBIOTIC RESISTANCE AND ITS ROLE IN NATURE itating the emergence of multidrug-resistant bacteria (Kohanski Antibiotic resistance can be defined in different ways: from the et al., 2010). Induction of prophage in animal feed by antibiotics microbiological point of view, the resistance is defined as a pheno- was evidenced. It may contribute to dissemination of antibiotic type which makes the microorganism less susceptible than other resistance (Allen et al., 2011). Induction of shigella toxin-encoding members of the same species irrespective of any level of resistance. bacteriophage by ciprofloxacin and enhanced Shiga toxin (Stx) On the contrary, when the resistance reaches a certain critical level production from Escherichia coli O157:H7 was demonstrated in so as to interfere with the pharmacotherapy of a clinical problem

www.frontiersin.org March 2013 | Volume 4 | Article 47 | 89

“fmicb-04-00047” — 2013/3/12 — 11:18 — page7—#7 Sengupta et al. Ecological role of antibiotics in nature

caused by the bacterium, it is called clinical resistance (Cantón and to antibiotics but also to a number of structurally unrelated Morosini, 2011). compounds viz, ethidium bromide, quaternary ammonium com- In the present scenario, acquired clinical resistance of pathogens pounds, the DNA-intercalating mutagen acridine, the anionic is a major concern in the healthcare management which results detergent sodium dodecyl sulfate and uncouplers. Hence they from genetic changes involving mutation or HGT. Besides appear to have a greater role in bacterial physiology. They acquired resistance dealt with in this article, some other types also confer resistance to some chemical substances produced by of antibiotic resistances are also observed in bacteria. For example the host, e.g., bile acids. It is speculated that antibiotic resis- in some cases tolerance of a bacterium to a certain antibiotic is tance is an offshoot of some hitherto undefined physiological dependent on its metabolic state. It exhibits susceptibility to the roles played by the determinants in these bacteria (Martinez and antibiotic in growing state but become resistant to the same antibi- Rojo, 2011). otic in stationary phase. The micro-environments in a biofilm Most antibiotic-producing strains carry genes encoding resis- also contribute to the differential susceptibility of bacteria embed- tance to the antibiotics that they produce (Hopwood, 2007; Tahlan ded in different parts of the polysaccharide matrix. Swarming, a et al., 2007) and these genes are usually found in the same gene phenomenon induced in some bacteria in response to nitrogen cluster as the antibiotic biosynthesis pathway genes (Benveniste limitation, confers resistance to some antibiotics (Martinez and and Davies, 1973; Martin and Liras, 1989). The proximity of the Rojo, 2011). Resistance to a variety of antibiotics was observed in antibiotic resistance gene with the antibiotic biosynthetic gene as swarming cells of some bacteria. This observation together with evidenced in Streptomyces coelicolor (where the genes encoding the resistance observed in biofilm clearly indicates that antibiotic export proteins are embedded in the actinorhodin biosynthesis resistance is a manifestation of social behavior of bacteria (Lai pathway) led to the postulation that these resistance determinants et al., 2009). Accumulation of the alarmone (p) ppGpp was also could have some role in the biosynthetic pathway of antibiotics reported to modulate antibiotic resistance. Involvement of this (Allen et al., 2010). Antibiotic resistance genes are frequently alarmone in persistence (resistance occurring in a small fraction present even in non-producer strains (Yamashita et al., 1985). In of naturally occurring bacteria) is postulated (Jayaraman, 2009). consequence, bacteria have evolved with a diverse pool of genes This type of resistance, known as phenotypic resistance, signifi- (the “resistome”) that protect them against the therapeutic dose cantly contributes to some problems in clinical management of of antibiotics. Gene orthologous to these have been identified on some bacterial infections. mobile genetic elements in resistant pathogens in clinical settings. The most ancient resistance also known as intrinsic resistance These genes that make up this environmental resistome have the (Sheldon, 2005) is species-specific and results from impermeabil- potential to be transferred to pathogens and indeed there is some ity of the cells to the antibiotic or lack of target of an antibiotic to a evidence that at least some clinically relevant resistance genes have certain organism. However, besides these passive factors, intrinsic originated in environmental microbes (Wright, 2010). resistance is also caused by some antibiotic-detoxifying determinants encoding chromosomally encoded β-lactamases, efflux ANCIENT ORIGIN OF ANTIBIOTIC RESISTANCE GENES pumps or target-protecting proteins. This antibiotic resistance is It is well-established documented that the antibiotic resistance is a widespread in nature and this huge intrinsic resistome of bacte- long-evolved trait in prokaryotes and the diverse pool of resistant ria consists of genes of varied phylogenetic origin This intrinsic genes co-evolved with antibiotics in non-clinical (natural) envi- resistome has a high degree of non-specificity and many of the ronment much before these have been used in human therapy. proteins encoded by these genes are involved in the basic phys- Most of the antibiotic producer organisms carry the respective iological functions in the natural ecosystem as demonstrated in antibiotic resistance genes and both the resistance and the antibi- Pseudomonas aeruginosa, the novel nosocomial Gram-negative otic biosynthesis pathway genes are usually found in the same gene pathogen of environmental origin (Fajardo et al., 2008). The pres- cluster (Allen et al., 2010). ence of antibiotics acts as a switch in physiological function to In the evolutionary scale many antibiotics or their structurally the antibiotic-resistance phenotype. These resistance genes per- related precursors are believed to be as old as amino acids. It has sist in bacteria even in antibiotic-free environment thus further been suggested that antibiotics are more than 500 million years old, substantiating their alternative role in bacterial physiology. Tet(L) dating back to the Cambrian period and probably evolved at the and Tet(K), the specific tetracycline c-resistance determinants are same time vertebrate fish emerged (Baltz, 2008). The components known to catalyze Na+ and K+ exchange for protons (Krulwich of the antibiotics are believed to be even older and the postulation et al., 2001). Tet(L), a tetracycline-efflux transporter, and MdfA, a is substantiated by the occurrence of non-protein amino acids multidrug-efflux transporter, both responsible for alkali tolerance of peptide antibiotics in meteorites and other primordial sources offer a striking example in support of this hypothesis (Krulwich (Johnson et al., 2008). Julian Davies at the University of British et al., 2005). In M. tuberculosis the P55 efflux pump plays an Columbia proposes that antibiotics could be some of the oldest important role in oxidative stress response and in vitro cell growth biomolecules (Amábile-Cuevas, 2003). in addition to contributing to its intrinsic resistance phenotype Metagenomic analyses of ancient DNA from 30,000-year- (Ramón-García et al., 2009). old to Beringian permafrost sediments dating back to the late It is obvious that bacteria evolve with antibiotic resistance Pleistocene age demonstrated the presence of different antibi- determinants get selective advantage over their antibiotic-sensitive otic resistance genes encoding resistance to β-lactam, tetracycline, counterparts in presence of antibiotics. However, some of the and glycopeptide antibiotics (D’Costa et al., 2011). Structural and multidrug resistance determinants confer resistance not only functional studies of one of these ancient resistance genes, the vanA

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 47 | 90

“fmicb-04-00047” — 2013/3/12 — 11:18 — page8—#8 Sengupta et al. Ecological role of antibiotics in nature

(vancomycin resistance operon) that encodes an ATP dependent diversified serine β-lactamases suggests an ancient root for the D-alanyl-D-lactate ligase confirmed that it is essentially indistin- antibiotic resistance genes (Hall and Barlow, 2003). guishable from VanA ligase associated with the recently discovered Even the Murray collection (procured before and after the vancomycin resistance in the clinic. introduction of antibiotics, i.e., between 1917 and 1952) exhibited Very recently, Lechuguilla Cave in New Mexico, cut off from ampicillin and tetracycline resistance in 11 out of 433 enterobacte- the human activity for over four million years, was found to rial strains and the resistance was non-conjugative. These studies harbor culturable microbiomes, highly resistant to antibiotics. reveal that the determinants of antibiotic resistance existed in Some strains were found to be resistant to as many as 14 differ- nature much before the human intervention. Millions of years ago, ent commercially available antibiotics. Resistance was detected to antibiotics and antibiotic biosynthetic pathways evolved suggest- a wide range of structurally different antibiotics including dap- ing that both antibiotics and the resistance genes are very ancient tomycin, an antibiotic of last resort in the treatment of drug (Wright and Poinar, 2012). The presence of multidrug-resistant resistant Gram-positive pathogens. Enzyme-mediated mecha- organisms even in this pristine environment reinforces the idea nisms (e.g., glycosylation, phosphorylation) leading to resistance that the antibiotic resistome is ancient and omnipresent in the against both natural and semi-synthetic antibiotics were also dis- microbial genome. covered in some ancient bacteria. Characterization of macrolide kinase obtained from one of these resistant organisms revealed CAN THE DOOMSDAY BE POSTPONED? it to be related to a known family of kinases circulating in drug In keeping with a policy package recommended by WHO in 2002 resistant pathogens at present (Bhullar et al., 2012). emphasis was given on the availability of proper facilities to ensure In another study, molecular analysis of a metagenomic library rapid testing of antibiotic resistance, regular documentation and from the cold-seep sediments of the deep sea Edison seamount sharing of the surveillance data, uninterrupted access to quality (about 10,000 years old) also demonstrated the presence of TEM- medical service, regulation on the sale of antibiotics, development type ESBLs (TEM-1 and TEM-116) suggesting that β-lactam of new diagnostic tests, and novel antimicrobials. But the problem resistance in microorganisms is likely to be present prior to the continues to be unabated till now (Leung et al., 2011). modern antibiotic era, and the diversity of TEM β-lactamases Recently, scientists (Marraffini and Sontheimer, 2008)fromthe is not a recent phenomenon, rather it is the result of a very Northwestern University have discovered a specific DNA sequence, ancient evolution (Song et al., 2005). Therefore, despite the fact called a CRISPR (clustered, regularly interspaced, short palin- that increase in the use of an antibiotic is directly associated dromic repeat) locus, which confer prokaryotes a type of acquired with increase in the frequency of resistant strains, it is obvi- immunity against the acquisition of resistance genes by encoding a ous that exposure to antibiotics is not a pre-requisite for the sequence-specific defense mechanism against bacteriophages and emergence of resistance. Even the diversification of the antibiotic thus conferring an immunity toward HGT. Members of recently resistance appears to occur much before the human intervention as potentially virulent strains of enterococcal have been shown to lack evidenced by the occurrence of the TEM-type ESBL in the 10,000- complete CRISPR loci. Expression of the CRISPR interference has year-old sediments of the Edison seamount. But the prevalence also been found to prevent the transfer of antibiotic resistance and of the resistance bacteria having TEM-1 and TEM-116 was as low virulent genes, not only in vitro, but also in vivo, during a pneu- as 0.3% (25 of 8,823) and 0.06% (5 of 8,823), respectively (Allen mococcal infection (Bikard et al., 2012). “If this mechanism could et al., 2009). Recent investigations revealed that the origin of both be manipulated in a clinical setting, it would provide a means to antibiotic resistance and the biosynthetic genes date back to the limit the spread of antibiotic resistance genes and virulence fac- ancient period much before the human civilization began. Both tors in Staphylococcus and other bacterial pathogens,” hopes Erik antibiotics and the resistance phenotype existed together in nature Sontheimer, Associate Professor at the Weinberg College of Arts for a long time. and Sciences. There are many other cases of occurrence of antibiotic resis- The new Eco-Evo drugs and strategies target prevention of the tance genes in apparently antibiotic-free environments. The evolution and emergence of resistant bacteria in the environment. metagenomic studies of remote Alaskan soil revealed the presence This might be achieved by using inhibitors against four P’s viz, of divergent β-lactamase genes and a bifunctional β-lactamase penetration (by using 7-valent conjugated vaccine, PC7 to prevent (Allen et al., 2009). These β-lactamases are more closely related colonization of resistant serotype in human), promiscuity (using to ancestral homologs compared to those isolated in clinical set- broad host range conjugation inhibitor), plasticity (recombinase tings and are capable of conferring resistance on Escherichia coli inhibitor), and persistence (decontamination of high risk clones despite the evolutionary distance. In another example of antibiotic or mobile genetic elements) of resistant bacteria. This strategy resistance in environments not impacted by antibiotics, the phe- appears implementable in view of the fact that preventive measures notype of more than 60% of the Enterobacteriaceae isolates from recommended in various steps are mostly adoptable. However, a pristine freshwater environment was found to be multidrug- their long-term impact on the environment warrants rigorous resistant. An example of this evidence exists in the two strains of testing before implementation (Baquero et al., 2011). In order to Citrobacter freundii that were collected prior to the antibiotic era combat the looming crisis, a number of different other approaches (the 1920s) which carried ampC β-lactamase genes. The encoded have been adopted. Pharmaceutical companies are tapping new AmpC β-lactamases were as active as the recent plasmid borne sources (samples obtained from tropical rain forests, myxobac- AmpC β-lactamases (class C) that were found in the antibiotic era teria, marine bacteria, extremophilic bacteria) for novel antibi- (Barlow and Hall, 2002). Phylogenetic analysis of the genetically otics. Development of antimicrobials against bacterial molecules,

www.frontiersin.org March 2013 | Volume 4 | Article 47 | 91

“fmicb-04-00047” — 2013/3/12 — 11:18 — page9—#9 Sengupta et al. Ecological role of antibiotics in nature

which were not targeted earlier (e.g., bacteria DNA polymerase to co-evolve with antibiotics to safe guard the producer organism III, the cell division protein FtsZ, fibronectin binding proteins) from antibiotic threat. is aimed at. Genes indispensable for survival of pathogens are While it is obvious that dissemination of resistance-conferring shortlisted in search of new targets. In another strategy, molecular genes has been facilitated following introduction of antibiotics techniques are being used to clone the antibiotic biosynthetic genes into the clinical practice, we are yet to get sufficient evidence to cluster of a strain into a different strain, ultimately to get a hybrid conclude that human intervention is the only contributing fac- molecule with antibiotic activity. In another approach called tor behind the spread of resistance. Until and unless we generate directed evolution, libraries are developed with randomly mixed required amount of information to fill up the gap of knowledge and matched gene clusters obtained from different antibiotic pro- in this aspect, we cannot expect to devise measures which would ducer strains. These libraries are transformed into protoplasts effectively arrest the progress of the crisis. and recombinants are screened for improved spectrum of antibi- Antibiotic resistance is a complex and continually evolving otic activity. Large number of new compounds synthesized on problem. A vast body of information has been engendered during a solid support, are screened for activity in another technique the past 70 years on the antibiotic resistance and its dissemina- called combinatorial chemistry. These new strategies are expected tion. Comparative genomics, molecular genetics, combinatorial to generate new compounds with novel antimicrobial activities chemistry, and structural biology are being applied to explore new (Strohl, 1997). antibiotics but still the struggle is on. Several secondary approaches Several secondary approaches like reduction of antibiotic con- like reduction of antibiotic consumption, preservation of existing sumption, preservation of existing therapeutics, development of therapeutics, development of new antibiotics, and development of new antibiotics, and development of new strategies like sensiti- new strategies like sensitization of antibiotic-resistant organisms zation of pathogens (Toney et al., 1998)havebeenadopted.The using QS inhibitors have been adopted but without much success. formulation, augmentin, a combination of β-lactamase inhibitor Relatively rare genes that happened to confer antibiotic resis- (clavulanic acid) and amoxicillin, is being successfully used against tance were once involved in other cellular functions, but were a variety of penicillin-resistant pathogens. selected for the resistance phenotype and mobilized from the environmental genomic reservoirs, with the rapid dissemination into CONCLUSION taxonomically divergent commensal and pathogenic bacteria. This It is apparent that antibiotics are endowed with multifaceted process was very rapid on an evolutionary scale and HGT, medi- activities and the cell modulates their role. The dual nature of ated by mobile genetic elements, played a prominent role in it. So antibiotic acting both as signaling molecule as well as growth it would be wise to plan a strategy to control the process of HGT inhibitory molecule suggests that antibiotic resistance genes have to control subsequent transfer of resistance genes to pathogens.

REFERENCES L. (2012). Fidaxomicin inhibits spore aureus. J. Antimicrob. Chemother. 17, acquisition during in vivo bacte- Adam, M., Murali, B., Glenn, N. O., and production in Clostridium difficile. 409–413. rial infection. Cell Host Microbe 12, Potter, S. S. (2008). Epigenetic inher- Clin. Infect. Dis. 55(Suppl. 2), S162– Bassler, B. L. (1999). How bacteria talk 177–186. itance based evolution of antibiotic S169. to each other: regulation of gene Bryskier, A. (2005). Antimicrobial resistance in bacteria. BMC Evol. Biol. Bahl, M. I., Sorensen, S. J., Hansen, expression by quorum sensing. Curr. Agents: Antibacterials and Antifun- 8:52. doi: 10.1186/1471-2148-8-52 L. H., and Licht, T. R. (2004). Opin. Microbiol. 2, 582–587. gals. Washington, DC: ASM Press. Allen, H. K., Donato, J., Wang, H. Effect of tetracycline on transfer Beaber, J. W., Hochhut, B., and Waldor, Cafaro, M. J., and Currie, C. R. (2005). H., Cloud-Hansen, K. A., Davies, J., and establishment of the tetracycline- M. K. (2004). SOS response promotes Phylogenetic analysis of mutualistic and Handelsman, J. (2010). Call of inducible conjugative transposon horizontal dissemination of antibi- filamentous bacteria associated with the wild: antibiotic resistance genes Tn916 in the guts of gnotobiotic otic resistance genes. Nature 427, fungus-growing ants. Can. J. Micro- in natural environments. Nat. Rev. rats. Appl. Environ. Microbiol. 70, 72–74. biol. 51, 441–446. Microbiol. 8, 251–259. 758–764. Benveniste, R., and Davies, J. (1973). Calabrese, E. J. (2009). Getting the Allen, H. K., Looft, T., Bayles, D. Baltz, R. H. (2008). Renaissance in Aminoglycoside antibiotic inactivat- dose–response wrong: why hormesis O., Humphrey, S., Levine, U. Y., antibacterial discovery from actino- ing enzymes in actinomycetes similar became marginalized and the thresh- Alt, D., et al. (2011). Antibiotic in mycetes. Curr. Opin. Pharmacol. 8, to those present in clinical isolates old model accepted. Arch. Toxicol. 83, feed induce prophages in swine fecal 557–563. of antibiotic-resistant bacteria. Proc. 227–247. microbiomes. MBio 2, e00260-11. Baquero, F., Coque, T. M., and de Natl. Acad. Sci. U.S.A. 70, 2276– Cantón, R., and Morosini, M. I. (2011). Allen, H. K., Moe, L. A., Rodbum- la Cruz, F. (2011). Ecology and 2280. Emergence and spread of antibi- rer, J., Gaarder, A., and Handelsman, evolution as targets: the need for Bhullar, K., Waglechner, N., Pawlowski, otic resistance following exposure to J. (2009). Functional metagenomics novel eco-evo drugs and strategies A., Koteva, K., Banks, E. D., Johnston, antibiotics. FEMS Microbiol. Rev. 35, reveals diverse beta-lactamases in a to fight antibiotic resistance. Antimi- M. D., et al. (2012). Antibiotic resis- 977–991. remote Alaskan soil. ISME J. 3, 243– crob. Agents Chemother. 55, 3649– tance is prevalent in an isolated cave Celli, J., and Trieu-Cuot, P. (1998). 251. 3660. microbiome. PLoS ONE 7:e34953. Circularization of Tn916 is Amábile-Cuevas, C. F. (2003). New Barlow, M., and Hall, B. G. (2002). doi: 10.1371/journal.pone.0034953 required for expression of the antibiotics and new resistance. Am. Origin and evolution of the AmpC Bibb, M. (2005). Regulation transposon-encoded transfer func- Sci. 91, 138–149. β-lactamases of Citrobacter freundii. of secondary metabolism in tions: characterization of long Aminov, R. I. (2009). The role of Antimicrob. Agents Chemother. 46, streptomycetes. Curr. Opin. tetracycline-inducible transcripts antibiotics and antibiotic resistance 1190–1198. Microbiol. 8, 208–215. reading through the attach- in nature. Environ. Microbiol. 11, Barr, V., Barr, K., Miller, M. R., and Bikard, D., Hatoum-Aslan, A., Mucida, ment site. Mol. Microbiol. 28, 2970–2988. Lacey, R. W. (1986). Beta-lactam D., and Marraffini, L. A. (2012). 103–117. Babakhani, F., Bouillaut, L., Gomez, A., antibiotics increase the frequency of CRISPR interference can prevent nat- Chait, R., Vetsigian, K., and Kishony, Sears, P.,Nguyen, L., and Sonenshien, plasmid transfer in Staphylococcus ural transformation and virulence R. (2011). What counters antibiotic

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 47 | 92

“fmicb-04-00047” — 2013/3/12 — 11:18 — page 10 — #10 Sengupta et al. Ecological role of antibiotics in nature

resistance in nature? Nat. Chem. Biol. Dubern, J. F., and Diggle, S. P. Henderson-Begg, S. K., Livermore, D. T., and Ohta, M. (2000). Effect 8, 2–5. (2008). Quorum sensing by 2-alkyl- M., and Hall, L. M. (2006). Effect of subinhibitory concentrations of Chattopadhyay, M. K., and Grossart, 4-quinolones in Pseudomonas aerugi- of subinhibitory concentrations of macrolides on expression of flag- H.-P. (2010). Antibiotic resistance: nosa and other bacterial species. Mol. antibiotics on mutation frequency in ellin in Pseudomonas aeruginosa and intractable and here’s why. BMJ 341, Biosyst. 4, 882–888. Streptococcus pneumoniae. J. Antimi- Proteus mirabilis. Antimicrob. Agents c6848. Evans, J., and Dyke, K. G. (1988). crob. Chemother. 57, 849–854. Chemother. 44, 2869–2872. Colomer-Lluch, M., Jofre, J., and Characterization of the conjugation Herrera-Insúa, I., Jacques-Palaz, K., Kohanski, M. A., DePristo, M. A., Muniesa, M. (2011). Antibiotic resis- system associated with the Staphylo- Murray, B. E., and Rakita, R. and Collins, J. J. (2010). Sub- tance genes in the bacteriophage coccus aureus plasmid pJE1. J. Gen. M. (1997). The effect of antibi- lethal antibiotic treatment leads to DNA fraction of environmental sam- Microbiol. 134, 1–8. otic exposure on adherence to multidrug resistance via radical- ples. PLoS ONE 6:e17549. doi: Fajardo, A., Martínez-Martín, N., Mer- neutrophils of Enterococcus faecium induced mutagenesis. Mol. Cell 37, 10.1371/journal.pone.0017549 cadillo, M., Galán, J. C., Ghysels, resistant to phagocytosis. J. Antimi- 311–320. Cortes, P. R., Piñas, G. E., Albarracin B., Matthijs, S., et al. (2008). The crob. Chemother. 39(Suppl. A), Krulwich, T. A., Jin, J., Guffanti, A. A., Orio, A. G., and Echenique, J. R. neglected intrinsic resistome of bac- 109–113. and Bechhofer, H. (2001). Functions (2008). Subinhibitory concentrations terial pathogens. PLoS ONE 3:e1619. Hoffman, L. R., D’Argenio, D. A., Mac- of tetracycline efflux proteins that of penicillin increase the mutation doi: 10.1371/journal.pone.0001619 Coss, M. J., Zhang, Z., Jones, R. do not involve tetracycline. J. Mol. rate to optochin resistance in Strep- Fleming, A. (1929). Classics in infec- A., and Miller, S. I. (2005). Amino- Microbiol. Biotechnol. 3, 237–246. tococcus pneumoniae. J. Antimicrob. tious diseases: on the antibacterial glycoside antibiotics induce bacterial Krulwich, T. A., Lewinson, O., Padan, Chemother. 62, 973–977. action of cultures of a penicillium, biofilm formation. Nature 436, 1171– E., and Bibi, E. (2005). Do phys- Cunha, B. A. (1995). Vancomycin. Med. with special reference to their use in 1175. iological roles foster persistence of Clin. North Am. 79, 817–831. the isolation of B. influenzae. Br. J. Hong, R. W., Shchepetov, M., Weiser, J. drug/multidrug-efflux transporters? Currie, C. R., Scott, J. A., Sum- Exp. Pathol. 10, 226–236. N., and Axelsen, P. H. (2003). Tran- A case study. Nat. Rev. Microbiol. 3, merbell, R. C., and Malloch, D. Forsberg, K. J., Reyes, A., Wang, B., Sell- scriptional profile of the Escherichia 566–572. (1999). Fungus-growing ants use eck, E. M., Sommer, M. O., and Dan- coli response to the antimicrobial Kumarasamy, K. K., Toleman, M. A., antibiotic producing bacteria to con- tas, G. (2012). The shared antibiotic insect peptide cecropin A. Antimi- Walsh, T. R., Bagaria, J, Butt, F., trol garden parasites. Nature 398, resistome of soil bacteria and human crob. Agents Chemother. 47, 1–6. Balakrishnan, R., et al. (2010). Emer- 701–704. pathogens. Science 337, 1107–1111. Hopwood, D. A. (2007). How do gence of a new antibiotic resistance Datta, N., and Hughes, V. M. (1983). Gerber, M., Walch, C., Löffler, B., antibiotic-producing bacteria ensure mechanism India, Pakistan and the Plasmids of the same Inc groups in Tischendorf, K., Reischl, U., and their self-resistance before antibiotic UK: a molecular biological and epi- Enterobacteria before and after the Ackermann, G. (2008). Effect of biosynthesis incapacitates them? Mol. demiological study. Lancet Infect. Dis. medical use of antibiotics. Nature sub-MIC concentrations of metron- Microbiol. 63, 937–940. 10, 597–602. 306, 616–617. idazole, vancomycin, clindamycin Hsiao, N. H., Gottelt, M., and Takano, Kümmerer, K. (2003). Significance of Davies, J. (1990). What are antibi- and linezolid on toxin gene tran- E. (2009). Regulation of antibiotic antibiotics in the environment. J. otics? Archaic functions for modern scription and production in Clostrid- production by bacterial hormones. Antimicrob. Chemother. 52, 5–7. activities. Mol. Microbiol. 4, 1227– ium difficile. J. Med. Microbiol. 57, Methods Enzymol. 458, 143–157. Kümmerer, K. (2004). Resistance in 1232. 776–783. Hughes, V. M., and Datta, N. (1983). the environment. J. Antimicrob. Davies, J. (2006). Are antibiotics nat- Girgis, H. S., Hottes, A. K., and Conjugative plasmids in bacteria of Chemother. 54, 311–320. urally antibiotics? J. Ind. Microbiol. Tavazoie, S. (2009). Genetic archi- the ‘pre-antibiotic’ era. Nature 302, Kurihara, Y., Hitomi, S., Oishi, T., Biotechnol. 33, 496–499. tecture of intrinsic antibiotic sus- 725–726. Kondo, T., Ebihara, T., Funayama, Davies, J. (2009). Darwin and micro- ceptibility. PLoS ONE 4:e5629. doi: Ichimiya, T., Takeoka, K., Hiramatsu, Y., et al. (2013). Characteristics of biomes. EMBO Rep. 10, 805. 10.1371/journal.pone.0005629 K., Hirai, K., Yamasaki, T., and bacteremia caused by extended- Davies, J., Spiegelman, G. B., and Goh, E. B.,Yim, G., Tsui,W.,McClure, J., Nasu, M. (1996). The influence of spectrum beta-lactamase-producing Yim, G. (2006). The world of Surette, M. G., and Davies, J. (2002). azithromycin on the biofilm forma- Proteus mirabilis. J. Infect. Chemother. subinhibitory antibiotic concentra- Transcriptional modulation of bacte- tion of Pseudomonas aeruginosa in doi: 10.1007/s10156-013-0563-3 tions. Curr. Opin. Microbiol. 9, rial gene expression by subinhibitory vitro. Chemotherapy 42, 186–191. [Epub ahead of print]. 445–453. concentrations of antibiotics. Proc. Jayaraman, R. (2009). Antibiotic resis- Kurylo-Borowska, Z., and Szer, W. D’Costa, V. M., King, C. E., Kalan, L., Natl. Acad. Sci. U.S.A. 99, 17025– tance: an overview of mechanisms (1972). Inhibition of bacterial DNA Morar, M., Sung, W. W., Schwarz, C., 17030. and a paradigm shift. Curr. Sci. 96, synthesis by edeine. Effect on et al. (2011). Antibiotic resistance is Guerin, E., Cambray, G., Sanchez- 1475–1484. Escherichia coli mutants lacking DNA ancient. Nature 477, 457–461. Alberola, N., Campoy, S., Erill, I., Johnson, A. P. (2011). Methicillin- polymerase I. Biochim. Biophys. Acta Doern, G. V., Heilmann, K. P., Huynh, Da Re, S., et al. (2009). The SOS resistant Staphylococcus aureus: the 287, 236–245. H. K., Rhomberg, P. R., Coffman, S. response controls integron recombi- European landscape. J. Antimicrob. Lai, S., Tremblay, J., and Deziel, E. L., and Brueggemann, A. B. (2001). nation. Science 324, 1034. Chemother. 66(Suppl. 4), iv43–iv48. (2009). Swarming motility: a multi- Antimicrobial resistance among clin- Gullberg, E., Cao, S., Berg, O G., Illback, Johnson, A. P., Cleaves, H. J., Dworkin, cellular behaviour conferring antimi- ical isolates of Streptococcus pneu- C., Sandegren, L., Hughes, D., et al. J. P., Glavin, D. P., Lazcano, A., and crobial resistance. Environ. Microbiol. moniae in the United States during (2011). Selection of resistant bacte- Bada, J. L. (2008). The Miller volcanic 11, 129–136. 1999–2000, including a comparison ria at very low antibiotic concentra- spark discharge experiment. Science Leung, E., Weil, D. E., Raviglione, M., of resistance rates since 1994–1995. tions. PLoS Pathog. 7:e1002158. doi: 322, 404. and Nakatani, H. on behalf of the Antimicrob. Agents Chemother. 45, 10.1371/journal.ppat.1002158 Kaufmann, G. F., Sartorio, R., Lee, S. World Health Organization World 1721–1729. Hall, B. G., and Barlow, M. (2003). H., Rogers, C. J., Meijler, M. M., Health Day Antimicrobial Resistance Doucet-Populaire, F., Trieu-Cuot, P., Structure-based phylogenies of the Moss, J. A., et al. (2005). Revisiting Technical Working Group. (2011). Dosbaa, I., Andremont, A., and Cour- serine beta-lactamases. J. Mol. Evol. quorum sensing: discovery of addi- The WHO policy package to combat valin, P. (1991). Inducible transfer of 57, 255–260. tional chemical and biological func- antimicrobial resistance. Bull. World conjugative transposon Tn1545 from Heeb, S., Fletcher, M. P., Chhabra, tions for 3-oxo-N-acylhomoserine Health Organ. 89, 390–392. Enterococcus faecalis to Listeria mono- S. R., Diggle, S. P., Williams, P., lactones. Proc. Natl. Acad. Sci. U.S.A. Levy, S. B. (1992). The Antibiotic Para- cytogenes in the digestive tracts of and Cámara, M. (2011). Quinolones: 102, 309–314. dox: How the Miracle Drugs are gnotobiotic mice. Antimicrob. Agents from antibiotics to autoinducers. Kawamura-Sato, K., Iinuma, Y., Destroying Their Miracle.NewYork: Chemother. 35,185–187. FEMS Microbiol. Rev. 35, 247–274. Hasegawa, T., Horii, T., Yamashino, Plenum Press.

www.frontiersin.org March 2013 | Volume 4 | Article 47 | 93

“fmicb-04-00047” — 2013/3/12 — 11:18 — page 11 — #11 Sengupta et al. Ecological role of antibiotics in nature

Linares, J. F., Gustafsson, I., Baquero, mirabilis by subinhibitory concentra- Rothstein, J. D., Patel, S., Regan, Davidson, A. R., et al. (2007). Initia- F., and Martinez, J. L. (2006). tions of azithromycin. Eur. J. Clin. M. R., Haenggeli, C., Huang, Y. tion of actinorhodin export in Strep- Antibiotics as intermicrobial signal- Microbiol. Infect. Dis. 11, 469–471. H., Bergles, D. E., et al. (2005). tomyces coelicolor. Mol. Microbiol. 63, ing agents instead of weapons. Proc. Murakami, T., Holt, T. G., Beta-lactam antibiotics offer neuro- 951–961. Natl. Acad. Sci. U.S.A. 103, 19484– and Thompson, C. J. (1989). protection by increasing glutamate Tanimoto, K., Tomita, H., Fujimoto, 19489. Thiostrepton-induced gene expres- transporter expression. Nature 433, S., Okuzumi, K., and Ike, Y. (2008). Liras, P., Gomez-Escribano, J. P., sion in Streptomyces lividans. J. 73–77. Fluoroquinolone enhances the muta- and Santamarta, I. (2008). Reg- Bacteriol. 171, 1459–1466. Ryan, R. P., and Dow, J. M. (2008). Dif- tion frequency for meropenem- ulatory mechanisms controlling Neeno-Eckwall, E. C., Kinkel, L. L., and fusible signals and interspecies com- selected carbapenem resistance in antibiotic production in Strepto- Schottel, J. L. (2001). Competition munication in bacteria. Microbiology Pseudomonas aeruginosa, but use of myces clavuligerus. J. Ind. Microbiol. and antibiosis in the biological con- 154, 1845–1858. the high-potency drug doripenem Biotechnol. 35, 667–676. trol of potato scab. Can. J. Microbiol. Rybak, M. J. (2006). Pharmacodynam- inhibits mutant formation. Antimi- Liu, B., and Pop, M. (2009). 47, 332–340. ics: relation to antimicrobial resis- crob. Agents Chemother. 52, 3795– ARDB-Antibiotic Resistance Genes Norrby, S. R., Nord, C. E., and Finch, tance. Am. J. Med. 119(Suppl. 1), 3800. Database. Nucleic Acids Res. 37, R. (2005). Lack of development of S37–S44; discussion S62–S70. Thomashow, L. S., and Weller, D. M. D443–D447. new antimicrobial drugs: a potential Sheldon, A. T. Jr. (2005). Antibiotic (1988). Role of a phenazine antibiotic López, E., Elez, M., Matic, serious threat to public health. Lancet resistance: a survival strategy. Clin. from Pseudomonas fluorescens in bio- I., and Blázquez, J. (2007). Infect. Dis. 5, 115–119. Lab. Sci. 18, 170–180. logical control of Gaeumannomyces Antibiotic-mediated recombi- Ochi, K., and Freese, E. (1983). Effect of Shoemaker, N. B., Vlamakis, H., Hayes, graminis var. tritici. J. Bacteriol. 170, nation: ciprofloxacin stimulates antibiotics on sporulation caused by K., and Salyers, A. A. (2001). Evi- 3499–3508. SOS-independent recombination of stringent response in Bacillus subtilis. dence for extensive resistance gene Toney, J. H., Fitzgerald, P. M., divergent sequences in Escherichia Microbiology 129, 3709–3720. transfer among Bacteroides spp. and Grover-Sharma, N., Olson, S. H., coli. Mol. Microbiol. 64, 83–93. Ohlsen, K., Ziebuhr, W., Koller, among Bacteroides and other genera May, W. J., Sundelof, J. G., Marraffini, L. A., and Sontheimer, E. K. P., Hell, W., Wichelhaus, T. in the human colon. Appl. Environ. et al. (1998). Antibiotic sensitiza- J. (2008). CRISPR interference limits A., and Hacker, J. (1998). Effects Microbiol. 67, 561–568. tion using biphenyl tetrazoles as horizontal gene transfer in staphylo- of subinhibitory concentrations of Showsh, S. A., and Andrews, R. E. potent inhibitors of Bacteroides frag- cocci by targeting DNA. Science 322, antibiotics on alpha-toxin (hla) gene Jr. (1992). Tetracycline enhances ilis metallo-beta-lactamase. Chem. 1843–1845. expression of methicillin-sensitive Tn916-mediated conjugal transfer. Biol. 5, 185–196. Martin, M. F., and Liras, P. (1989). and methicillin-resistant Staphylo- Plasmid 28, 213–224. Torres, O. R., Korman, R. Z., Zahler, Organization and expression of genes coccus aureus isolates. Antimicrob. Skindersoe, M. E., Alhede, M., Phipps, S. A., and Dunny, G. M. (1991). involved in the biosynthesis of antibi- Agents Chemother. 42, 2817–2823. R., Yang, L., Jensen, P. O., Ras- The conjugative transposon Tn925: otics and other secondary metabo- Pedro, L., Banos, R. C., Aznar, S., mussen, T. B., et al. (2008). Effects enhancement of conjugal transfer by lites. Annu.Rev.Microbiol.43, Madrid, C., Balsalobre, C., and of antibiotics on quorum sensing in tetracycline in Enterococcus faecalis 173–206. Juarez, A. (2011). Antibiotics shaping Pseudomonas aeruginosa. Antimicrob. and mobilization of chromosomal Martinez, J. L., and Rojo, F. (2011). bacterial genome: deletion of an IS91 Agents Chemother. 52, 3648–3663. genes in Bacillus subtilis and E. fae- Metabolic regulation of antibiotic flanked virulence determinant upon Song, J. S., Jeon, J. H., Lee, J. H., Jeong, calis. Mol. Gen. Genet. 225, 395–400. resistance. FEMS Microbiol. Rev. 35, exposure to subinhibitory antibiotic S. H., Jeong, B. C., Kim, S. J., et al. Tsui, W. H., Yim, G., Wang, H. H., 768–789. concentrations. PloS ONE 6:e27606. (2005). Molecular characterization McClure, J. E., Surette, M. G., and Matsushita, O., Okabe, A., Hayashi, doi: 10.1371/journal.pone.0027606 of TEM-type beta-lactamases iden- Davies, J. (2004). Dual effects of MLS H., and Kanemasa, Y. (1989). Lin- Qazi, S., Middleton, B., Muharram, S. tified in cold-seep sediments of Edi- antibiotics: transcriptional modula- comycin increases the half-life of H., Cockayne, A., Hill, P., O’Shea, son Seamount (south of Lihir Island, tion and interactions on the ribo- beta-lactamase mRNA. Antimicrob. P., et al. (2006). N-acylhomoserine Papua New Guinea). J. Microbiol. 43, some. Chem. Biol. 11, 1307–1316. Agents Chemother. 33, 805–809. lactones antagonize virulence gene 172–178. Ubeda, C., Maiques, E., Knecht, E., McPhee, J. B., Lewenza, S., and expression and quorum sensing in Spellberg, B., Guidos, R., Gilbert, D., Lasa, I., Novick, R. P.,and Penades, R. Hancock, R. E. (2003). Cationic Staphylococcus aureus. Infect. Immun. Bradley, J., Boucher, H. W., Scheld, (2005). Antibiotic-induced SOS antimicrobial peptides activate a 74, 910–919. W. M., et al. (2008). The epidemic response promotes horizontal two-component regulatory system, Quinones, M., Kimsey, H. H., and of antibiotic-resistant infections: a dissemination of pathogenicity PmrA–PmrB, that regulates resis- Waldor, M. K. (2005). LexA cleav- call to action for the medical com- island-encoded virulence factors in tance to polymyxin B and cationic age is required for CTX prophage munity from the Infectious Diseases staphylococci. Mol. Microbiol. 56, antimicrobial peptides in Pseu- induction. Mol. Cell 17, 291–300. Society of America. Clin. Infect. Dis. 836–844. domonas aeruginosa. Mol. Microbiol. Raghunath, D. (2008). Emerging antibi- 46, 155–164. Valentine, P. J., Shoemaker, N. B., and 50, 205–217. otic resistance in bacteria with spe- Srinivasan, A., Dick, J. D., and Perl, T. Salyers, A. A. (1988). Mobilization of Melhus, A. (2001). Effects of amoxicillin cial reference to India. J. Biosci. 33, M. (2002). Vancomycin resistance in Bacteroides plasmids by Bacteroides on the expression of cytokines dur- 593–603. staphylococci. Clin. Microbiol. Rev. conjugal elements. J. Bacteriol. 170, ing experimental acute otitis media Ramón-García, S., Martín, C., Thomp- 15, 430–438. 1319–1324. caused by non-typeable Haemophilus son, C. J., and Aínsa, J. A. (2009). Stevens, A. M., Shoemaker, N. B., Li, L. Ventura, M., Canchaya, C., Tauch, A., influenzae. J. Antimicrob. Chemother. Role of the Mycobacterium tuber- Y., and Salyers, A. A. (1993). Tetra- Chandra, G., Fitzgerald, G. F., Chater, 48, 397–402. culosis P55 efflux pump in intrin- cycline regulation of genes on Bac- K. F., et al. (2007). Genomics of Acti- Mesak, L. R., Miao, V., and Davies, sic drug resistance, oxidative stress teroides conjugative transposons. J. nobacteria: tracing the evolutionary J. (2008). Effects of subinhibitory responses, and growth. Antimi- Bacteriol. 175, 6134–6141. history of an ancient phylum. Micro- concentrations of antibiotics on SOS crob. Agents Chemother. 53, 3675– Strohl, W. R. (1997). “Industrial antibi- biol. Mol. Biol. Rev. 71, 495–548. and DNA repair gene expression 3682. otics: today and the future,” in Waksman, S. A. (1961). The role of in Staphylococcus aureus. Antimi- Retzlaff, L., and Distler, J. (1995). Biotechnology of Antibiotics, 2nd Edn, antibiotics in nature. Perspect. Biol. crob. Agents Chemother. 52, 3394– The regulator of streptomycin gene Vol. 82, Drugs and the Pharmaceutical Med. 4, 271–286. 3397. expression, StrR, of Streptomyces Sciences, ed. W. R. Strohl (New York: Whittle, G., Shoemaker, N. B., and Saly- Molinari, G., Paglia, P., and Schito, G. griseus is a DNA binding activa- Marcel Dekker), 1–47. ers, A. A. (2002). Identification of two C. (1992). Inhibition of motility of tor protein with multiple recognition Tahlan, K., Ahn, S. K., Sing, A., genes involved in the modulation of Pseudomonas aeruginosa and Proteus sites. Mol. Microbiol. 18, 151–162. Bodnaruk, T. D., Willems, A. R., conjugal transfer of the Bacteroides

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 47 | 94

“fmicb-04-00047” — 2013/3/12 — 11:18 — page 12 — #12 Sengupta et al. Ecological role of antibiotics in nature

conjugative transposon CTnDOT. J. Yarmolinsky, M. B., and Haba, G. L. Zhang, X., McDaniel, A. D., Wolf, Citation: Sengupta S, Chattopadhyay Bacteriol. 184, 3839–3847. (1959). Inhibition by puromycin of L. E., Keusch, G. T., Waldor, M. MK and Grossart H-P (2013) The Wright, G. D. (2010). Antibiotic resis- amino acid incorporation into pro- K., and Acheson, D. W. (2000). multifaceted roles of antibiotics and tance in the environment: a link to tein. Proc. Natl. Acad. Sci. U.S.A. 45, Quinolone antibiotics induce Shiga antibiotic resistance in nature. Front. the clinic? Curr. Opin. Microbiol. 13, 1721–1729. toxin-encoding bacteriophages, toxin Microbiol. 4:47. doi: 10.3389/fmicb.2013. 589–594. Yim, G., McClure, J., Surette, M. G., production, and death in mice. J. 00047 Wright, G. D., and Poinar, H. and Davies, J. E. (2011). Modula- Infect. Dis. 181, 664–670. This article was submitted to Fron- (2012). Antibiotic resistance is tion of Salmonella gene expression tiers in Antimicrobials, Resistance and ancient: implications for drug discov- by subinhibitory concentrations of Chemotherapy, a specialty of Frontiers in ery. Trends Microbiol. 20, 157–159. quinolones. J. Antibiot. (Tokyo) 64, Microbiology. Yamashita, F., Hotta, K., Kurasawa, 73–78. Conflict of Interest Statement: The Copyright © 2013 Sengupta, Chattopad- S., Okami, Y., and Umezawa, H. Yim, G., Wang, H. H., and Davies, J. authors declare that the research was hyay and Grossart. This is an open- (1985). New antibiotic-producing (2006a). The truth about antibiotics. conducted in the absence of any com- access article distributed under the terms streptomycetes, selected by antibi- Int. J. Med. Microbiol. 296, 163–170. mercial or ﬁnancial relationships that of the Creative Commons Attribution otic resistance as a marker. I. New Yim, G., de la Cruz, F., Spiegelman, could be construed as a potential con- License, which permits use, distribution antibiotic production generated by G., and Davies, J. (2006b). Transcrip- ﬂict of interest. and reproduction in other forums, pro- protoplast fusion treatment between tion modulation of S. typhimurium vided the original authors and source Streptomyces griseus and S. tenji- promoters by sub-MIC levels of Received: 12 October 2012; accepted: are credited and subject to any copy- mariensis. J. Antibiot. (Tokyo) 38, rifampicin. J. Bacteriol. 188, 7988– 20 February 2013; published online: 12 right notices concerning any third-party 58–63. 7991. March 2013. graphics etc.

www.frontiersin.org March 2013 | Volume 4 | Article 47 | 95

“fmicb-04-00047” — 2013/3/12 — 11:18 — page 13 — #13 MINI REVIEW ARTICLE published: 07 March 2013 doi: 10.3389/fmicb.2013.00044 Broad-host-range IncP-1 plasmids and their resistance potential

Magdalena Popowska* and Agata Krawczyk-Balska Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland

Edited by: The plasmids of the incompatibility (Inc) group IncP-1, also called IncP,as extrachromosomal Fiona Walsh, Agroscope Changins genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are Wädenswil, Switzerland composed of backbone genes that encode a variety of essential functions and accessory Reviewed by: genes that have implications for human health and environmental bioremediation. Broad- Holger Heuer, Julius Kühn-Institut, Federal Research Centre for host-range IncP plasmids are known to spread genes between distinct phylogenetic groups Cultivated Plants, Germany of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, Yoshimi Matsumoto, Osaka heavy metals, and quaternary ammonium compounds used as disinfectants.The backbone University, Japan of these plasmids carries modules that enable them to effectively replicate, move to *Correspondence: a new host via conjugative transfer and to be stably maintained in bacterial cells. The Magdalena Popowska, Department of Applied Microbiology, Institute of adaptive, resistance, and virulence genes are mainly located on mobile genetic elements Microbiology, Faculty of Biology, integrated between the functional plasmid backbone modules. Environmental studies have University of Warsaw, Ilji Miecznikowa demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater 1, 02-096 Warsaw, Poland. e-mail: [email protected] treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad-host- range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

Keywords: IncP plasmid, antibiotic resistance, heavy metals, xenobiotic, catabolism, horizontal gene transfer, bioremediation

CHARACTERISTICS OF IncP-1 PLASMIDS in a broad range of hosts (Shintani et al., 2010a). These plasmids A characteristic feature of all plasmids, including IncP-1 plas- demonstrategreatdiversity–aphylogeneticstudyhasshownthe mids, is that they belong to specific incompatibility (Inc) groups. presence of 45 backbone genes on IncP-1 plasmids, but only 33 Plasmids are classified into Inc groups based on their replication are shared by all the plasmids that have been completely sequenced and partitioning systems: two plasmids belonging to the same to date. Inc group are unable to coexist in a single bacterial cell (Shintani A detailed phylogenetic analysis and study of the amino acid et al., 2010b). sequence of protein TrfA, which initiates plasmid replication, Members of the Enterobacteriaceae family and the genus Pseu- have allowed the classification of group IncP-1 into six subgro- domonas carry plasmids belonging to more than 30 Inc groups. ups: -α,-β,-γ,-ε,-δ (Bahl, 2009), and -ζ (Norberg et al., 2011) Plasmids from four of these groups (IncP,W, N, and Q) can trans- and also into an unnamed subgroup (Pachulec and van der Does, fer between and maintain themselves in both enteric bacteria and 2010). Unpublished data also indicate the existence of a η sub- strains of Pseudomonas (Dröge et al., 2000). In particular, IncP group (Sen et al., 2012). IncP-1 plasmids have been identified plasmids are widely distributed in Gram-negative bacteria, e.g., in in clinical and environmental, phylogenetically very distant, host Escherichia coli, Pseudomonas spp., Klebsiella aerogenes, and Sphin- bacterial species from around the world. Bacteria carrying these gomonas (Thomas, 2000; Shintani et al., 2010a). It should be added plasmids have been isolated from soil in areas contaminated by that IncP-1 in the Pseudomonas plasmid classification corres- industry, pig manure, wastewater of industrial origin, river sedi- ponds to IncP in the E. coli plasmid classification (Thomas and ment, and fresh water (Bahl, 2009; Norberg et al., 2011; Sen et al., Haines, 2004). 2012). Comparative analysis of the nucleotide sequences of plas- The plasmids of the subgroup IncP-1 are of considerable inter- mids belonging to the IncP-1 subgroups α and β demonstrated est to both molecular biologists and environmentalists due to the high similarity of their gene organization and also led to the their highly efficient conjugative transfer and ability to replicate identification of adaptive genes called “load genes.” The backbone

www.frontiersin.org March 2013 | Volume 4 | Article 44 | 96

“fmicb-04-00044” — 2013/3/6 — 11:20 — page1—#1 Popowska and Krawczyk-Balska IncP plasmids and antibiotic resistance

sequences of these plasmids are responsible for the regulation of There are four basic mechanisms of plasmid-encoded antibi- replication, stable maintenance in a bacterial cell and efficient con- otic resistance: (i) enzymatic hydrolysis of the antibiotic molecule, jugative transfer. In plasmids of the IncP-1β subgroup, the Tra1 which results in inactivation of the compound, (ii) enzymatic and Tra2 regions of the backbone, that mediate conjugative trans- modification of the antibiotic to prevent interaction with the cel- fer, are always separated by regions of clustered restriction sites, lular target, (iii) efflux, which removes the antibiotic from the which may represent hotspots for the integration of various deter- interior of the cell via specific cell membrane pumps, and (iv) a minants. These regions can be a site for the insertion of mobile reaction called “bypass,” which is the formation of an alternative genetic elements (MGE) carrying catabolic genes, and antibiotic metabolic pathway that allows bypassing of the blocked stage and and heavy metal resistance determinants. The capacity for MGE modification of the antibiotic target (Alekshun and Levy, 2007). integration is highly variable among IncP-1 plasmids. Plasmids The genes encoding the proteins responsible for these mechanisms pJP4, pSS60, and pSS50, belonging to the subgroup IncP-1β,con- are found on plasmids present in both clinical and environmen- tain one site for the integration of catabolic genes in their plasmid tal bacterial isolates (Aminov and Mackie, 2007; Allen et al., 2010; backbone, which is located between the oriV and trfA system repli- Wright, 2010). cation sequences. The number of insertions in the backbone varies Table 1 shows plasmids from IncP-1 group, well characterized among IncP-1 plasmids (Trefault et al., 2004). pB4 is the only plas- in terms of phenotype and molecular mechanisms of observed mid known to have three insertion sites: catabolic genes grouped resistance to antibiotics and heavy metals. Resistance plasmids into an operon are located near the oriV sequence, and antibiotic classified within subgroup IncP-1α were first identified in hos- resistance determinants are located at both ends of region Tra2 pital isolates of Pseudomonas aeruginosa and K. aerogenes. These (Adamczyk and Jagura-Burdzy, 2003). were found to encode multi-drug resistance (MDR) against antibi- There are currently only three known representatives of the sub- otics including penicillin, kanamycin, and tetracycline (Adamczyk group IncP-1δ, namely plasmids pEST4011, pIJB1, and pAKD4. and Jagura-Burdzy, 2003). Using the exogenous plasmid isolation Plasmid pAKD4 is regarded as the prototype of this subgroup method Dröge et al. (2000) found resistance plasmids, including because it is the only one to possess the complete backbone those belonging to the subgroup IncP-1α, in bacterial communi- sequence. Like other IncP-1 plasmids, those of subgroup δ carry ties from activated sludge derived from wastewater treatment plant specific genes that are located on transposons integrated within (WWTP). Twelve plasmids were identified carrying an antibiotic the plasmid backbone (Sen et al., 2010, 2012). and heavy metal resistance determinant, and ten of these could More than 50 plasmids belonging to the subgroup IncP-1ε have be classified to group IncP. These studies confirmed the wide recently been identified and characterized. These plasmids confer distribution of this Inc group in these environments. resistance to a broad spectrum of antibiotics. This is due to the To date, the complete nucleotide sequences of six resistance presence of a class 1 integron that carries the gene sul1 responsible plasmids belonging to subgroup IncP-1α have been determined. for sulphonamide resistance at its 5 end, plus gene cassettes carry- These plasmids, RP4/RK2, pTB11, pBS228, pB5, pB11, and pSP21, ing several determinants of resistance to other antibiotics (Heuer show a high degree of similarity with respect to elements of their et al., 2012). Plasmids belonging to subgroup IncP-1ε exhibit great plasmid backbone, while they are distinguished by the nature of diversity (Sen et al., 2011). the genes located within their adaptive modules (e.g., ISTB11, ISSP21, Tn402 derivatives, Tn1,Tn7). Plasmid pB5 contains an ANTIBIOTIC RESISTANCE GENES PRESENT ON IncP integron of class 1 carrying a resistance cassette with aacC1 and PLASMIDS aacA4 genes, which encode enzymes that provide resistance to The extensive use of antibiotics for the treatment of humans kanamycin, gentamicin, and tobramycin (Szczepanowski et al., and animals, and to stimulate the growth of livestock, has led 2011). The IncP-1α plasmids contain different variants of the inte- to the rapid emergence and spread of resistance determinants gron aadA gene cassette (aadA1, aadA2, aadA4, and aadA5), which to a broad spectrum of antibiotic substances (Allen et al., 2010; encode streptomycin/spectinomycin-300-O-adenylyltransferases Aminov, 2010; Popowska et al., 2010, 2012; Tello et al., 2012). conferring resistance to the aminoglycosides streptomycin and These determinants are located mainly on MGE such as plas- spectinomycin (Tennstedt et al., 2003, 2005). The aadA2 gene cas- mids and conjugative transposons, which ensures their spread sette is widely distributed, having been identified in the integrons by horizontal gene transfer. Horizontal gene transfer involving of many bacteria belonging to at least 11 genera. Numerous studies the acquisition of foreign DNA by transduction, transformation, have demonstrated that different environmental and pathogenic or conjugation, is quite common among bacteria and plasmids bacteria share a common pool of integron-specific resistance gene play a key role in this process (Aminov, 2011; Martinez, 2009; cassettes (Tennstedt et al., 2003, 2005). Stokes and Gillings, 2011). Among the plasmids conferring resis- In the case of plasmids pTB11 and pSP21, a class 1 integron car- tance to antibiotics, representatives of the incompatibility groups rying genes that determine bacterial resistance to aminoglycosides P, Q, N, and W have been identified. These are characterized by is located within transposon Tn402. In addition, these plasmids their wide host range, including pathogenic bacteria, and so pose possess a region containing genes for tetracycline resistance (tetA a serious threat to human health (Dröge et al., 2000). Numerous and tetR; Tennstedt et al., 2005; Szczepanowski et al., 2011). studies have demonstrated that the prevalence of such plasmids The IncP-1α plasmid pTB11 carries the aphA-orf3 module, in different environments is very high, e.g., in soil, surface waters, encoding aminoglycoside-30-phosphotransferase responsible for wastewaters, and natural fertilizers (Götz et al., 1996; Haines et al., aminoglycoside resistance, that shares the highest degree of 2006; Binh et al., 2008; Sen et al., 2011; Heuer et al., 2012). similarity with the corresponding gene of plasmid RP4, originating

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 44 | 97

“fmicb-04-00044” — 2013/3/6 — 11:20 — page2—#2 Popowska and Krawczyk-Balska IncP plasmids and antibiotic resistance (Continued) Szczepanowski et al. Szczepanowski et al. Sen et al. (2012) Tauch et al. (2003) Dröge et al. (2000) ; Dröge et al. (2000) , /JX469826.1 /NC_003430.1 /NC_019020.1 (2011) /CP002152.1 (2011) Dröge et al. (2000) , Dröge et al. (2000) Schlüter et al. (2005)/NC_007502.1 Schlüter et al. (2003)/NC_004840.1 Norberg et al./AY950444.1 (2011) Pansegrau et al./L27758.1 (1994) Harayama et al. (1980) Bahl et al. (2007)/NC_008272.1 Sen et al./JN106164.1 (2011) Heuer et al. (2004) /NC_006388.1 Dröge et al. (2000) , Tennstedt et al. (2005)/NC_006352.1 Thorsted et al. (1998) , family) 501 Tn 501 ), Tn 1721, /(Tn 3 1721, 5719 QAC 1 “cryptic” Tn, Tn 402/ 402 (Tn 5090 Tn 5090 ,Tn Tn 5053 Tn 402 nd nd nd Tn 5501, Tn 5090 ,Tn /Tn 21 Tn 501 Tn 5393c ,Tn Tn 21 ,Tn Tn 501, Tn 402/ Tn 5053 Tn 4371 ,Tn Tn 402 Tn 402/ Tn 21 -like transposon Tn- tet, Tn 5393c ,Tn , cmlA1, sul1, NPS_2 , strAB, mexCD-oprJ, chr NPS_1 aacA4, aacC1, tetA, tetR, aphA, sul1, qacE 1 aadA4, oxa2, sul1, qacE 1, qacF oxa2, sul1, strAB, tetA, qacE 1, mer tetA, aacA4, oxa2, sul1, qacE 1 nd tetA, tetR, aphA, merE aphA, aadA1, aacA4, oxa2, tetA, tetR oprN, merE tetA, aph nd tetA, tetR, dfrA1, aadA11b, sul1, qacE 1 dhfrIIc aadA, sul1, merE bla tetA(C), tetR(C), qacE 1 aadA2, bla am, Tc -lact β β -lactam, Cm, Su, β -lactam / Su, β -lactam, Su, quaternary -lactam, Su, Sm, Tc, quaternary -lactam, tripartite multi-drug Sm, Tc, Km, Gm, Su, quaternary ammonium compounds ammonium compounds, Hg Sm, Sp, ammonium compounds quaternary ammonium compounds Multi-drug efﬂux (MDE) outer membrane prot. NodT family, Hg quaternary ammonium compounds ResistancesSp, Sm, Su, Hg Resistance genes Detected transposons Reference/accession No. resistance (MDR) efﬂux system, Sm, Em, Chr compounds Aminoglycosides, Aminoglycosides, Tc, Sm, Sp, Cm, Su Tc, Ap, Km, Hg Tc, Sm, Sp, Em, Tc, Km, Ap Tc, Km, Ap Tp Tc, Tp, aminoglycosides, Su, Sp, Tc, quaternary ammonium β IncP-1 α IncP-1 subgroup 60,732/56,167β IncP-1 General features of IncP-1 plasmids carrying resistance genes. # | pTB11 68,869α IncP-1 pB5 64,696α IncP-1 pB6pB8 58 57,198pB10β IncP-1 64,508pB11 66,911pB12ββ IncP-1 64,393α IncP-1 β IncP-1 pMCBF1 62,689ζ IncP-1 RP4/RK2 60,099α IncP-1 pTH10 70 R751 53,423β IncP-1 pKJK5 54,383ε IncP-1 Plasmid Size (kb)pAKD1 IncP-1 58,246β IncP-1 pB2/pB3 pB4 79,370ββ IncP-1 Table 1

www.frontiersin.org March 2013 | Volume 4 | Article 44 | 98

“fmicb-04-00044” — 2013/3/6 — 11:20 — page3—#3 Popowska and Krawczyk-Balska IncP plasmids and antibiotic resistance Szczepanowski et al./NC_019021.1 (2011) Unpublished/CP003505 Haines et al. (2007)/NC_008357.1 Sen et al./JX469830.1 (2012) Sen et al./JX469833.1 (2012) Sen et al./JX469831.1 (2012) Pansegrau et al., (1994) Sen et al.JX469825.1 (2012) Sen et al./JQ432564.1 (2012) 7 1 ,Tn /Tn 402 family) 1721 5053 ,Tn 5718 /(Tn 3 7 ,Tn ,Tn nd nd Tn 1013 Tn 6305 Tn 3 ,Tn Tn 3 Tn 402 ,Tn Tn 1525 , tetA, , aph, − 67 1 − 67 TEM TEM bla bla , aphA, merE − 67 TEM bla aadA1, aphA, sph, tetA, merE aacA4, strAB, sul1, qacE 1, merE tetR aac(6)-Ib, sul1, merE aadA5, oxa2, sul1, qacE tetA, tetR, aph, aadA1, aacA4, oxa2 aadA, dhfr, tetA, aac(6)-Ib, aphA1, pEC-IMPQ_139 β -lactam β -lactam β -lactam, Su, β -lactam, Km, Hg Resistancesβ -lactam, Tc Resistance genes Detected transposons Reference/accession No. ammonium compounds, Hg quaternary ammonium compounds β -lactam, Hg Aminoglycosides, Km, Sm, Aminoglycosides, Su, quaternary Aminoglycosides, Sp, Tp, Tc, Tc, Km, aminoglycosides, subgroup Continued | Plasmids pB2 and pB3 differ only by a duplication of a tetA(C)-tetR-tnpAIS26 fragment in pB2. Plasmid Size (kb) IncP-1 pG527 80,762α IncP-1 # Hg, inorganic mercury; nd, not determined. pWEC911 74,056 IncP-1 Tc, BRA100 56,265β IncP-1 pSP21 72,683α IncP-1 pKSP212 54,342pBRSB222 36,880 IncP-1 IncP-1 Aminoglycosides, Su , Hg Aminoglycosides, pBS228 89,147α IncP-1 pKS208 50,604 IncP-1 Aminoglycosides, Km, Ap, ampicillin; Cb, carbenicillin; Cm, chloramphenicol; Gm, gentamicin; Km, kanamycin; Nx, nalidixic acid; Sm, streptomycin; Sp, spectinomycin; Su, sulfanilamide;Tc, tetracycline;Tp, trimethoprim;Tm, tobramycin; Table 1

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 44 | 99

“fmicb-04-00044” — 2013/3/6 — 11:20 — page4—#4 Popowska and Krawczyk-Balska IncP plasmids and antibiotic resistance

from a clinical isolate. Another aminoglycoside resistance gene sulphonamides, tetracycline, trimethoprim, disinfectants, and cassette located on pTB11 and pSp32 encodes an aminoglycoside- mercury ions. The greatest MGE diversity has been observed 60-N-acetyltransferase mediating resistance to different amino- in plasmids of this subgroup isolated from bacteria living in glycosides such as tobramycin, amikacin, and gentamicin. WWTP (Szczepanowski et al., 2004). WWTP bacteria represent Two other aminoglycoside resistance gene cassettes, aadB and a reservoir for β-lactamase genes that have yet to be isolated aacA29b, which respectively, code for an aminoglycoside-200-O- from clinical strains. Mobile integron gene cassettes seem to adenylyltransferase and an aminoglycoside-60-N-acetyltransferase, function in the dissemination of β-lactamase genes in sewage have also been identified on IncP-1α plasmids. The aadB gene was habitats. found on a plasmid from a clinical isolate of Acinetobacter bau- An important role in the spread of resistance genes in agricul- mannii, while an aacA29b gene cassette, present on a plasmid, tural systems is played by representatives of subgroup IncP-1ε. represents a new variant of aacA29a previously found on In59 in The study of Heuer et al. (2012) showed that fifty plasmids a clinical P. aeruginosa isolate (Siarkou et al., 2009). belonging to this subgroup, isolated from samples of soil and Resistance plasmids are also represented in subgroup IncP-1β. manure, contain integrons of class 1 with gene cassettes plus the These include the fully sequenced plasmids pB2/pB3, PB4, PB8, gene sul1 (sulphonamide resistance). The nucleotide sequences of and PB10. These plasmids carry a class 1 integron or, in the case four of these plasmids have been determined (pKS77, pHH3414, of PB4, a fragment of Tn402. Besides sul1 that confers resistance pHH128, and pHH3408) and they differ mainly in the organi- to sulfonamides, these plasmids may also contain genes for resis- zation of the elements in transposon Tn402. These elements are tance to other antibiotics and chemotherapeutics, e.g., oxa (PB8, a class 1 integron IS1326 and, in the case of plasmids pHH3414, PB10, and pB11) and bla (PB4 and PB3), encoding β-lactamase pKS77, and pHH128, transposon Tn1721 carrying the tetracycline enzymes responsible for resistance to β-lactam antibiotics. Parts resistance genes tetA and tetR. The resistance integrons contain the of the oxa1 gene cassette in plasmid pSp33 and blaNPS1 on the gene cassettes aadA, (pHH3414), aadB (pKS77), aadA1b, dfrA1b, Tn402 remnant in this plasmid are nearly identical to corre- and two copies of catB (pHH128; Heuer et al., 2012). The dif- sponding regions identified in different Korean clinical isolates ferent aad gene variants encode adenyltransferases that modify of Salmonella enterica ssp. enterica (Lee et al., 2003, 2004) and the streptomycin and spectinomycin molecules, resulting in their P. aeruginosa (Jacoby and Matthew, 1979; Pai and Jacoby, 2001), inactivation. A similar mechanism is employed by the chloram- respectively. However, some bla genes identified in this subgroup phenicol acetyltransferase encoded by the cat genes to confer of plasmids have novel sequences. All of the aforementioned IncP- resistance to chloramphenicol. The dfr gene permits bacterial 1β plasmids also carry genes encoding resistance to streptomycin, cells to survive in an environment with high concentrations of a member of the aminoglycoside antibiotic family. Plasmids PB4 trimethoprim (Schlüter et al., 2007). and PB10 contain transposon Tn5393c, which includes the genes Interestingly, many studies have demonstrated a close relation- strA and strB,encoding aminoglycoside-3-O-phosphotransferase ship between the occurrence of MDR and resistance/tolerance to and aminoglycoside-6-O-phosphotransferase, respectively, that heavy metal ions (Lazar et al., 2002). Bacterial strains isolated from modify the streptomycin molecule, thus causing its inactivation. agricultural soil receiving wastewater can exhibit high levels of Identical or almost identical copies of Tn5393 were recently iden- both metal and antibiotic resistance. Shafiani and Malik (2003) tified in different human, fish, and plant pathogens. Plasmid pB10 isolated 64 bacteria belonging to the genera Pseudomonas, Azo- also possesses the tetracycline resistance transposon Tn1721, that tobacter, and Rhizobium from wastewater-irrigated soil. All the was first described in E. coli (Allmeier et al., 1992). A very simi- Pseudomonas isolates were resistant to heavy metals and cloxacillin, lar transposon has been found in conjugative plasmids from the and more than half were also resistant to methicillin. Among these fish pathogen Aeromonas salmonicida (Sørum et al., 2003), the strains were MDR isolates. Ansari et al. (2008) found that 40 strains human pathogen Salmonella enterica ssp. enterica (Chiu et al., isolated from agricultural soils irrigated with wastewater showed 2005) and a clinical isolate of E. coli (Boyd et al., 2004). With resistance to most of the tested heavy metals (Ni, Cu, Zn, Pb). the use of plasmid pB10, carrying amoxicillin, streptomycin, sul- Moreover, around 75% of these strains were resistant to tetracy- fonamide, and tetracycline resistance genes, is has been shown that cline, 57.5% to doxycycline, and 50% to ampicillin and nalidixic this broad-host-range IncP-1 plasmid can be transferred to food- acid. The plasmids isolated from these resistant strains all belonged borne pathogens (Salmonella spp. and E. coli O157:H7) under to the IncP group. Out of 12 pAKD plasmids (IncP-1 group) iso- laboratory conditions (Van Meervenne et al., 2012). These results lated from Norwegian soils only one, pAKD1, encoded antibiotic show that the antibiotic resistance genes encoded on plasmid resistance in addition to mercury resistance. This plasmid car- pB10 can be expressed in the new hosts (pathogenic organ- ries a MDR Tn21-like transposon with a class 1 integron (Sen isms) making these pathogens resistant to multiple antibiotics. et al., 2011). These data suggest heavy metals treatment might have These studies have shown that there is a serious risk to human co-selected for MDR genes. This is consistent with previous and health in cases of such transfer. The plasmid PB3 carries the present observations that pollution with heavy metals can cause a gene clmA1 conferring resistance to chloramphenicol. The prod- rise in the abundance of drug resistance genes (Baker-Austin et al., uct of the clmA1 gene is a protein that acts as an efflux pump, 2006; Seiler and Berendonk, 2012). which removes the drug from the bacterial cell (Schlüter et al., 2007). The genetic modules on these IncP-1β plasmids con- CONCLUSION sist mainly of MGE that carry diverse determinants conferring The increasing use of antibiotics in human and veterinary resistance to different aminoglycosides, β-lactams, macrolides, medicine and agricultural production systems has caused the

www.frontiersin.org March 2013 | Volume 4 | Article 44 |100

“fmicb-04-00044” — 2013/3/6 — 11:20 — page5—#5 Popowska and Krawczyk-Balska IncP plasmids and antibiotic resistance

increasing development of high levels of and novel antibiotic resis- Escherichia, Salmonella, Shigella, Klebsiella, Aeromonas, and Pseu- tances and the rapid global spread of antibiotic resistance genes domonas. Of course, in order to determine the level of risk of (Aminov, 2009, 2010; Martinez, 2009; Schlüter et al., 2007; Sen the spread of resistance genes located on these plasmids extensive et al., 2011). A consequence of the horizontal transfer of the genes experimental studies are required. However, the demonstrated located on MGE, especially on Broad-host-range plasmids is the ability to transfer multiresistance pB10 plasmid, isolated from a rising detection and levels of resistant bacteria, particularly of WWTP, to the foodborne pathogens Salmonella spp. or E. coli MDR bacteria, both in clinical and natural environments (Dröge O157: H7 with expression of most of the transferred resistance et al., 2000; Aminov and Mackie, 2007). genes in these strains poses a real threat to the life and health of IncP-1 plasmids are characterized by their wide distribution people (Van Meervenne et al., 2012). in the environment and ability to efficiently replicate in the cells Detailed analysis of IncP-1 plasmid genomes can shed light of diverse hosts. In addition, the presence of transposons car- on the adaptive mechanisms underlying bacterial evolution and rying both antibiotic and heavy metal resistance genes on these this information may be used to develop effective methods of plasmids permits the host bacteria to survive and multiply in bioremediation of contaminated land with xenobiotic and antibi- environments contaminated with these compounds. The broad- otics and heavy metals. Intensive research on the biodegradation host-range IncP-1 plasmids carry determinants for resistance to at of compounds that are a serious threat to the environment has least one heavy metal (Ni, Cd, Co, Cu, Hg, Pb, Zn), and antibiotics led to the isolation and characterization of bacterial strains that of different groups, i.e., tetracyclines, quinolones, aminoglyco- can use these substances as a source of carbon and energy. In sides, sulfonamides, β-lactams, and chemotherapeutics. This fact many cases, the adaptation of these microorganisms is due to plas- is not surprising in light of recent data showing that heavy met- mids (mainly of Inc group P) that carry genes encoding enzymes als present in the environment might have co-selected for MDR involved in the degradation of xenobiotics. It is known, that the genes (Seiler and Berendonk, 2012). The two main mechanisms degradative plasmids (IncP-1, P-7, and P-9) contain catabolic of antibiotic resistance encoded on IncP-1 plasmids are mediated genes, often arranged as operons, encoding enzymes required for by efflux pumps and enzymatic modification or hydrolysis of the the degradation and utilization of many toxic compounds includ- antibiotic molecule. IncP-1 plasmid-containing bacteria are also ing naphthalene, toluene, chlorobenzene, p-toluenesulfonate, key players in the horizontal transfer of antibiotic resistance in 2,4-D, haloacetate, and atrazine (Williams, 2004; Shintani et al., agricultural systems. It has been suggested that the spread of multi- 2010a,b). The majority of bacterial genes encoding enzymes of resistant bacteria in soil, water, and WWTPs is mainly due to their catabolic pathways are located on MGE such as plasmids, inser- possession of IncP-1 plasmids (Schlüter et al., 2003, 2005; Aminov tion sequences (e.g., as a form of composite transposons), trans- and Mackie, 2007; Bahl et al., 2007; Ansari et al., 2008; Norberg posons, integrons, genomic islands, and phage genomes (Top et al., et al., 2011; Sen et al., 2011). 2002; Top and Springael, 2003; Nojiri et al., 2004; Springael and Based on their DNA sequences, it can be concluded that the Top, 2004). vast majority of these resistance genes are located on MGE such Theoretically, genetic modules present in this type of ele- as transposons and/or integrons (Tn402-like transposon with a ment (both in catabolic and resistance plasmids) may be used class 1 integron), embedded within these transposons. In addition, in the construction of genetically modified strains that are opti- the presence of different combinations of MGE and integrons in mized for use in bioremediation and biotechnology (Vidali, 2001; IncP plasmids as well as of characteristic hot spots for the inte- Urgun-Demirtas et al., 2006; Singh et al., 2011). This is of great gration of accessory elements creates unlimited possibilities for importance due to the fact that these antimicrobial agents are pow- rearrangements of the genetic material of these plasmids. It there- erful inhibitors of xenobiotic biodegradation activities, mainly due fore appears that the spread of resistance genes would be possible to their bacteriostatic or bactericidal activities. However, before the and quite hazardous in infection control. Literature data show that broader implementation of such microorganisms, further research many representatives of this group of plasmids carrying resistance is required to thoroughly characterize them in order to limit the genes have been identified in pathogenic bacteria of the genus spread of antibiotic resistance genes.

REFERENCES Allmeier, H., Cresnar, B., Greck, Aminov, R. I. (2011). Horizontal gene a Danish wastewater treatment plant. Adamczyk, M., and Jagura-Burdzy, M., and Schmitt, R. (1992). exchange in environmental micro- Plasmid 62, 134–139. G. (2003). Spread and survival Complete nucleotide sequence of biota. Front. Microbiol. 2:158. doi: Bahl, M. I., Hansen, L. H., Goes- of promiscuous IncP-1 plas- Tn1721: gene organization and a 10.3389/fmicb.2011.00158 mann, A., and Sørensen, S. J. (2007). mids. Acta Biochim. Pol. 50, novel gene product with features Aminov, R., and Mackie, R. I. (2007). The multiple antibiotic resistance 425–453. of a chemotaxis protein. Gene 111, Evolution and ecology of antibiotic IncP-1 plasmid pKJK5 isolated from Alekshun, M. N., and Levy, S. B. (2007). 11–20. resistance genes. FEMS Microbiol. a soil environment is phylogenet- Molecular mechanisms of antibacte- Aminov, R. I. (2009). The role of Lett. 271, 147–161. ically divergent from members of rial multidrug resistance. Cell 128, antibiotics and antibiotic resistance Ansari, M. I., Grohmann, E., and Malik, the previously established alpha, beta 1037–1050. in nature. Environ. Microbiol. 11, A. (2008). Conjugative plasmids in and delta sub-groups. Plasmid 58, Allen, H. K., Donato, J., Wang, H. 2970–2988. multi-resistant bacterial isolates from 31–43. H., Cloud-Hansen, K. A., Davies, J., Aminov, R. I. (2010). A brief his- Indian soil. J. Appl. Microbiol. 104, Baker-Austin, C., Wright, M. S., and Handelsman, J. (2010). Call of tory of the antibiotic era: lessons 1774–1781. Stepanauskas, R., and McArthur, J. V. the wild: antibiotic resistance genes learned and challenges for the Bahl, M. I. (2009). All IncP-1 plasmid (2006). Co-selection of antibiotic and in natural environments. Nat. Rev. future. Front. Microbiol. 1:134. doi: subgroups, including the novel e sub- metal resistance. Trends Microbiol. 14, Microbiol. 8, 251–259. 10.3389/fmicb.2010.00134 group, are prevalent in the inﬂuent of 176–182.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 44 |101

“fmicb-04-00044” — 2013/3/6 — 11:20 — page6—#6 Popowska and Krawczyk-Balska IncP plasmids and antibiotic resistance

Binh, C. T., Heuer, H., Kaupenjohann, complete sequences of plasmids pB2 Popowska, M., Rzeczycka, M., Miernik, Shintani, M., Takahashi, Y., Yamane, M., and Smalla, K. (2008). Piggery and pB3 provide evidence for a recent A., Krawczyk-Balska, A., Walsh, H., and Nojiri, H. (2010a). The manure used for soil fertilization is ancestor of the IncP-1β group with- F., and Duffy, B. (2012). Influ- behavior and significance of degrada- a reservoir for transferable antibiotic out any accessory genes. Microbiology ence of soil use on prevalence tive plasmids belonging to Inc groups resistance plasmids. FEMS Microbiol. 150, 3591–3599. of tetracycline, streptomycin, in Pseudomonas within natural envi- Ecol. 66, 25–37. Jacoby, G. A., and Matthew, M. and erythromycin resistance and ronments and microcosms. Microbes Boyd, D. A., Tyler, S., Christian- (1979). The distribution of β-lact- associated resistance genes. Antimi- Environ. 25, 253–265. son, S., McGeer, A., Muller, M. amase genes on plasmids found in crob. Agents Chemother. 56, 1434– Shintani, M., Yamane,H., and Nojiri, H. P., Willey, B. M., et al. (2004). Pseudomonas. Plasmid 2, 41–47. 1443. (2010b). Behaviour of various hosts Complete nucleotide sequence of Lazar, V.,Cernat, R., Balotsen, C., Cotar, Schlüter, A., Heuer, H., Szczepanowski, of the IncP-7 carbazole-degradative a 92-kilobase plasmid harboring A., Coipan, E., and Cojocaru, C. R., Forney, L. J., Thomas, C. plasmid pCAR1 in artificial micro- the CTX-M-15 extended-spectrum (2002). Correlation between multiple M., Puhler, A., et al. (2003). The cosms. Biosci. Biotechnol. Biochem. beta-lactamase involved in an out- antibiotic resistance and heavy metal 64508 bp IncP-1β antibiotic multire- 74, 343–349. break in long-term-care facilities in tolerance among some E. coli strains sistance plasmid pB10 isolated from a Siarkou, V. I., Vitti, D., Protonotariou, Toronto, Canada. Antimicrob. Agents isolated from polluted waters. Bacte- waste-water treatment plant provides E., Ikonomidis, A., and Sofianou, D. Chemother. 48, 3758–3764. riol. Virusol. Parazitol. Epidemiol. 47, evidence for recombination between (2009). Molecular epidemiology of Chiu, C. H., Tang, P., Chu, C., 155–160. members of different branches of outbreak-related Pseudomonas aerug- Hu, S., Bao, Q., Yu, J., et al. Lee, K., Yong, D., Yum, J. H., the IncP-1β group. Microbiology 149, inosa strains carrying the novel vari- (2005). The genome sequence of Kim, H. H., and Chong, Y. (2003). 3139–3153. ant blaVIM-17 metallo-β-lactamase Salmonella enterica serovar Cholerae- Diversity of TEM-52 extended- Schlüter, A., Heuer, H., Szczepanowski, gene. Antimicrob. Agents Chemother. suis, a highly invasive and resistant spectrum beta-lactamase-producing R., Poler, S. M., Schneiker, S., Püh- 53, 1325–1330. zoonotic pathogen. Nucleic Acids Res. nontyphoidal Salmonella isolates in ler, A., et al. (2005). Plasmid pB8 Singh, J. S., Abhilash, P. C., Singh, H. 33, 1690–1698. Korea. J. Antimicrob. Chemother. 52, is closely related to the prototype B., Singh, R. P., and Singh, D. P. Dröge, M., Puhler, A., and Selbitschka, 493–496. IncP-1β plasmid R751 but trans- (2011). Genetically engineered bac- W. (2000). Phenotypic and molec- Lee, K., Yong, D., Yum, J. H., Lim, fers poorly to Escherichia coli and teria: an emerging tool for envi- ular characterization of conjugative Y. S., Kim, H. S., Lee, B. K., et al. carries a new transposon encod- ronmental remediation and future antibiotic resistance plasmids iso- (2004). Emergence of multidrug- ing a small multidrug resistance research perspectives. Gene 480, lated from bacterial communities of resistant Salmonella enterica serovar (SMR) efflux protein. Plasmid 54, 1–9. activated sludge. Mol. Gen. Genet. typhiinKorea.Antimicrob. Agents 135–148. Sørum, H., L’Abe’e-Lund, T. M., Sol- 263, 471–482. Chemother. 48, 4130–4135. Schlüter, A., Szczepanowski, R., Pühler, berg, A., and Wold, A. (2003). Götz, A., Pukall, R., Smit, E., Tietze, Martinez, J. L. (2009). Environmen- A., and Top, E. M. (2007). Genomics Integron-containing IncU R plas- E., Prager, R., Tschäpe, H., et al. tal pollution by antibiotics and by of IncP-1 antibiotic resistance plasmids pRAS1 and pAr-32 from the (1996). Detection and characteriza- antibiotic resistance determinants. mids isolated from wastewater treat- fish pathogen Aeromonas salmoni- tion of broad-host-range plasmids in Environ. Pollut. 157, 2893–2902. ment plants provides evidence for cida. Antimicrob. Agents Chemother. environmental bacteria by PCR. Appl. Nojiri, H., Shintani, M., and Omori, T. a widely accessible drug resistance 47, 1285–1290. Environ. Microbiol. 62, 2621–2628. (2004). Divergence of mobile genetic gene pool. FEMS Microbiol. Rev. 31, Springael, D., and Top, E. M. Haines, A. S., Akhtar, P.,Stephens, E. R., elements involved in the distribu- 449–477. (2004). Horizontal gene transfer and Jones, K., Thomas, C. M., Perkins, tion of xenobiotic-catabolic capac- Seiler, C., and Berendonk, T. U. (2012). microbial adaptation to xenobiotics: C. D., et al. (2006). Plasmids from ity. Appl. Microbiol. Biotechnol. 64, Heavy metal driven co-selection of new types of mobile genetic ele- freshwater environments capable of 154–174. antibiotic resistance in soil and water ments and lessons from ecologi- IncQ retrotransfer are diverse and Norberg, P., Bergstrom, M., Jethava, bodies impacted by agriculture and cal studies. Trends Microbiol. 12, include pQKH54, a new IncP-1 sub- V., Dubhashi, D., and Hermansson, aquaculture. Front. Microbiol. 3:399. 53–58. group archetype. Microbiology 152, M. (2011). The IncP-1 plasmid back- doi: 10.3389/fmicb.2012.00399 Stokes, H. W., and Gillings, M. R. 2689–2701. bone adapts to different host bacterial Sen, D., Brown, C. J., Top, E. M., and (2011). Gene flow, mobile genetic Haines, A. S., Jones, K., Batt, S. M., species and evolves through homolo- Sullivan, J. (2012). Inferring the evo- elements and the recruitment of Kosheleva, I. A., and Thomas, C. M. gous recombination. Nat. Commun. lutionary history of IncP-1 plasmids antibiotic resistance genes into Gram (2007). Sequence of plasmid pBS228 2, 1–11. despite incongruence among back- negative pathogens. FEMS Microbiol. and reconstruction of the IncP-1α Pachulec, E., and van der Does, bone gene trees. Mol. Biol. Evol. 30, Rev. 35, 790–819. phylogeny. Plasmid 58, 76–83. C. (2010). Conjugative plasmids 154–166. Szczepanowski, R., Eikmeyera, F., Harf- Harayama, S., Tsuda, M., and Iino, of Neisseria gonorrhoeae. PLoS Sen, D., Van Der Auwera, G., Rogers, manna, J., Blomb, J., Rogersc, T. (1980). High frequency mobi- ONE 5:e9962. doi: 10.1371/jour- L., Thomas, C. M., Brown, C. J., and L. M., Top, E. M., et al. (2011). lization of the chromosome of nal.pone.0009962 Top, E. M. (2011). Broad-host-range Sequencing and comparative analysis Escherichia coli by a mutant of plas- Pai, H., and Jacoby, G. A. (2001). plasmids from agricultural soils have of IncP-1 antibiotic resistance plasmid RP4 temperature-sensitive for Sequences of the NPS-1 and TLE-1 IncP-1 backbones with diverse acces- mids reveal a highly conserved back- maintenance. Mol. Gen. Genet. 180, blactamase genes. Antimicrob. Agents sory genes. Appl. Environ. Microbiol. bone and differences within acces- 47–56. Chemother. 45, 2947–2948. 77, 7975–7983. sory regions. J. Biotechnol. 155, Heuer, H., Binh, C. T., Jechalke, Pansegrau, W., Lanka, E., Barth, P. T., Sen, D., Yano, H., Suzuki, H., Krol, J. E., 95–103. S., Kopmann, C., Zimmerling, U., Figurski, D. H., Guiney, D. G., Haas, Rogers, L., Brown, C. J., et al. (2010). Szczepanowski, R., Krahn, I., Linke, Krögerrecklenfort, E., et al. (2012). D., et al. (1994). Complete nucleotide Comparative genomics of pAKD4, B., Goesmann, A., Pühler, A., and IncP-1ε plasmids are important vec- sequence of Birmingham IncP a plas- the prototype IncP-1δ plasmid with Schlüter, A. (2004). Antibiotic mul- tors of antibiotic resistance genes in mids. Compilation and comparative a complete backbone. Plasmid 63, tiresistance plasmid pRSB101 iso- agricultural systems: diversification analysis. J. Mol. Biol. 239, 623–663. 98–107. lated from a wastewater treatment driven by class 1 integron gene cas- Popowska, M., Miernik, A., Rzeczycka, Shafiani, S., and Malik, A. (2003). plant is related to plasmids resid- settes. Front. Microbiol. 3:2. doi: M., and Łopaciuk, A. (2010). The Tolerance of pesticides and antibi- ing in phytopathogenic bacteria 10.3389/fmicb.2012.00002 impact of environmental contami- otic resistance in bacteria iso- and carries eight different resistance Heuer, H., Szczepanowski, R., nation with antibiotics on levels of lated from wastewater-irrigated soil. determinants including a multidrug Schneiker, S., Pühler, A., Top, E. resistance in soil bacteria. J. Environ. World J. Microbiol. Biotechnol. 19, transport system. Microbiology 150, M., and Schlüter, A. (2004). The Qual. 39, 1679–1687. 897–901. 3613–3630.

www.frontiersin.org March 2013 | Volume 4 | Article 44 |102

“fmicb-04-00044” — 2013/3/6 — 11:20 — page7—#7 Popowska and Krawczyk-Balska IncP plasmids and antibiotic resistance

Tauch, A., Schlüter, A., Bischoff, N., integron and other transposable ele- et al. (2004). Genetic organiza- the clinic? Curr. Opin. Microbiol. 13, Goesmann, A., Meyer, F., and Pühler, ments. Plasmid 53, 218–238. tion of the catabolic plasmid pJP4 589–594. A. (2003). The 79,370-bp conjugative Thomas, C. M. (2000). The Horizon- from Ralstonia eutropha JMP134 plasmid pB4 consists of an IncP-1β tal Gene Pool: Bacterial Plasmids and (pJP4) reveals mechanisms of Conflict of Interest Statement: The backbone loaded with a chromate Gene Spread. Amsterdam: Harwood adaptation to chloroaromatic authors declare that the research was resistance transposon, the strA-strB Academic Publishers. pollutants and evolution of spe- conducted in the absence of any com- streptomycin resistance gene pair, Thomas, C. M., and Haines, A. S. cialized chloroaromatic degradation mercial or financial relationships that the oxacillinase gene blaNPS-1, and (2004). “Plasmids of the genus Pseu- pathways. Environ. Microbiol. 6, could be construed as a potential con- a tripartite antibiotic efflux system domonas,” in Pseudomonas, ed. J.-L. 655–668. flict of interest. of the resistance-nodulation-division Ramos (London: Kluwer/Plenum), Urgun-Demirtas, M., Stark, B., and family. Mol. Gen. Genomics 268, 197–231. Pagilla, K. (2006). Use of genetically Received: 15 November 2012; accepted: 570–584. Thorsted, P.B., Macartney, D. P.,Akhtar, engineered microorganisms (GEMs) 19 February 2013; published online: 07 Tello, A., Austin, B., and Telfer, P., Haines, A. S., Ali, N., Davidson, P., for the bioremediation of contam- March 2013. T. C. (2012). Selective pressure of et al. (1998). Complete sequence of inants. Crit. Rev. Biotechnol. 26, Citation: Popowska M and Krawczyk- antibiotic pollution on bacteria of the IncP-1β plasmid R751: implica- 145–164. Balska A (2013) Broad-host-range IncP-1 importance to public health. Env- tions for evolution and organisation Van Meervenne, E., Van Coillie, E., plasmids and their resistance potential. iron. Health Perspect. 120, 1100– of the IncP backbone. J. Mol. Biol. Kerckhof, F.-M., Devlieghere, F., Front. Microbiol. 4:44. doi: 10.3389/ 1106. 282, 969–990. Herman, L., De Gelder, L. S. P., fmicb.2013.00044 Tennstedt, T., Szczepanowski, R., Braun, Top, E., Springael, D., and Boon, N. et al. (2012). Strain-specific trans- This article was submitted to Fron- S., Pühler, A., and Schlüter, A. (2003). (2002). Catabolic mobile genetic ele- fer of antibiotic resistance from an tiers in Antimicrobials, Resistance and Occurrence of integron-associated ments and their potential use in environmental plasmid to foodborne Chemotherapy, a specialty of Frontiers in resistance gene cassettes located on bioaugmentation of polluted soils pathogens. J. Biomed. Biotechnol. Microbiology. antibiotic resistance plasmids iso- and waters. FEMS Microbiol. Ecol. 42, 2012, 834598. Copyright © 2013 Popowska and lated from a wastewater treatment 199–208. Vidali, M. (2001). Bioremediation. An Krawczyk-Balska. This is an open- plant. FEMS Microbiol. Ecol. 45, Top, E. M., and Springael, D. (2003). overview. Pure Appl. Chem. 73, 1163– access article distributed under the terms 239–252. The role of mobile genetic elements 1172. of the Creative Commons Attribution Tennstedt, T.,Szczepanowski, R., Krahn, in bacterial adaptation to xenobi- Williams, P. A. (2004). Catabolic plas- License, which permits use, distribution I., Pühler, A., and Schlüter, A. (2005). otic organic compounds. Curr. Opin. mids: fast-track bacterial evolution to and reproduction in other forums, pro- Sequence of the 68,869 bp IncP- Biotechnol. 14, 262–269. combat pollution. Microbiol. Today vided the original authors and source 1α plasmid pTB11 from a waste- Trefault, N., De la Iglesia, R., 31, 168–170. are credited and subject to any copy- water treatment plant reveals a highly Molina, A. M., Manzano, M., Wright, G. D. (2010). Antibiotic resis- right notices concerning any third-party conserved backbone, a Tn402-like Ledger, T., Pérez-Pantoja, D., tance in the environment: a link to graphics etc.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy March 2013 | Volume 4 | Article 44 |103

“fmicb-04-00044” — 2013/3/6 — 11:20 — page8—#8 REVIEW ARTICLE published: 05 February 2013 doi: 10.3389/fmicb.2013.00007 RND multidrug efﬂux pumps: what are they good for?

Carolina Alvarez-Ortega*, Jorge Olivares and José L. Martínez* Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientíﬁcas, Madrid, Spain

Edited by: Multidrug efflux pumps are chromosomally encoded genetic elements capable of mediating Fiona Walsh, Agroscope Changins resistance to toxic compounds in several life forms. In bacteria, these elements are involved Wädenswil, Switzerland in intrinsic and acquired resistance to antibiotics. Unlike other well-known horizontally Reviewed by: acquired antibiotic resistance determinants, genes encoding for multidrug efflux pumps Stefania Stefani, University of Catania, Italy belong to the core of bacterial genomes and thus have evolved over millions of years. Thomas Maddox, University of The selective pressure stemming from the use of antibiotics to treat bacterial infections Liverpool, UK is relatively recent in evolutionary terms. Therefore, it is unlikely that these elements *Correspondence: have evolved in response to antibiotics. In the last years, several studies have identified Carolina Alvarez-Ortega and José L. numerous functions for efflux pumps that go beyond antibiotic extrusion. In this review Martínez, Departamento de Biotecnología Microbiana, Centro we present some examples of these functions that range from bacterial interactions with Nacional de Biotecnología, Consejo plant or animal hosts, to the detoxification of metabolic intermediates or the maintenance Superior de Investigaciones of cellular homeostasis. Científicas, Darwin 3, Cantoblanco, 28049 Madrid, Spain. Keywords: multidrug efflux pumps, host/bacteria interactions, plant/bacteria interactions, quorum sensing, e-mail: [email protected]; antibiotic resistance, bacterial homeostasis, bacterial virulence [email protected]

INTRODUCTION 2011). The activity of an efflux pump depends on the different Multidrug resistance (MDR) efflux pumps are relevant elements types of energy source each system uses: ABC transporters are that contribute to both intrinsic and acquired resistance to toxic fueled by ATP hydrolysis; MFS, RND, and SMR use the proton- + + compounds in diverse life forms, including humans where they motive force and MATE transporters consist of Na /H drug have a role in resistance to anti-cancer drugs (Wu et al., 2011), antiport systems (Piddock, 2006a). to bacteria, where they are involved in resistance to antibiotics The RND family includes several members that are relevant to (Webber and Piddock, 2003; Li and Nikaido, 2004, 2009; Poole, antibiotic resistance in Gram-negative bacteria, whereas the MATE 2005, 2007). Unlike well-known horizontally acquired antibi- family has been mainly associated to resistance in Gram-positive otic resistance determinants, MDR efflux pumps are usually microorganisms (Piddock, 2006a; Vila and Martinez, 2008). This chromosomally encoded and the structural components of dif- review will focus exclusively on RND efflux systems. ferent systems are highly conserved in all members of a given The crystallographic analysis of AcrB, a model member of bacterial species (Saier et al., 1998; Paulsen et al., 2001; Saier the RND family, revealed that this protein forms a homotrimer and Paulsen, 2001; Paulsen, 2003; Baquero, 2004; Andam et al., (Murakami et al., 2002; Nakashima et al., 2011), that associates 2011). MDR systems are ancient elements, present in bacterial into a tripartite complex along with an outer membrane protein genomes long before the use of antibiotics for the treatment (OMP, TolC) and a periplasmic membrane-fusion protein (MFP, of human infections (Martinez et al., 2009a). This, along with AcrA; Figure 1). Usually the genes encoding for these proteins are their ubiquity in different organisms, suggests that the main found in a single operon, however, the gene encoding for the OMP function of these elements goes beyond providing resistance to can also be found elsewhere in the chromosome, as it happens with antibiotics. The fact that quinolones, a family of synthetic antibi- TolC in Escherichia coli (Koronakis et al., 2000, 2004); or is part of otics, constitute a common substrate of MDR efflux pumps an operon encoding for a different efflux pump (Figure 2). The supports this notion (Alonso et al., 1999; Hernandez et al., 2011b). Pseudomonas aeruginosa RND efflux pump MexXY is an example These observations also suggest that the recent selective pres- of the latter, where the system uses the OprM porin encoded in sure imposed by the use of antibiotics is not the main evoluti- the mexAB-OprM operon (Figure 2; Mine et al., 1999). onary driver for MDR efflux pumps (Alonso et al., 2001; Martinez In this review we will address the different functional roles that et al., 2009b). RND efflux pumps may have in addition to mediating antibi- Bacterial MDR efflux pumps can be grouped into five different otic resistance. Exhaustive information on structure, regulatory structural families: the adenosine triphosphate (ATP)-bindingcas- aspects, and antibiotic resistance can be found elsewhere (Saier sette (ABC) superfamily (Lubelski et al., 2007), the multidrug and et al., 1998; Paulsen et al., 2001; Saier and Paulsen, 2001; Paulsen, toxic compound extrusion (MATE) family (Kuroda and Tsuchiya, 2003; Webber and Piddock, 2003; Li and Nikaido, 2004, 2009; 2009), the major facilitator superfamily (MFS) (Pao et al., 1998), Poole, 2004, 2007; Piddock, 2006a; Blair and Piddock, 2009; the small multidrug resistance (SMR) family (Chung and Saier, Nikaido, 2009, 2011; Nikaido and Takatsuka, 2009). 2001), and the resistance/nodulation/division (RND) superfam- Some of the most relevant roles so far identified include involve- ily (Murakami et al., 2006; Nikaido and Takatsuka, 2009; Nikaido, ment in bacterial virulence (Piddock, 2006b), plant–bacteria

www.frontiersin.org February 2013 | Volume 4 | Article 7 |104

“fmicb-04-00007” — 2013/2/2 — 20:45 — page1—#1 Alvarez-Ortega et al. Ecological role of MDR pumps

FIGURE 1 | Structure of an RND efflux pump. The figure shows a scheme the AcrB RND protein is coupled to the proton gradient. It has been of the structure of the E. coli AcrAB-TolC system. As shown, the system is a shown that these efflux pumps can extrude different compounds tripartite complex formed by the inner membrane AcrB protein, the outer form the bacterial cytoplasm and the periplasm. Adapted from Blair and membrane protein TolC and the membrane fusion protein AcrA. The activity of Piddock (2009). interactions (Maggiorani Valecillos et al., 2006), trafficking of (Figure 2). This local regulator usually keeps expression of quorum sensing molecules (Evans et al., 1998; Kohler et al., 2001), the efflux pump at a very low-level. High-level expression can and detoxification processes from metabolic intermediates, and be achieved either through an effector-mediated release of the toxic compounds such as heavy metals, solvents, or antimicro- repressed state or through mutations in one or more regulators bials produced by other microorganisms (Aendekerk et al., 2002, (Hernandez et al., 2009, 2011a). Activation may occur at differ- 2005; Ramos et al., 2002; Nies, 2003; Sekiya et al., 2003; Burse et al., ent levels: (1) By inactivation of the local repressor that blocks 2004a). A comprehensive review of all potential functions identi- the expression of the pump’s structural genes such as AcrR in E. fied to date for all RND efflux pumps is beyond the scope of this coli (Ma et al., 1996), MexR in P. aeruginosa (Poole et al., 1996; review. Instead, we would like to discuss some selected examples Sanchez et al., 2002c), or SmeT in Stenotrophomonas maltophilia of the ecological role that these systems may have in the absence (Sanchez et al., 2002a); (2) By activation of a global transcriptional of antibiotics. As stated above, we believe that the evolution of regulator like SoxS, RobA, or RamA in E. coli (Martin et al., 2008; bacterial RND efflux pumps has been primarily driven by their Zhang et al., 2008; Perez et al., 2012); (3) By switching on–off one physiological functions and not by the selective pressure imposed or more steps that interlink regulatory cascades such as MtrR of by the relatively recent human use of antibiotics. We consider Neisseria gonorrhoeae (Johnson et al., 2011); and (4) Through the the important role RND efflux pumps currently play in antibi- emergence and selection of mutations in key genes like mexT in P. otic resistance to be an evolutionary novelty stemming from the aeruginosa (Kohler et al., 1999). aforementioned use of antibiotics by humankind (Martinez, 2008; Multidrug efflux pumps extrude a wide range of substrates. Baquero et al., 2009). However, the number of effectors regulating them is lower in comparison. Understanding the mechanisms of regulation may help in REGULATION OF RND EFFLUX SYSTEMS BY NATURAL deciphering the function of RND efflux pumps, since it is expected EFFECTORS that different effectors trigger expression only when a given pump The regulation of bacterial RND efflux systems is often mediated is required. RND efflux systems whose expression is controlled by by global and local regulators, resulting in a multilayered control natural inducers normally encountered during the course of infec- to optimize gene expression in response to specific cues. A number tive processes have been studied in detail. Induction of expression of positive and negative regulators along with their known mecha- by bile salts and fatty acids in enteric bacteria are perhaps the best nism of action have been reviewed elsewhere (Grkovic et al., 2002; studied examples of substances capable of modulating expression Li and Nikaido, 2009). of these systems. In most cases a transcriptional regulator (typically a repres- Expression of the acrAB system in E. coli is induced by sor) is encoded upstream the operon coding for the efflux pump decanoate and unconjugated bile salts usually encountered by the

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 7 |105

“fmicb-04-00007” — 2013/2/2 — 20:45 — page2—#2 Alvarez-Ortega et al. Ecological role of MDR pumps

FIGURE 2 | Representative examples of transcriptional regulation may use the OMP from another system, such as in the MexXY system and genetic organization of RND efflux systems. Local regulators using OprM (c). The OMP component may be located elsewhere in the can be either transcriptional activators, such as MexT (a) or transcriptional chromosome, such as TolC (f) and can be used by one or more different repressors, such as MexR, MexZ, AcrR, or AcrS (b, c, d, and e). The three systems as in the case of AcrAB (d) and AcrEF (e). MexEF-OprN, structural components may be organized in a single operon, such as in the MexAB-OprM, and MexXY belong to P.aeruginosa; AcrAB and AcrEF MexEF-OprN (a) or MexAB-OprM (b) systems; alternatively, a given system belong to E. coli. organism in the intestinal tract (Ma et al., 1995). The mechanism whereas the FarAB-MtrE efflux pump (Lee and Shafer, 1999)is involves binding of these effectors to the Rob transcriptional reg- not needed to colonize this environment. It has been suggested ulator (Rosenberg et al., 2003). Bile salts also induce expression that FarAB-MtrE is important for the resistance of N. gonorrhoeae of acrAB in Salmonella, however, in this case the effector binds to certain long-chained fatty acids that are present in the rectum the RamA transcriptional regulator (Nikaido et al., 2008). Inter- (Lee and Shafer, 1999). Altogether these studies indicate that N. estingly, in both cases the inductor is also a substrate for the gonorrhoeae harbors efflux pumps each one responding to dif- efflux system, thus allowing the cell to respond quickly to dele- ferent environmental cues that enable adaptation for survival in terious environmental substances. Additional examples of bile different ecosystems. salts-mediated induction include the cmeABC system in Campy- Metal cations are another example of natural compounds capa- lobacter jejuni, the vexD gene in Vibrio cholerae and various ble of inducing expression of RND efflux pumps. Metals are RND-type efflux system genes in Bacteroides fragilis (Lin et al., required as cofactors in several bacterial processes. However, they 2005b; Pumbwe et al., 2007). These examples strongly suggest that are toxic at high concentrations. Consequently, bacteria harbor these systems are relevant to bacterial adaptation for surviving in systems to maintain the cellular metal homeostasis. In some cases, the gut and that this may be their original function. this regulation implies that the efflux pump is involved in the In this regard, it has been suggested that some efflux pumps extrusion of these toxic effectors (Nies, 2003). However, in other from human commensals and pathogens have evolved to overcome cases the situation is more complex and the effector is simply the innate immunity of the host (Blair and Piddock, 2009). For an environmental cue that indicates the type of ecosystem sur- instance, the susceptibility to vertebrate antibacterial peptides in rounding the organism. The cusCBA system in E. coli and mtrCDE N. gonorrhoeae depends on the activity of the MtrCDE RND efflux system in N. gonorrhoeae constitute two of the most studied exam- pump (Shafer et al., 1998). Notably, this efflux pump is required to ples of metal-induced regulation among pathogenic bacteria. The achieve mutation-driven resistance to penicillin in N. gonorrhoeae CusCBA system confers tolerance to copper and silver ions (Franke (Veal et al., 2002) and overexpression mutants present reduced et al., 2001; Grass and Rensing, 2001). Both substrates serve susceptibility to several antibiotics and show an increase in in vivo as natural inducers for cusCBA expression (Franke et al., 2001; fitness (Warner et al., 2007). MtrCDE (Jerse et al., 2003), enhances Yamamoto and Ishihama, 2005), suggesting that this RND efflux experimental gonococcal genital tract infections in female mice, pump may have been first selected to overcome the toxicity of these

www.frontiersin.org February 2013 | Volume 4 | Article 7 | 106

“fmicb-04-00007” — 2013/2/2 — 20:45 — page3—#3 Alvarez-Ortega et al. Ecological role of MDR pumps

metals. As stated above, the MtrCDE system is involved in resis- pumps encoded in the genome of a given bacterial species may tance to host-derived antibacterial peptides (Shafer et al., 1998). have a different function. This adaptation does not rely exclu- It was recently reported that mtrCDE expression is indirectly reg- sively on the extrusion of toxic antimicrobial plant exudates. For ulated by free levels of iron through the regulation of its major instance, salicylic acid, an important signaling molecule produced transcriptional repressor, MtrR, by the MpeR transcriptional by plants (Loake and Grant, 2007), induces the expression of the regulator (Mercante et al., 2012). Under the proposed model, E. chrysanthemi efflux pumps acrAB and emrAB (Ravirala et al., expression of the efflux system would increase under iron-limited 2007). This indicates that RND efflux pumps are relevant ele- conditions, a situation that bacteria can encounter over the course ments mediating bacteria/plant interactions at different levels that of the infection process (Martinez et al., 1990). The P. aeruginosa include the response to toxic compounds, host specificity and CzcABC efflux system confers tolerance to zinc, cadmium, and interspecies signal trafficking. This functional role is not confined cobalt and constitutes another example of metal-induced expres- to plant-infective bacteria. Mutants of the mutualistic symbiont sion. The regulation occurs through the metal-inducible CzcRS Rhizobium etli lacking the RmrAB efflux pump form fewer nodules two-component system that is activated in the presence of the on its host Phaseolus vulgaris than the corresponding wild-type system’s substrates or indirectly in the presence of copper (Caille strain (Gonzalez-Pasayo and Martinez-Romero, 2000). Similarly, et al., 2007; Dieppois et al., 2012). the SmeAB efflux pump plays an important role in the nodulation competitiveness in Sinorhizobium meliloti (Eda et al., 2011). The THE ROLE OF EFFLUX PUMPS IN PLANT–BACTERIA effect of efflux pumps on plant–bacteria interactions can be host- INTERACTIONS specific. For instance, BdeAB from Bradyrhizobium japonicum is The rhizosphere is a complex ecosystem characterized by a high needed for the symbiotic nitrogen-fixation activity on soybean, microbial activity that results in a bacterial population density that but not on other host plants such as mung bean and cowpea can be two orders of magnitude higher than in bulk soil (Matilla (Lindemann et al., 2010). et al., 2007). The structure of the rhizosphere’s microbiota is gov- erned by the release of nutrients through plant root exudates and THE ROLE OF EFFLUX PUMPS IN BACTERIAL VIRULENCE by the ecological relationships of the microorganisms present in From a clinical point of view, antibiotic resistance could be consid- this ecosystem. A transcriptomic analysis of Pseudomonas putida ered as a colonization factor since only those organisms surviving grown in the rhizosphere of maize revealed that the expression within a treated patient will be able to cause an infection (Mar- of different efflux pumps is induced in this ecosystem (Matilla tinez and Baquero, 2002). However, in this section we would like et al., 2007), thus suggesting a relevant function for the coloniza- to address the direct role that RND efflux pumps play in the viru- tion of this environment. Plant exudates have been identified lence of different human pathogens. As mentioned in a previous as good effectors of RND efflux pumps, and it has been shown section, the expression of different RND efflux pumps is trig- that these secondary metabolites bind regulators of RND efflux gered by human-produced compounds, and they contribute to pumps such as TtgR (Alguel et al., 2007), the local repressor of the colonization of different environments in the human host. the TtgABC system in P. putida (Teran et al., 2003). Some com- Although the role of efflux pumps on virulence has been studied pounds produced by plants have antibacterial effects and it has for several organism (Piddock, 2006b), only in a few cases compre- been described the RND efflux pumps are required from the first hensive studies including different systems from a single bacterial steps of bacterial plant colonization (Espinosa-Urgel et al., 2000) species have been performed. Below we discuss some of these to survival in plant tissues (Barabote et al., 2003), possibly due to examples their involvement in protection against these compounds. This is the case of Erwinia amylovora, the cause of fire blight disease in Vibrio cholerae RND EFFLUX PUMPS AND VIRULENCE rosaceous plants (Eastgate, 2000). The plantlet toxic metabolites Vibrio cholerae possesses six different operons encoding for RND- naringenin and phloretin are good inducers of the efflux pump type efflux systems: vexAB, vexCD (breAB), vexEF, vexGH, vexIJK, acrAB in this bacterial species, and E. amylovora acrAB mutants and vexLM (Kitaoka et al., 2011). While different RND efflux sys- are much less virulent that their wild-type counterpart (Burse tems often share an OMP,it is rather common that operons encode et al., 2004b). A similar situation occurs in Agrobacterium tume- for a cognate OMP for each system (Figure 2). In the case of V. faciens. Coumestrol, an antimicrobial root-exudated flavonoid, cholerae, it seems that all six different RND efflux systems operate is both a substrate and an inducer of expression of the ifeABR with the same OMP, encoded by the tolC gene (Bina et al., 2008; efflux system (Palumbo et al., 1998). The fact that this system Cerda-Maira et al., 2008). is needed for effective root colonization indicates that it plays During the course of V. cholerae infections, bacteria colonize an important role in A. tumefaciens resistance to plant-produced primarily the small intestine, where they penetrate the mucus antimicrobials. lining coating the intestinal epithelium. In addition to factors pro- Comprehensive analyses on Erwinia chrysanthemi RND efflux duced by the innate immune system, the intestinal environment pumps revealed that each system may differentially contribute is rich in substances such as bile salts and organic acids that are to host specificity. Mutants defective in each of the pumps were capable of inhibiting bacterial growth (Reidl and Klose, 2002). differently affected in their virulence in diverse hosts and the sus- Predictably, four V. cholerae RND efflux systems have been impli- ceptibility to plant-produced antimicrobials was specific for each cated in in vitro resistance to bile salts and detergents similar to pump (Maggiorani Valecillos et al., 2006). As discussed in the case detergent-like molecules the organism is likely to encounter during of N. gonorrhoeae, this suggests that each of the several efflux colonization of the intestinal epithelium.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 7 |107

“fmicb-04-00007” — 2013/2/2 — 20:45 — page4—#4 Alvarez-Ortega et al. Ecological role of MDR pumps

Susceptibility studies with single and multiple mutant com- isoniazid resistance when overexpressed in Mycobacterium smeg- binations revealed that VexB has broad substrate specificity and matis (Pasca et al., 2005; De Rossi et al., 2006; da Silva et al., 2011). that it is the primary RND efflux system responsible for resis- Moreover, deletion mutants created in 11 mmpL genes failed to tance to bile salts in vitro (Bina et al., 2008). VexD, VexK, and exhibit significantly altered drug susceptibility in M. tuberculosis VexH have also been implied in resistance to bile salts, which (Domenech et al., 2005). denotes redundancy among the different RND efflux systems (Tay- The primary role of most MmpL proteins appears to be lor et al., 2012). Moreover, the expression of vexD is induced in the transport of lipids to be incorporated on the cell enve- the presence of bile salts (Cerda-Maira et al., 2008). The VexK lope. The complex mycobacterial cell wall is composed of and VexH contribution to bile salts resistance is only evident in a peptidoglycan, arabinogalactan, and mycolic acids, the surface ΔvexBD double mutant background, which suggests a supportive of which is covered by non-covalently associated lipids that role for VexK and VexH. However, as Taylor et al. (2012) point include trehalose monomycolate (TMM), trehalose dimycolate out, this hierarchy might be limited to their in vitro experimen- (TDM), sulfolipids, phenolic glycolipids, and phthiocerol dimyco- tal conditions. In fact, the increasing attenuation levels displayed cerosates (PDIMs; Tahlan et al., 2012). These lipids play important by combination mutants in in vivo colonization experiments roles in protection against host-derived toxic molecules, bear (ΔvexBDK < ΔvexBDH < ΔvexBDHK < ΔRND), suggest that an immunomodulatory activity and contribute to M. tuberculo- VexH plays a more relevant role than VexK during the infection sis pathogenicity (Neyrolles and Guilhot, 2011). Lipid transport process. functions have been ascribed to MmpL3, MmpL7, and MmpL8, RND efflux systems are also required for optimal expression and in some cases deletion mutants have demonstrated the con- of the genes encoding for two of the most important V. cholerae tribution of additional MmpL proteins to host survival and virulence factors: cholera toxin (CT) and the toxin-coregulated pathogenicity. pilus (TCP). A ΔRND mutant exhibited decreased transcription The inability to create an mmpL3 deletion mutant combined of the tcpA and toxT genes, the latter encoding for a transcriptional with its absence in transposon mutant collections suggests that activator responsible for transcription of the genes encoding for this gene is essential to M. tuberculosis (Domenech et al., 2005; CT, and a concomitant decrease in CT and TCP production (Bina Lamichhane et al., 2005). A recent study aimed at identifying the et al., 2008). While VexB is able to complement this phenotype, a target of a novel M. tuberculosis antibiotic found data that suggests vexBDHK still exhibits a decrease in CT and TCP,thus suggesting a that MmpL3 transports TMM out of the cell and that its inhibition role for VexM and VexF in virulence factor production (Bina et al., prevents the incorporation of de novo-synthesized mycolic acids 2008; Taylor et al., 2012). into the cell envelope (Tahlan et al., 2012). The mechanism through which the V. cholerae RND efflux sys- MmpL7 is required for PDIM transport to the cell surface and tems modulate the production of virulence factors has not been was the first MmpL protein implicated in lipid transport in M. elucidated. However, it has been proposed that deletion of sys- tuberculosis (Cox et al., 1999). In addition, MmpL7 appears to tems with redundant functions could lead to the accumulation function as a scaffold for the PpsE polyketide synthase required of a low molecular weight molecule that normally functions as a for the final step of phthiocerol synthesis, thus coupling trans- negative effector molecule involved in fine-tuning the expression port and synthesis (Jain and Cox, 2005). At least two different of the affected virulence factors (Taylor et al., 2012). V. cholerae studies have determined that mmpL7 mutants display an atten- inhabits aquatic environments where it normally grows associ- uation phenotype in murine virulence models (Cox et al., 1999; ated with zooplankton or egg masses (Reidl and Klose, 2002). It is Domenech et al., 2005). MmpL8 has been implicated in the trans- possible that some of the RND efflux systems have dedicated func- port of the SL-N, a precursor of the SL-1 sulfolipid, with a tions specific to this portion of the organism’s life cycle. This may similar mechanism to that of MmpL7 where synthesis and trans- be particularly true for VexM and VexF, for which no function port appear to be coupled (Converse et al., 2003; Domenech in resistance to bile salts and antimicrobials has been identified et al., 2004). mmpL8 mutants also display attenuated lethality in to date. murine virulence models (Converse et al., 2003; Domenech et al., 2004, 2005). Mycobacterium tuberculosis RND EFFLUX PUMPS AND Domenech et al. (2005) determined that an mmpL4 mutant VIRULENCE has both impaired growth kinetics and impaired lethality in a vir- The M. tuberculosis genome possesses 13 different genes encod- ulence murine model. The same study determined that while an ing for RND proteins (Cole et al., 1998). Several domains in mmpL11 mutant shows a growth pattern similar to that of the these proteins are unique to mycobacteria and are thus desig- wild-type during the active growth phase, the mutant is attenu- nated as MmpL (Mycobacterial membrane protein Large). Four ated during the course of chronic infections in an in vivo model. mmpL genes appear to be in operons also containing an mmpS No substrate has been identified for these transporters. A role in gene. The latter are predicted to encode for proteins equi- heme uptake has been recently proposed for MmpL11 and such a valent to the MFPs in other bacterial RND systems (Domenech function would be in line with the attenuated virulence phenotype et al., 2005). observed with an mmpL11 mutant (Tullius et al., 2011). Further- In spite of the documented M. tuberculosis resistance against more, a role in extrusion of host-derived antimicrobials similar to first and second line antimicrobial therapy, none of the RND sys- that observed for V. cholerae RND efflux systems cannot be ruled tems have been associated with antibiotic efflux to date, the only out for those MmpL proteins that appear to be involved in the M. possible exception being MmpL7, which is capable of conferring tuberculosis infection process.

www.frontiersin.org February 2013 | Volume 4 | Article 7 |108

“fmicb-04-00007” — 2013/2/2 — 20:45 — page5—#5 Alvarez-Ortega et al. Ecological role of MDR pumps

Helicobacter pylori RND EFFLUX PUMPS AND VIRULENCE hp1326 (renamed as crdA), encoding for a secreted protein, is The gastric colonizer Helicobacter pylori possesses three different strongly induced in the presence of copper and growth of an operons encoding for RND efflux systems (Tomb et al., 1997). hp1326 mutant was also impaired in the presence of copper. Over the years the systems have received different nomenclatures hp1326 is transcribed as a monocistronic unit, but is believed that may often lead to confusion when revising the literature: to constitute a copper resistance system along with hp1327–1329. hp0605–hp0607 is also referred to as hefABC; hp0969–hp0971 A follow up study revealed that copper-mediated expression of was originally denominated as hefFDE and is currently known hp1326 requires the CrdRS two-component system (Waidneret al., as cznABC; finally, the system encoded by hp1329–hp1327 was 2005); the study did not address expression of hp1327–1229. originally named hefIHG and currently hp1329 and hp1328 are Mutants lacking the CrdRS system are unable to colonize the stom- known as czcA and czcB, respectively, while hp1327 is known achofmice(Panthel et al., 2003). This suggests that hp1326 and as crdB. hp1327–1329 might play an important role during the infective Bina et al. (2000) initially assessed in vitro and in vivo expres- process of H. pylori. sion profiles of each system as well as the individual contribution to The RND efflux system encoded by hp0969–0971 (renamed as intrinsic antibiotic susceptibility. The study revealed that hp0607 cznABC) has been implicated in cadmium, zinc, and nickel resis- (hefC) and hp0969 (hefF) are expressed both in in vivo and in vitro, tance (Stahler et al., 2006). Stahler et al. (2006) showed growth while hp1329 (hefI) is only expressed in vivo. Knockoutsineach inhibition of individual mutants in the presence of these metals. system failed to identify a contribution to intrinsic antibiotic sus- The H. pylori urease, a nickel-containing enzyme, is an essen- ceptibility with 19 different compounds. However, overexpression tial colonization factor that enables survival in acidic conditions. of selected components has been associated with antibiotic resis- Urease activity and expression is regulated in response to nickel tance and different studies revisiting the contribution of each availability (van Vliet et al., 2001), accordingly, cznC and cznA system to antibiotic susceptibility determined that hp0607 (hefC) mutants exhibited enhanced urease activity (Stahler et al., 2006). and hp0605 (hefA) are involved in intrinsic antibiotic resistance to The authors propose that the cznABC system plays an impor- diverse antibiotics (Kutschke and de Jonge, 2005; Liu et al., 2008; tant role in fine-tuning urease activity, as nickel efflux reduces Hirata et al., 2010; Tsugawa et al., 2011). activity, while cadmium and zinc efflux prevents inhibition of H. pylori is exposed to bile salts resulting from reflux into the this enzyme. High urea concentrations are toxic at neutral pH, human stomach; bile salts have an inhibitory effect on H. pylori therefore, untimely activation of this enzyme resulting from per- growth, yet the ability to thrive in the presence of a bile gradient turbations in metals homeostasis can be detrimental to the cell suggests that this organism has bile resistance mechanisms in place (Meyer-Rosberg et al., 1996; Rektorschek et al., 1998). The inabil- (Worku et al., 2004; Shao et al., 2008). HefC was recently found to ity of cznA, cznB, and cznC mutants to achieve gastric colonization play a role in resistance to bile salts and ceragenins (synthetic bile in a gerbil animal model and the failure of a cznA mutant to sur- salt derivatives with antimicrobial activity; Trainor et al., 2011). vive in acidic conditions might be linked to urease activity (Bijlsma A hefC mutant exhibited increased susceptibility to deoxycholate, et al., 2000; Stahler et al., 2006). cholate, glycodeoxycholate, taurodeoxycholate, taurocholate and to ceragenin 11(CSA 11); while no changes in susceptibility were EFFLUX PUMPS AND GLOBAL BACTERIAL PHYSIOLOGY observed with mutants in the other two efflux systems. Moreover, One of the putative functions of RND efflux pumps is detox- HefC appears to have substrate specificity for bile salts, since no ification from detrimental intermediates derived from bacterial change in susceptibility was observed with detergents. Although metabolism (Neyfakh, 1997). Studies on this subject have been direct efflux of bile salts through HefC has not been experimen- mainly performed using mutants that overproduce RND efflux tally demonstrated yet, it is likely that this system contributes pumps. It is conceivable that overexpression of these elements to H. pylori successful colonization of bile-containing envi- might cause a metabolic burden on bacterial populations (Mar- ronments. tinez et al., 2007, 2011; Andersson and Hughes, 2010). Indeed, During the course of gastric colonization, H. pylori is exposed different publications have shown that overproduction of RND to additional environmental stresses, including low pH gradi- efflux pumps may impact bacterial physiology (Sanchez et al., ents (4.0–6.0) and acid shock. Acidic environments impact the 2002b; Ruiz-Diez et al., 2003; Alonso et al., 2004; Linares et al., bioavailability of metals like iron and nickel, which play an essen- 2005; Lertpiriyapong et al., 2012; Olivares et al., 2012). Moreover, tial role in bacterial metabolism. In addition, environmental the uncontrolled production of these elements can affect the abil- metal fluctuations are expected to arise from damaged epithelium ity of pathogenic bacteria to infect experimental animal models, and diffusion from ingested food (Stoof et al., 2008). Maintain- seriously impairing their virulence (Cosson et al., 2002; Hirakata ing a cytoplasmic metal homeostasis is crucial to bacteria, as et al., 2002; Warner et al., 2007; Lertpiriyapong et al., 2012; Perez excessive concentrations can lead to severe cellular damage. The et al., 2012). other two H. pylori RND efflux systems are involved in metal The energy expenditure required to constantly maintain the efflux. activity of an efflux pump could lead to a fitness reduction The system encoded by hp1327–1329 (crdB, czcB, and czcA) upon overproduction of these elements. However, our group has constitutes a novel copper efflux pump. Expression of hp1329 recently shown that overproduction of the P. aeruginosa MexEF- is induced in the presence of copper and growth of hp1327 OprN efflux system does not produce a fitness cost as measured in and hp1328 mutants is inhibited in the presence of this metal classical competition tests, although it alters several physiological (Waidner et al., 2002). The same study found that expression of aspects, including elements relevant for P. aeruginosa virulence

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 7 |109

“fmicb-04-00007” — 2013/2/2 — 20:45 — page6—#6 Alvarez-Ortega et al. Ecological role of MDR pumps

such as Type III and Type VI secretion (Tian et al., 2009a,b; Oli- alleviating the entrance through the T6SS (Lin et al., 2005b). The vares et al., 2012). Notably, this effect is specific to each pump and functional interaction between the T6SS and CmeABC might be might be associated to their functional role, as overexpression of crucial for intestinal colonization by C. jejuni, thus playing a either MexAB-OprM of MexXY does not produce the same effect key role in the virulence of this bacterial pathogen (Lertpiriyapong (Linares et al., 2005). et al., 2012). As mentioned before, efflux pumps might be involved in the Given the integration of RND efflux systems in bacterial elimination of endogenous toxic compounds. The P. aeruginosa metabolic networks, it is not surprising that their regulation is MexGHI-OpmD efflux system might be implicated in the extru- also incorporated in global regulatory networks. Global regula- sion of anthranilate, a toxic intermediate of the Pseudomonas tors such as MarA, RamA, and SoxS can activate the expression quinolone signal (PQS) synthetic pathway (Aendekerk et al., 2002, of efflux pumps such as AcrAB-TolC in E. coli and in addi- 2005; Sekiya et al., 2003), whereas MexEF-OprN extrudes kynure- tional Enterobacteriaceae (Davin-Regli et al., 2008). Similarly, the nine, another intermediate in the same pathway (Olivares et al., pleiotropic regulator MgrA (Luong et al., 2006) controls autol- 2012). A recent study has shown that kynurenine and its deriva- ysis, virulence, biofilm formation, and efflux pump activity tives have relevant effects in different human diseases, including in Staphylococcus aureus (Ingavale et al., 2003, 2005; Truong- modulation of the activation of glutamate and nicotinic receptors, Bolduc et al., 2003, 2005; Trotonda et al., 2008). The control of the modification of the immune response in situations of inflam- efflux pumps by this global regulator is specific for each pump. mation and infection, and the generation and removal of reactive Increased expression of mgrA in vivo in a subcutaneous abscess oxygen species (Stone et al., 2012). Any potential impact that the model upregulates expression of the norB and tet38 efflux pumps, constant extrusion of kynurenine by a MexEF-OprN overexpres- whereas expression of norA and norC is downregulated (Ding sion mutant may have on the pathogenic behavior of P. aeruginosa et al., 2008). The relevance of these pumps for the in vivo growth remains to be established. of S. aureus has been studied; norB and tet38 defective mutants Pseudomonas quinolone signal is one of the quorum sensing present a growth defect in a mice abscess model and the pheno- (QS) signals produced by P. aeruginosa (Mcknight et al., 2000). type was not attributable to a staphylococcal stress response (Deng Strains overexpressing MDR efflux pumps capable of extruding et al., 2012). QS signals or their intermediates are likely to be impaired in the QS MexT,the transcriptional activator of MexEF-OprN in P.aerug- response. Indeed, overexpression of MexEF-OprN impairs the QS inosa (Figure 2), constitutes another example of global regulation. response of P. aeruginosa (Kohler et al., 2001; Olivares et al., 2012). MexT regulates the expression of several P. aeruginosa genes Previous studies also showed that MexAB-OprM likely extrudes (Tian et al., 2009a). A portion of this regulation is mediated the 3O12-HSL QS signal (Evans et al., 1998; Pearson et al., 1999), by the activity of the pump through the extrusion of a pre- and that overproduction of this efflux pump reduced the expres- cursor of the PQS QS signal, and the concomitant impairment sion of selected QS-regulated genes. The P. aeruginosa QS regulon of the QS response (Olivares et al., 2012). However, the expres- comprises approximately 5% of this organism’s genome (Schuster sion of other genes is directly regulated by MexT (Tian et al., et al., 2003); including several genes involved in virulence. Expres- 2009a). A recent study demonstrated that MexT functions as a sion of some of these genes might be energetically costly. However, redox-responsive regulator (Fargier et al., 2012), indicating that once the QS signals reach a specific threshold, expression of the it might be involved in controlling cellular redox homeostasis. regulon is maintained. It has been suggested that being signal- The fact that a local transcriptional regulator of an efflux pump blind can be a good adaptive strategy to avoid this energetic burden behaves as a global regulator further supports the involvement of (Haas, 2006). Whether the efflux pump-mediated extrusion of QS these elements in general processes of bacterial physiology and signals may be beneficial to P. aeruginosa under specific conditions not simply as a response to the presence of antibiotics in the remains to be determined. environment. Efflux pumps may also compensate for the effects that other bacterial elements may have on the organism. This might be CONCLUDING REMARKS the case of C. jejuni, a leading cause of food-borne enterocol- The emergence of antibiotic resistance in bacterial human itis worldwide (Ruiz-Palacios, 2007). As an intestinal pathogen pathogens is a very recent process in the evolutionary timescale. this bacterium must overcome the antimicrobial effects of the It is often assumed that resistance genes have been mainly origi- bile salts secreted into the intestinal tract (Hofmann and Eck- nated in antibiotic producers where they play a detoxification role mann, 2006). The RND-type efflux pump CmeABC confers (Benveniste and Davies, 1973; Webb and Davies, 1993; Davies, resistance to a broad range of antibacterial substances includ- 1997). However, in the few cases where the origin of resistance ing bile salts, fatty acids, and detergents (Lin et al., 2005a). On genes has been tracked, the original hosts are not antibiotic the other hand, it has been demonstrated that the type VI secre- producers. The QnrA gene from Shewanella algae constitutes a tion system (T6SS) plays a key role in the colonization of the prime example, as it confers resistance to quinolones, which are intestinal tract (Lertpiriyapong et al., 2012). The activation of the synthetic antibiotics (Poirel et al., 2005). This indicates that, at T6SS may enable bile salts to enter inside the bacterium through least in some cases, antibiotic resistance would be an emergent the open secretion channel (Bidlack and Silverman, 2004); and function that has been recently selected due to the use of antibi- this can compromise bacterial viability and infective capability. otics for treating infections (Martinez, 2008, 2009a,b; Baquero Bile salts trigger the expression of the CmeABC efflux pump; et al., 2009; Fajardo et al., 2009). As we have seen in this review, which extrudes the bile salts immediately outside the cell thus MDR efflux pumps also fall within this category, since they

www.frontiersin.org February 2013 | Volume 4 | Article 7 |110

“fmicb-04-00007” — 2013/2/2 — 20:45 — page7—#7 Alvarez-Ortega et al. Ecological role of MDR pumps

exhibit multiple functions relevant to bacterial physiology in addi- ACKNOWLEDGMENTS tion to mediating antibiotic resistance. A complete understanding Work in our laboratory is supported by grants BIO2011- of these functions is important in order to deﬁne the networks 25255 from the Spanish Ministry of Science and Innovation, that connect antibiotic resistance with other basic physiologi- PROMPT from CAM, and HEALTH-F3-2011-282004 (EVOTAR) cal processes (Linares et al., 2010; Martinez and Rojo, 2011), and HEALTH-F3-2010-241476 (PAR) from the European Union. both during the course of infections and in natural, non-clinical Jorge Olivares is the recipient of a fellowship from the Government ecosystems. of Chile.

REFERENCES Barabote, R. D., Johnson, O. L., Zetina, Caille, O., Rossier, C., and Perron, Expression of multidrug resistance Aendekerk, S., Diggle, S. P., Song, Z., E., San Francisco, S. K., Fralick, K. (2007). A copper-activated two- efflux pump gene norA is iron Hoiby, N., Cornelis, P., Williams, J. A., and San Francisco, M. J. component system interacts with zinc responsive in Staphylococcus aureus. P., et al. (2005). The MexGHI- (2003). Erwinia chrysanthemi tolC is and imipenem resistance in Pseu- J. Bacteriol. 194, 1753–1762. OpmD multidrug efflux pump con- involved in resistance to antimicro- domonas aeruginosa. J. Bacteriol. 189, De Rossi, E.,Ainsa, J.A., and Riccardi, G. trols growth, antibiotic susceptibility bial plant chemicals and is essential 4561–4568. (2006). Role of mycobacterial efflux and virulence in Pseudomonas aerugi- for phytopathogenesis. J. Bacteriol. Cerda-Maira, F. A., Ringelberg, C. transporters in drug resistance: an nosa via 4-quinolone-dependent cell- 185, 5772–5778. S., and Taylor, R. K. (2008). The unresolved question. FEMS Micro- to-cell communication. Microbiology Benveniste, R., and Davies, J. (1973). bile response repressor BreR regu- biol. Rev. 30, 36–52. 151, 1113–1125. Aminoglycoside antibiotic-inactiva- lates expression of the Vibrio cholerae Dieppois, G., Ducret, V., Caille, O., Aendekerk, S., Ghysels, B., Cornelis, ting enzymes in actinomycetes simi- breAB efflux system operon. J. Bacte- and Perron, K. (2012). The tran- P., and Baysse, C. (2002). Char- lar to those present in clinical isolates riol. 190, 7441–7452. scriptional regulator CzcR modulates acterization of a new efflux pump, of antibiotic-resistant bacteria. Proc. Chung, Y. J., and Saier, M. H. Jr. antibiotic resistance and quorum MexGHI-OpmD, from Pseudomonas Natl. Acad. Sci. U.S.A. 70, 2276–2280. (2001). SMR-type multidrug resis- sensing in Pseudomonas aerugi- aeruginosa that confers resistance to Bidlack, J. E., and Silverman, P. M. tance pumps. Curr. Opin. Drug Dis- nosa. PLoS ONE 7:e38148. doi: vanadium. Microbiology 148, 2371– (2004). An active type IV secretion cov. Dev. 4, 237–245. 10.1371/journal.pone.0038148 2381. system encoded by the F plasmid sen- Cole, S. T., Brosch, R., Parkhill, J., Gar- Ding, Y., Onodera, Y., Lee, J. C., and Alguel, Y., Meng, C., Teran, W., Krell, sitizes Escherichia coli to bile salts. J. nier, T., Churcher, C., Harris, D., et al. Hooper, D. C. (2008). NorB, an T., Ramos, J. L., Gallegos, M. T., Bacteriol. 186, 5202–5209. (1998). Deciphering the biology of efflux pump in Staphylococcus aureus et al. (2007). Crystal structures of Bijlsma, J. J., Lie, A. L. M., Nooten- Mycobacterium tuberculosis from the MW2, contributes to bacterial fit- multidrug binding protein TtgR in boom, I. C., Vandenbroucke-Grauls, complete genome sequence. Nature ness in abscesses. J. Bacteriol. 190, complex with antibiotics and plant C. M., and Kusters, J. G. (2000). 393, 537–544. 7123–7129. antimicrobials. J. Mol. Biol. 369, Identification of loci essential for the Converse, S. E., Mougous, J. D., Leavell, Domenech, P., Reed, M. B., and 829–840. growth of Helicobacter pylori under M. D., Leary, J. A., Bertozzi, C. R., Barry, C. E. III (2005). Contribu- Alonso, A., Morales, G., Escalante, acidic conditions. J. Infect. Dis. 182, and Cox, J. S. (2003). MmpL8 is tion of the Mycobacterium tuberculo- R., Campanario, E., Sastre, L., and 1566–1569. required for sulfolipid-1 biosynthesis sis MmpL protein family to virulence Martinez, J. L. (2004). Overexpres- Bina, J. E., Alm, R. A., Uria-Nickelsen, and Mycobacterium tuberculosis vir- and drug resistance. Infect. Immun. sion of the multidrug efflux pump M., Thomas, S. R., Trust, T. J., and ulence. Proc. Natl. Acad. Sci. U.S.A. 73, 3492–3501. SmeDEF impairs Stenotrophomonas Hancock, R. E. (2000). Helicobac- 100, 6121–6126. Domenech, P., Reed, M. B., Dowd, C. maltophilia physiology. J. Antimicrob. ter pylori uptake and efflux: basis Cosson, P., Zulianello, L., Join- S., Manca, C., Kaplan, G., and Barry, Chemother. 53, 432–434. for intrinsic susceptibility to antibi- Lambert, O., Faurisson, F., Gebbie, C. E. III (2004). The role of MmpL8 Alonso, A., Rojo, F., and Martinez, J. otics in vitro. Antimicrob. Agents L., Benghezal, M., et al. (2002). Pseu- in sulfatide biogenesis and virulence L. (1999). Environmental and clinical Chemother. 44, 248–254. domonas aeruginosa virulence ana- of Mycobacterium tuberculosis. J. Biol. isolates of Pseudomonas aeruginosa Bina, X. R., Provenzano, D., Nguyen, lyzedinaDictyostelium discoideum Chem. 279, 21257–21265. show pathogenic and biodegradative N., and Bina, J. E. (2008). Vib- host system. J. Bacteriol. 184, 3027– Eastgate, J. (2000). Erwinia amylovora: properties irrespective of their origin. rio cholerae RND family efflux sys- 3033. the molecular basis of fireblight dis- Environ. Microbiol. 1, 421–430. tems are required for antimicrobial Cox, J. S., Chen, B., Mcneil, M., and ease. Mol. Plant Pathol. 1, 325–329. Alonso, A., Sanchez, P., and Martinez, J. resistance, optimal virulence factor Jacobs, W. R. Jr. (1999). Complex Eda, S., Mitsui, H., and Minamisawa, L. (2001). Environmental selection of production, and colonization of the lipid determines tissue-specific repli- K. (2011). Involvement of the smeAB antibiotic resistance genes. Environ. infant mouse small intestine. Infect. cation of Mycobacterium tuberculosis multidrug efflux pump in resistance Microbiol. 3, 1–9. Immun. 76, 3595–3605. in mice. Nature 402, 79–83. to plant antimicrobials and contribu- Andam, C. P., Fournier, G. P., and Gog- Blair, J. M., and Piddock, L. J. (2009). da Silva, P. E., Von Groll, A., Martin, tion to nodulation competitiveness in arten, J. P. (2011). Multilevel popula- Structure, function and inhibition of A., and Palomino, J. C. (2011). Efflux Sinorhizobium meliloti. Appl. Envi- tions and the evolution of antibiotic RND efflux pumps in Gram-negative as a mechanism for drug resistance ron. Microbiol. 77, 2855–2862. resistance through horizontal gene bacteria: an update. Curr. Opin. in Mycobacterium tuberculosis. FEMS Espinosa-Urgel, M., Salido, A., and transfer. FEMS Microbiol. Rev. 35, Microbiol. 12, 512–519. Immunol. Med. Microbiol. 63, 1–9. Ramos, J. L. (2000). Genetic analy- 756–767. Burse, A., Weingart, H., and Ullrich, Davies, J. E. (1997). Origins, acquisi- sis of functions involved in adhesion Andersson, D. I., and Hughes, D. (2010). M. S. (2004a). NorM, an Erwinia tion and dissemination of antibiotic of Pseudomonas putida to seeds. J. Antibiotic resistance and its cost: is amylovora multidrug efflux pump resistance determinants. Ciba Found. Bacteriol. 182, 2363–2369. it possible to reverse resistance? Nat. involved in in vitro competition with Symp. 207, 15–27. Evans, K., Passador, L., Srikumar, Rev. Microbiol. 8, 260–271. other epiphytic bacteria. Appl. Envi- Davin-Regli, A., Bolla, J. M., James, C. R., Tsang, E., Nezezon, J., and Baquero, F. (2004). From pieces to pat- ron. Microbiol. 70, 693–703. E., Lavigne, J. P., Chevalier, J., Gar- Poole, K. (1998). Influence of the terns: evolutionary engineering in Burse, A., Weingart, H., and Ullrich, notel, E., et al. (2008). Membrane MexAB-OprM multidrug efflux sys- bacterial pathogens. Nat. Rev. Micro- M. S. (2004b). The phytoalexin- permeability and regulation of drug tem on quorum sensing in Pseu- biol. 2, 510–518. inducible multidrug efflux pump “influx and efflux” in enterobacte- domonas aeruginosa. J. Bacteriol. 180, Baquero, F., Alvarez-Ortega, C., and AcrAB contributes to virulence in rial pathogens. Curr. Drug Targets 9, 5443–5447. Martinez, J. L. (2009). Ecology and the fire blight pathogen, Erwinia 750–759. Fajardo, A., Linares, J. F., and Martinez, evolution of antibiotic resistance. amylovora. Mol. Plant Microbe Inter- Deng, X., Sun, F., Ji, Q., Liang, H., J. L. (2009). Towards an ecological Environ. Microbiol. Rep. 1, 469–476. act. 17, 43–54. Missiakas, D., Lan, L., et al. (2012). approach to antibiotics and antibiotic

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 7 |111

“fmicb-04-00007” — 2013/2/2 — 20:45 — page8—#8 Alvarez-Ortega et al. Ecological role of MDR pumps

resistance genes. Clin. Microbiol. Ingavale, S., Van Wamel, W., Luong, Lamichhane, G., Tyagi, S., and Lubelski, J., Konings, W. N., and Infect. 15(Suppl. 1), 14–16. T. T., Lee, C. Y., and Cheung, A. Bishai, W. R. (2005). Designer Driessen, A. J. (2007). Distribution Fargier, E., MacAogáin, M., Mooij, M. J., L. (2005). Rat/MgrA, a regulator of arrays for defined mutant analysis to and physiology of ABC-type trans- Woods,D. F.,Morrissey, J. P.,Dobson, autolysis, is a regulator of virulence detect genes essential for survival of porters contributing to multidrug A. D. W., et al. (2012). MexT func- genes in Staphylococcus aureus. Infect. Mycobacterium tuberculosis in mouse resistance in bacteria. Microbiol. Mol. tions as a redox-responsive regulator Immun. 73, 1423–1431. lungs. Infect. Immun. 73, 2533–2540. Biol. Rev. 71, 463–476. modulating disulfide stress resistance Ingavale, S. S., VanWamel,W., and Che- Lee, E. H., and Shafer,W.M. (1999). The Luong, T. T., Dunman, P. M., Murphy, in Pseudomonas aeruginosa. J. Bacte- ung, A. L. (2003). Characterization of farAB-encoded efflux pump medi- E., Projan, S. J., and Lee, C. Y. (2006). riol. 194, 3502–3511. RAT, an autolysis regulator in Staphy- ates resistance of gonococci to long- Transcription profiling of the mgrA Franke, S., Grass, G., and Nies, D. H. lococcus aureus. Mol. Microbiol. 48, chained antibacterial fatty acids. Mol. regulon in Staphylococcus aureus. J. (2001). The product of the ybdE gene 1451–1466. Microbiol. 33, 839–845. Bacteriol. 188, 1899–1910. of the Escherichia coli chromosome Jain, M., and Cox, J. S. (2005). Interac- Lertpiriyapong, K., Gamazon, E. R., Ma, D., Alberti, M., Lynch, C., Nikaido, is involved in detoxification of silver tion between polyketide synthase and Feng, Y., Park, D. S., Pang, J., Botka, H., and Hearst, J. E. (1996). The ions. Microbiology 147, 965–972. transporter suggests coupled synthe- G., et al. (2012). Campylobacter jejuni local repressor AcrR plays a modu- Gonzalez-Pasayo, R., and Martinez- sis and export of virulence lipid in M. type VI secretion system: roles in lating role in the regulation of acrAB Romero, E. (2000). Multiresistance tuberculosis. PLoS Pathog. 1:e2. doi: adaptation to deoxycholic acid, host genes of Escherichia coli by global genes of Rhizobium etli CFN42. Mol. 10.1371/journal.ppat.0010002 cell adherence, invasion, and in vivo stress signals. Mol. Microbiol. 19, Plant Microbe Interact. 13, 572–577. Jerse, A. E., Sharma, N. D., Simms, A. N., colonization. PLoS ONE 7:e42842. 101–112. Grass, G., and Rensing, C. (2001). Crow, E. T., Snyder, L. A., and Shafer, doi: 10.1371/journal.pone.0042842 Ma, D., Cook, D. N., Alberti, M., Genes involved in copper homeosta- W. M. (2003). A gonococcal efflux Li, X. Z., and Nikaido, H. (2004). Efflux- Pon, N. G., Nikaido, H., and Hearst, sis in Escherichia coli. J. Bacteriol. 183, pump system enhances bacterial sur- mediated drug resistance in bacteria. J. E. (1995). Genes acrA and acrB 2145–2147. vival in a female mouse model of Drugs 64, 159–204. encode a stress-induced efflux system Grkovic, S., Brown, M. H., and Skurray, genital tract infection. Infect. Immun. Li, X. Z., and Nikaido, H. (2009). Efflux- of Escherichia coli. Mol. Microbiol. 16, R. A. (2002). Regulation of bacterial 71, 5576–5582. mediated drug resistance in bacteria: 45–55. drug export systems. Microbiol. Mol. Johnson, P. J., Stringer, V.A., and Shafer, an update. Drugs 69, 1555–1623. Maggiorani Valecillos, A., Rodriguez Biol. Rev. 66, 671–701. W. M. (2011). Off-target gene reg- Lin, J., Akiba, M., Sahin, O., and Palenzuela, P., and Lopez-Solanilla, Haas, D. (2006). Cost of cell–cell sig- ulation mediated by transcriptional Zhang, Q. (2005a). CmeR func- E. (2006). The role of several mul- nalling in Pseudomonas aeruginosa: repressors of antimicrobial efflux tions as a transcriptional repressor for tidrug resistance systems in Erwinia why it can pay to be signal-blind. Nat. pump genes in Neisseria gonorrhoeae. the multidrug efflux pump CmeABC chrysanthemi pathogenesis. Mol. Rev. Microbiol. 4, 562. Antimicrob. Agents Chemother. 55, in Campylobacter jejuni. Antimicrob. Plant Microbe Interact. 19, 607–613. Hernandez, A., Mate, M. J., Sanchez- 2559–2565. Agents Chemother. 49, 1067–1075. Martin, R. G., Bartlett, E. S., Rosner, J. Diaz, P. C., Romero, A., Rojo, F., Kitaoka, M., Miyata, S. T., Unterweger, Lin, J., Cagliero, C., Guo, B., Barton, L., and Wall, M. E. (2008). Activation and Martinez, J. L. (2009). Structural D., and Pukatzki, S. (2011). Antibi- Y. W., Maurel, M. C., Payot, S., et al. of the Escherichia coli marA/soxS/rob and functional analysis of SmeT, the otic resistance mechanisms of Vib- (2005b). Bile salts modulate expres- regulon in response to transcriptional repressor of the Stenotrophomonas rio cholerae. J. Med. Microbiol. 60, sion of the CmeABC multidrug efflux activator concentration. J. Mol. Biol. maltophilia multidrug efflux pump 397–407. pump in Campylobacter jejuni. J. 380, 278–284. SmeDEF. J. Biol. Chem. 284, 14428– Kohler, T., Epp, S. F., Curty, L. K., and Bacteriol. 187, 7417–7424. Martinez, J. L. (2008). Antibiotics and 14438. Pechere, J. C. (1999). Characteriza- Linares, J. F., Lopez, J. A., Camafeita, antibiotic resistance genes in natural Hernandez, A., Ruiz, F. M., Romero, tion of MexT, the regulator of the E., Albar, J. P., Rojo, F., and Mar- environments. Science 321, 365–367. A., and Martinez, J. L. (2011a). MexE-MexF-OprN multidrug efflux tinez, J. L. (2005). Overexpression of Martinez, J. L. (2009a). Environmen- The binding of triclosan to SmeT, system of Pseudomonas aeruginosa. J. the multidrug efflux pumps MexCD- tal pollution by antibiotics and by the repressor of the multidrug efflux Bacteriol. 181, 6300–6305. OprJ and MexEF-OprN is associated antibiotic resistance determinants. pump SmeDEF, induces antibiotic Kohler, T., Van Delden, C., Curty, L. K., with a reduction of type III secre- Environ. Pollut. 157, 2893–2902. resistance in Stenotrophomonas mal- Hamzehpour, M. M., and Pechere, tion in Pseudomonas aeruginosa. J. Martinez, J. L. (2009b). The role of nat- tophilia. PLoS Pathog. 7:e1002103. J. C. (2001). Overexpression of the Bacteriol. 187, 1384–1391. ural environments in the evolution of doi: 10.1371/journal.ppat.1002103 MexEF-OprN multidrug efflux sys- Linares, J. F., Moreno, R., Fajardo, A., resistance traits in pathogenic bacte- Hernandez, A., Sanchez, M. B., and tem affects cell-to-cell signaling in Martinez-Solano, L., Escalante, R., ria. Proc. R. Soc. Lond. B Biol. Sci. 276, Martinez, J. L. (2011b). Quinolone Pseudomonas aeruginosa. J. Bacteriol. Rojo, F., et al. (2010). The global 2521–2530. resistance: much more than pre- 183, 5213–5222. regulator Crc modulates metabolism, Martinez, J. L., and Baquero, F. dicted. Front. Microbiol. 2:22. doi: Koronakis, V., Eswaran, J., and Hughes, susceptibility to antibiotics and vir- (2002). Interactions among strate- 10.3389/fmicb.2011.00022 C. (2004). Structure and function of ulence in Pseudomonas aeruginosa. gies associated with bacterial infec- Hirakata, Y., Srikumar, R., Poole, K., TolC: the bacterial exit duct for pro- Environ. Microbiol. 12, 3196–3212. tion: pathogenicity, epidemicity, and Gotoh, N., Suematsu, T., Kohno, S., teins and drugs. Annu. Rev. Biochem. Lindemann, A., Koch, M., Pessi, G., antibiotic resistance. Clin. Microbiol. et al. (2002). Multidrug efflux sys- 73, 467–489. Muller, A. J., Balsiger, S., Hennecke, Rev. 15, 647–679. tems play an important role in the Koronakis, V., Sharff, A., Koronakis, H., et al. (2010). Host-specific symbi- Martinez, J. L., Baquero, F., and Ander- invasiveness of Pseudomonas aerugi- E., Luisi, B., and Hughes, C. (2000). otic requirement of BdeAB, a RegR- sson, D. I. (2007). Predicting antibi- nosa. J. Exp. Med. 196, 109–118. Crystal structure of the bacterial controlled RND-type efflux system otic resistance. Nat. Rev. Microbiol. 5, Hirata, K., Suzuki, H., Nishizawa, T., membrane protein TolC central to in Bradyrhizobium japonicum. FEMS 958–965. Tsugawa, H., Muraoka, H., Saito, Y., multidrug efflux and protein export. Microbiol. Lett. 312, 184–191. Martinez, J. L., Baquero, F., and Ander- et al. (2010). Contribution of efflux Nature 405, 914–919. Liu, Z. Q., Zheng, P. Y., and Yang, P. sson, D. I. (2011). Beyond serial pumps to clarithromycin resistance Kuroda, T., and Tsuchiya, T. (2009). C. (2008). Efflux pump gene hefA of passages: new methods for predicting in Helicobacter pylori. J. Gastroenterol. Multidrug efflux transporters in the Helicobacter pylori plays an important the emergence of resistance to novel Hepatol. 25(Suppl. 1), S75–S79. MATE family. Biochim. Biophys. Acta role in multidrug resistance. World J. antibiotics. Curr. Opin. Pharmacol. Hofmann, A. F., and Eckmann, L. 1794, 763–768. Gastroenterol. 14, 5217–5222. 11, 439–445. (2006). How bile acids confer gut Kutschke, A., and de Jonge, B. L. Loake, G., and Grant, M. (2007). Sali- Martinez, J. L., Delgado-Iribarren, A., mucosal protection against bacteria. (2005). Compound efflux in Heli- cylic acid in plant defence-the players and Baquero, F. (1990). Mechanisms Proc. Natl. Acad. Sci. U.S.A. 103, cobacter pylori. Antimicrob. Agents and protagonists. Curr. Opin. Plant of iron acquisition and bacterial viru- 4333–4334. Chemother. 49, 3009–3010. Biol. 10, 466–472. lence. FEMS Microbiol. Rev. 6, 45–56.

www.frontiersin.org February 2013 | Volume 4 | Article 7 |112

“fmicb-04-00007” — 2013/2/2 — 20:45 — page9—#9 Alvarez-Ortega et al. Ecological role of MDR pumps

Martinez, J. L., Fajardo, A., Garmen- Neyrolles, O., and Guilhot, C. (2011). efflux and diffusion are involved in and into the host. FEMS Microbiol. dia, L., Hernandez, A., Linares, J. F., Recent advances in deciphering transport of Pseudomonas aeruginosa Rev. 26, 125–139. Martinez-Solano, L., et al. (2009a). A the contribution of Mycobacterium cell-to-cell signals. J. Bacteriol. 181, Rektorschek, M., Weeks, D., Sachs, G., global view of antibiotic resistance. tuberculosis lipids to pathogenesis. 1203–1210. and Melchers, K. (1998). Influence FEMS Microbiol. Rev. 33, 44–65. Tuberculosis 91, 187–195. Perez, A., Poza, M., Aranda, J., Latasa, of pH on metabolism and urease Martinez, J. L., Sanchez, M. B., Nies, D. H. (2003). Efflux-mediated C., Medrano, F. J., Tomas, M., activity of Helicobacter pylori. Gas- Martinez-Solano, L., Hernandez, A., heavy metal resistance in prokaryotes. et al. (2012). Effect of the tran- troenterology 115, 628–641. Garmendia, L., Fajardo, A., et al. FEMS Microbiol. Rev. 27, 313–339. scriptional activators SoxS, RobA and Rosenberg, E. Y., Bertenthal, D., Nilles, (2009b). Functional role of bacterial Nikaido, E., Yamaguchi, A., and RamA on expression of the multidrug M. L., Bertrand, K. P., and Nikaido, multidrug efflux pumps in microbial Nishino, K. (2008). AcrAB multidrug efflux pump AcrAB-TolC in Enter- H. (2003). Bile salts and fatty acids natural ecosystems. FEMS Microbiol. efflux pump regulation in Salmonella obacter cloacae. Antimicrob. Agents induce the expression of Escherichia Rev. 33, 430–449. enterica serovar Typhimurium by Chemother. 56, 6256–6266. coli AcrAB multidrug efflux pump Martinez, J. L., and Rojo, F. (2011). RamA in response to environmental Piddock, L. J. (2006a). Clinically rel- through their interaction with Rob Metabolic regulation of antibiotic signals. J. Biol. Chem. 283, 24245– evant chromosomally encoded mul- regulatory protein. Mol. Microbiol. resistance. FEMS Microbiol. Rev. 35, 24253. tidrug resistance efflux pumps in 48, 1609–1619. 768–789. Nikaido, H. (2009). Multidrug resis- bacteria. Clin. Microbiol. Rev. 19, Ruiz-Diez, B., Sanchez, P., Baquero, F., Matilla, M. A., Espinosa-Urgel, M., tance in bacteria. Annu. Rev. Biochem. 382–402. Martinez, J. L., and Navas, A. (2003). Rodriguez-Herva, J. J., Ramos, J. L., 78, 119–146. Piddock, L. J. (2006b). Multidrug- Differential interactions within the and Ramos-Gonzalez, M. I. (2007). Nikaido, H. (2011). Structure and resistance efflux pumps – not just Caenorhabditis elegans–Pseudomonas Genomic analysis reveals the major mechanism of RND-type multidrug for resistance. Nat. Rev. Microbiol. 4, aeruginosa pathogenesis model. J. driving forces of bacterial life in the efflux pumps. Adv. Enzymol. Relat. 629–636. Theor. Biol. 225, 469–476. rhizosphere. Genome Biol. 8, R179. Areas Mol. Biol. 77, 1–60. Poirel, L., Rodriguez-Martinez, J. M., Ruiz-Palacios, G. M. (2007). The health Mcknight, S. L., Iglewski, B. H., and Nikaido, H., and Takatsuka, Y. (2009). Mammeri, H., Liard, A., and Nord- burden of Campylobacter infection Pesci, E. C. (2000). The Pseudomonas Mechanisms of RND multidrug mann, P. (2005). Origin of plasmid- and the impact of antimicrobial resis- quinolone signal regulates rhl quo- efflux pumps. Biochim. Biophys. Acta mediated quinolone resistance deter- tance: playing chicken. Clin. Infect. rum sensing in Pseudomonas aerug- 1794, 769–781. minant QnrA. Antimicrob. Agents Dis. 44, 701–703. inosa. J. Bacteriol. 182, 2702–2708. Olivares, J., Alvarez-Ortega, C., Linares, Chemother. 49, 3523–3525. Saier, M. H. Jr., and Paulsen, I. T. (2001). Mercante, A. D., Jackson, L., John- J. F.,Rojo, F.,Kohler, T.,and Martinez, Poole, K. (2004). “Efflux pumps,” in Phylogeny of multidrug transporters. son, P. J., Stringer, V. A., Dyer, D. J. L. (2012). Overproduction of the Pseudomonas, ed. J.-L. Ramos (New Semin. Cell Dev. Biol. 12, 205–213. W., and Shafer, W. M. (2012). MpeR multidrug efflux pump MexEF-OprN York: Kluwer Academic/Plenum Pub- Saier, M. H. Jr., Paulsen, I. T., Sliwinski, regulates the mtr efflux locus in does not impair Pseudomonas aerug- lisher), 636–641. M. K., Pao, S. S., Skurray, R. A., and Neisseria gonorrhoeae and modulates inosa fitness in competition tests, but Poole, K. (2005). Efflux-mediated Nikaido, H. (1998). Evolutionary ori- antimicrobial resistance by an iron- produces specific changes in bac- antimicrobial resistance. J. Antimi- gins of multidrug and drug-specific responsive mechanism. Antimicrob. terial regulatory networks. Environ. crob. Chemother. 56, 20–51. efflux pumps in bacteria. FASEB J. 12, Agents Chemother. 56, 1491–1501. Microbiol. 14, 1968–1981. Poole, K. (2007). Efflux pumps 265–274. Meyer-Rosberg, K., Scott, D. R., Rex, Palumbo, J. D., Kado, C. I., and Phillips, as antimicrobial resistance mecha- Sanchez, P., Alonso, A., and Martinez, D., Melchers, K., and Sachs, G. D. A. (1998). An isoflavonoid- nisms. Ann. Med. 39, 162–176. J. L. (2002a). Cloning and character- (1996). The effect of environmental inducible efflux pump in Agrobac- Poole, K., Gotoh, N., Tsujimoto, H., ization of SmeT, a repressor of the pH on the proton motive force of terium tumefaciens is involved in Zhao, Q.,Wada,A.,Yamasaki,T.,et al. Stenotrophomonas maltophilia mul- Helicobacter pylori. Gastroenterology competitive colonization of roots. J. (1996). Overexpression of the mexC- tidrug efflux pump SmeDEF. Antimi- 111, 886–900. Bacteriol. 180, 3107–3113. mexD-oprJ efflux operon in nfxB- crob. Agents Chemother. 46, 3386– Mine, T., Morita, Y., Kataoka, A., Panthel, K., Dietz, P.,Haas, R., and Beier, type multidrug-resistant strains of 3393. Mizushima, T., and Tsuchiya, T. D. (2003). Two-component systems Pseudomonas aeruginosa. Mol. Micro- Sanchez, P., Linares, J. F., Ruiz- (1999). Expression in Escherichia coli of Helicobacter pylori contribute to biol. 21, 713–724. Diez, B., Campanario, E., Navas, of a new multidrug efflux pump, virulence in a mouse infection model. Pumbwe, L., Skilbeck, C. A., Nakano, V., A., Baquero, F., et al. (2002b). Fit- MexXY, from Pseudomonas aerugi- Infect. Immun. 71, 5381–5385. Avila-Campos, M. J., Piazza, R. M., ness of in vitro selected Pseudomonas nosa. Antimicrob. Agents Chemother. Pao, S. S., Paulsen, I. T., and Saier, M. and Wexler, H. M. (2007). Bile salts aeruginosa nalB and nfxB multidrug 43, 415–417. H. Jr. (1998). Major facilitator super- enhance bacterial co-aggregation, resistant mutants. J. Antimicrob. Murakami, S., Nakashima, R., family. Microbiol. Mol. Biol. Rev. 62, bacterial-intestinal epithelial cell Chemother. 50, 657–664. Yamashita, E., Matsumoto, T., 1–34. adhesion, biofilm formation and Sanchez, P., Rojo, F., and Martinez, and Yamaguchi, A. (2006). Crystal Pasca, M. R., Guglierame, P., De antimicrobial resistance of Bac- J. L. (2002c). Transcriptional regula- structures of a multidrug trans- Rossi, E., Zara, F., and Ric- teroides fragilis. Microb. Pathog. 43, tion of mexR, the repressor of Pseu- porter reveal a functionally rotating cardi, G. (2005). mmpL7 gene of 78–87. domonas aeruginosa mexAB-oprM mechanism. Nature 443, 173–179. Mycobacterium tuberculosis is respon- Ramos, J. L., Duque, E., Gallegos, M. multidrug efflux pump. FEMS Micro- Murakami, S., Nakashima, R., sible for isoniazid efflux in Mycobac- T., Godoy, P.,Ramos-Gonzalez, M. I., biol. Lett. 207, 63–68. Yamashita, E., and Yamaguchi, A. terium smegmatis. Antimicrob. Agents Rojas, A., et al. (2002). Mechanisms Schuster, M., Lostroh, C. P., Ogi, T., (2002). Crystal structure of bacterial Chemother. 49, 4775–4777. of solvent tolerance in gram-negative and Greenberg, E. P. (2003). Identi- multidrug efflux transporter AcrB. Paulsen, I. T. (2003). Multidrug efflux bacteria. Annu.Rev.Microbiol.56, fication, timing, and signal specificity Nature 419, 587–593. pumps and resistance: regulation and 743–768. of Pseudomonas aeruginosa quorum- Nakashima, R., Sakurai, K., Yamasaki, evolution. Curr. Opin. Microbiol. 6, Ravirala, R. S., Barabote, R. D., Wheeler, controlled genes: a transcriptome S., Nishino, K., and Yamaguchi, A. 446–451. D. M., Reverchon, S., Tatum, O., Mal- analysis. J. Bacteriol. 185, 2066– (2011). Structures of the multidrug Paulsen, I. T.,Chen, J., Nelson, K. E., and ouf, J., et al. (2007). Efflux pump gene 2079. exporter AcrB reveal a proximal mul- Saier, M. H. Jr. (2001). Comparative expression in Erwinia chrysanthemi Sekiya, H., Mima, T., Morita,Y.,Kuroda, tisite drug-binding pocket. Nature genomics of microbial drug efflux is induced by exposure to phenolic T., Mizushima, T., and Tsuchiya, 480, 565–569. systems. J. Mol. Microbiol. Biotechnol. acids. Mol. Plant Microbe Interact. 20, T. (2003). Functional cloning and Neyfakh, A. A. (1997). Natural func- 3, 145–150. 313–320. characterization of a multidrug tions of bacterial multidrug trans- Pearson, J. P., Van Delden, C., Reidl, J., and Klose, K. E. (2002). Vibrio efflux pump, mexHI-opmD, from porters. Trends Microbiol. 5, 309–313. and Iglewski, B. H. (1999). Active cholerae and cholera: out of the water a Pseudomonas aeruginosa mutant.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 7 | 113

“fmicb-04-00007” — 2013/2/2 — 20:45 — page 10 — #10 Alvarez-Ortega et al. Ecological role of MDR pumps

Antimicrob. Agents Chemother. 47, by the drug binding repressor TtgR. Mcmath, L. M., et al. (2011). Discov- pumps in bacterial antibiotic resis- 2990–2992. Antimicrob. Agents Chemother. 47, ery and characterization of a unique tance. J. Antimicrob. Chemother. 51, Shafer, W. M., Qu, X., Waring, A. 3067–3072. mycobacterial heme acquisition sys- 9–11. J., and Lehrer, R. I. (1998). Modu- Tian, Z. X., Fargier, E., Mac Aogain, M., tem. Proc. Natl. Acad. Sci. U.S.A. 108, Worku, M. L., Karim, Q. N., lation of Neisseria gonorrhoeae sus- Adams, C., Wang, Y. P., and O’Gara, 5051–5056. Spencer, J., and Sidebotham, R. ceptibility to vertebrate antibacterial F. (2009a). Transcriptome profiling van Vliet, A. H., Kuipers, E. J., Waidner, L. (2004). Chemotactic response of peptidesduetoamemberofthe defines a novel regulon modulated by B., Davies, B. J., De Vries, N., Penn, Helicobacter pylori to human plasma resistance/nodulation/division efflux the LysR-type transcriptional regula- C. W.,et al. (2001). Nickel-responsive and bile. J. Med. Microbiol. 53, pump family. Proc. Natl. Acad. Sci. tor MexT in Pseudomonas aeruginosa. induction of urease expression in 807–811. U.S.A. 95, 1829–1833. Nucleic Acids Res. 37, 7546–7559. Helicobacter pylori is mediated at the Wu, C. P., Hsieh, C. H., and Wu, Y. Shao, C., Zhang, Q., Sun, Y., Liu, Z., Tian, Z. X., Mac Aogain, M., O’Connor, transcriptional level. Infect. Immun. S. (2011). The emergence of drug Zeng, J., Zhou, Y., et al. (2008). Heli- H. F., Fargier, E., Mooij, M. J., 69, 4891–4897. transporter-mediated multidrug cobacter pylori protein response to Adams, C., et al. (2009b). MexT Veal, W. L., Nicholas, R. A., and Shafer, resistance to cancer chemotherapy. human bile stress. J. Med. Microbiol. modulates virulence determinants W. M. (2002). Overexpression of the Mol. Pharm. 8, 1996–2011. 57, 151–158. in Pseudomonas aeruginosa indepen- MtrC-MtrD-MtrE efflux pump due Yamamoto, K., and Ishihama, A. Stahler, F. N., Odenbreit, S., Haas, R., dent of the MexEF-OprN efflux to an mtrR mutation is required for (2005). Transcriptional response of Wilrich, J., Van Vliet, A. H., Kusters, pump. Microb. Pathog. 47, 237–241. chromosomally mediated penicillin Escherichia coli to external copper. J. G., et al. (2006). The novel Heli- Tomb, J. F., White, O., Kerlavage, A. R., resistance in Neisseria gonorrhoeae. J. Mol. Microbiol. 56, 215–227. cobacter pylori CznABC metal efflux Clayton, R. A., Sutton, G. G., Fleis- Bacteriol. 184, 5619–5624. Zhang, A., Rosner, J. L., and Mar- pump is required for cadmium, zinc, chmann, R. D., et al. (1997). The Vila, J., and Martinez, J. L. (2008). Clin- tin, R. G. (2008). Transcriptional and nickel resistance, urease modula- complete genome sequence of the ical impact of the over-expression activation by MarA, SoxS and Rob tion, and gastric colonization. Infect. gastric pathogen Helicobacter pylori. of efflux pump in nonfermentative of two tolC promoters using one Immun. 74, 3845–3852. Nature 388, 539–547. gram-negative bacilli, development binding site: a complex promoter Stone, T. W., Forrest, C. M., Stoy, Trainor, E. A., Horton, K. E., Savage, P. of efflux pump inhibitors. Curr. Drug configuration for tolC in Escherichia N., and Darlington, L. G. (2012). B., Testerman, T. L., and Mcgee, D. J. Targets 9, 797–807. coli. Mol. Microbiol. 69, 1450– Involvement of kynurenines in Hunt- (2011). Role of the HefC efflux pump Waidner, B., Melchers, K., Ivanov, I., 1455. ington’s disease and stroke-induced in Helicobacter pylori cholesterol- Loferer, H., Bensch, K. W., Kist, brain damage. J. Neural. Transm. 119, dependent resistance to ceragenins M., et al. (2002). Identification by Conflict of Interest Statement: The 261–274. and bile salts. Infect. Immun. 79, RNA profiling and mutational anal- authors declare that the research was Stoof, J., Belzer, C., and Van Vliet, A. 88–97. ysis of the novel copper resistance conducted in the absence of any com- H. M. (2008).“Metal metabolism and Trotonda, M. P., Tamber, S., Memmi, determinants CrdA (HP1326), CrdB mercial or financial relationships that transport in Helicobacter pylori,” in G., and Cheung, A. L. (2008). (HP1327), and CzcB (HP1328) in could be construed as a potential con- Helicobacter pylori: Molecular Genet- MgrA represses biofilm formation in Helicobacter pylori. J. Bacteriol. 184, flict of interest. ics and Cellular Biology,ed.Y. Staphylococcus aureus. Infect. Immun. 6700–6708. Yamaoka (Norfolk: Caister Academic 76, 5645–5654. Waidner, B., Melchers, K., Stahler, Received: 13 November 2012; paper Press), 165–177. Truong-Bolduc, Q. C., Dunman, P. M., F. N., Kist, M., and Bereswill, pending published: 04 December 2012; Tahlan, K., Wilson, R., Kastrinsky, D. Strahilevitz, J., Projan, S. J., and S. (2005). The Helicobacter pylori accepted: 07 January 2013; published B., Arora, K., Nair, V., Fischer, E., Hooper, D. C. (2005). MgrA is a CrdRS two-component regulation online: 05 February 2013. et al. (2012). SQ109 targets MmpL3, multiple regulator of two new efflux system (HP1364/HP1365) is required Citation: Alvarez-Ortega C, Olivares J a membrane transporter of trehalose pumps in Staphylococcus aureus. J. for copper-mediated induction of and Martínez JL (2013) RND multidrug monomycolate involved in mycolic Bacteriol. 187, 2395–2405. the copper resistance determinant efflux pumps: what are they good for? acid donation to the cell wall core of Truong-Bolduc, Q. C., Zhang, X., and CrdA. J. Bacteriol. 187, 4683– Front. Microbio. 4:7. doi: 10.3389/fmicb. Mycobacterium tuberculosis. Antimi- Hooper, D. C. (2003). Characteriza- 4688. 2013.00007 crob. Agents Chemother. 56, 1797– tion of NorR protein, a multifunc- Warner, D. M., Folster, J. P., Shafer, This article was submitted to Fron- 1809. tional regulator of norA expression W. M., and Jerse, A. E. (2007). tiers in Antimicrobials, Resistance and Taylor, D. L., Bina, X. R., and Bina, in Staphylococcus aureus. J. Bacteriol. Regulation of the MtrC-MtrD-MtrE Chemotherapy, a specialty of Frontiers in J. E. (2012). Vibrio cholerae VexH 185, 3127–3138. efflux-pump system modulates the Microbiology. encodes a multiple drug efflux pump Tsugawa, H., Suzuki, H., Muraoka, in vivo fitness of Neisseria gonor- Copyright © 2013 Alvarez-Ortega, Oli- that contributes to the production H., Ikeda, F., Hirata, K., Matsuzaki, rhoeae. J. Infect. Dis. 196, 1804– vares and Martínez. This is an open- of cholera toxin and the toxin co- J., et al. (2011). Enhanced bacte- 1812. access article distributed under the terms regulated pilus. PLoS ONE 7:e38208. rial efflux system is the first step Webb, V., and Davies, J. (1993). Antibi- of the Creative Commons Attribution doi: 10.1371/journal.pone.0038208 to the development of metronida- otic preparations contain DNA: a License, which permits use, distribution Teran, W., Felipe, A., Segura, A., Rojas, zole resistance in Helicobacter pylori. source of drug resistance genes? and reproduction in other forums, pro- A., Ramos, J. L., and Gallegos, M. T. Biochem. Biophys. Res. Commun. 404, Antimicrob. Agents Chemother. 37, vided the original authors and source (2003). Antibiotic-dependent induc- 656–660. 2379–2384. are credited and subject to any copy- tion of Pseudomonas putida DOT- Tullius, M. V., Harmston, C. A., Owens, Webber, M. A., and Piddock, L. J. right notices concerning any third-party T1E TtgABC efflux pump is mediated C. P., Chim, N., Morse, R. P., (2003). The importance of efflux graphics etc.

www.frontiersin.org February 2013 | Volume 4 | Article 7 | 114

“fmicb-04-00007” — 2013/2/2 — 20:45 — page 11 — #11 MINI REVIEW ARTICLE published: 14 February 2013 doi: 10.3389/fmicb.2013.00021 Extracellular DNA-induced antimicrobial peptide resistance mechanisms in Pseudomonas aeruginosa

Shawn Lewenza1,2* 1 Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada 2 Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, Canada

Edited by: Extracellular DNA (eDNA) is in the environment, bodily fluids, in the matrix of biofilms, and Fiona Walsh, Agroscope accumulates at infection sites. eDNA can function as a nutrient source, a universal biofilm Changins-Wädenswil, Switzerland matrix component, and an innate immune effector in eDNA traps. In biofilms, eDNA is Reviewed by: required for attachment, aggregation, and stabilization of microcolonies. We have recently Elaine Allan, University College London, UK shown that eDNA can sequester divalent metal cations, which has interesting implications + Charles W. Knapp, University of on antibiotic resistance. eDNA binds metal cations and thus activates the Mg2 -responsive Strathclyde, UK PhoPQ and PmrAB two-component systems. In Pseudomonas aeruginosa and many other *Correspondence: Gram-negative bacteria, the PhoPQ/PmrAB systems control various genes required for Shawn Lewenza, Department of virulence and resisting killing by antimicrobial peptides (APs), including the pmr genes Microbiology, Immunology and Infectious Diseases, University of (PA3552–PA3559) that are responsible for the addition of aminoarabinose to lipid A. Calgary, 3330 Hospital Drive, Health The PA4773–PA4775 genes are a second DNA-induced cluster and are required for the Research Innovation Centre, Room production of spermidine on the outer surface, which protects the outer membrane from 2C66, Calgary, AB, Canada T2N 4N1. e-mail: [email protected] AP treatment. Both modifications mask the negative surface charges and limit membrane damage by APs. DNA-enriched biofilms or planktonic cultures have increased antibiotic resistance phenotypes to APs and aminoglycosides. These dual antibiotic resistance and immune evasion strategies may be expressed in DNA-rich environments and contribute to long-term survival.

Keywords: antibiotic resistance, antimicrobial peptides, bioﬁlm, PhoPQ, PmrAB, Pseudomonas aeruginosa, immune evasion, extracellular DNA

SOURCE AND FUNCTIONS OF EXTRACELLULAR DNA physiology and serves many functions for bacteria. eDNA has Extracellular DNA (eDNA) is released from dead plant or microor- been shown to serve as a sole nutrient source of phosphate, nitro- ganisms and accumulates in soil, aquatic, and sediment envi- gen, and carbon for Pseudomonas aeruginosa, Escherichia coli, and ronments (Dell’Anno and Danovaro, 2005; Vlassov et al., 2007; Shewanella spp. (Finkel and Kolter, 2001; Palchevskiy and Finkel, Pietramellar et al., 2009). Bacteria actively release or secrete DNA, 2006; Pinchuk et al., 2008). We identified a secreted DNase (EddB) or it is released during bacterial lysis and outer membrane vesicle that is produced in the presence of low DNA concentrations and formation (Chiang and Tolker-Nielsen, 2010). eDNA is known to under limiting phosphate conditions (Mulcahy et al., 2010). The accumulate in many Gram-negative and Gram-positive bacterial EddB DNase is required for degradation of eDNA and utiliza- biofilms (Tetz et al., 2009; Chiang and Tolker-Nielsen, 2010). tion of DNA fragments or nucleotides as a sole source of carbon, Extracellular DNA is present in healthy body sites and fluids, nitrogen, and phosphate (Mulcahy et al., 2010). There is an alka- such as the gastrointestinal tract, blood, milk, secretions, and likely line phosphatase expressed upstream of the DNase, EddA, which on mucosal surfaces (Vlassov et al., 2007). During infection, eDNA may also be required for phosphorus acquisition from DNA. In can accumulate due to the heavy recruitment of host immune cells Shewanella oneidensis, a secreted DNase (ExeM) with significant and the production of neutrophil extracellular traps (NETs),as dis- homology to EddB (34% identity) is also required for utilization of cussed later. Chronic lung infections in persons challenged with DNA as a nutrient source (Godeke et al., 2011). A number of intra- cystic fibrosis (CF) are caused by polymicrobial biofilms that are cellular ssDNA exonucleases have also been shown to be required adapted for long-term survival. The sputum from CF patients has for growth using DNA as a sole carbon course (Palchevskiy and very high concentrations of eDNA and is the reason for the use Finkel, 2006). DNA uptake also facilitates lateral gene transfer of human recombinant deoxyribonuclease (DNase) as a mucolytic (LGT) and integration of foreign DNA sequences into the genome. treatment (Shak et al.,1990; Ranasinha et al.,1993). Inhaled DNase Palchevskiy and Finkel (2006) proposed that dsDNA was taken (Pulmozyme) has been shown to reduce sputum viscosity, inflam- into the cell, similar to the process of DNA uptake for LGT, con- mation, and exacerbations, as well as improve lung function and verted to ssDNA and then degraded by intracellular exonucleases survival (Jones and Wallis, 2010; Konstan and Ratjen, 2012). upon entry into the cytoplasm.

DNA IS A NUTRIENT SOURCE DNA IS A BIOFILM MATRIX POLYMER Given the abundance of eDNA in the environment, it is not Extracellular DNA is required and primarily acts to facilitate surprising that DNA has a significant influence on bacterial attachment, aggregation, stabilization, and maturation of biofilm

www.frontiersin.org February 2013 | Volume 4 | Article 21 | 115

“fmicb-04-00021” — 2013/2/13 — 19:01 — page1—#1 Lewenza DNA-induced antibiotic resistance genes

formation (Chiang and Tolker-Nielsen, 2010). DNase treatment CATION CHELATION AND ANTIMICROBIAL ACTIVITIES OF DNA of young P. aeruginosa biofilms results in biofilm dissolution, The focus of our initial work was to test the hypothesis that but mature biofilms resist DNase treatment, indicating a role in the matrix polymers influence bacterial gene expression. While early biofilm formation (Whitchurch et al., 2002). Accumulation biofilm polymers are known to have several protective immune of exopolysaccharide (EPS) in mature biofilms probably accounts evasion functions, we wondered if the matrix polymers also drive for the inability to degrade mature biofilms with exogenous DNase. unique gene expression profiles that contribute to the phenotypes Mutant strains that accumulated less eDNA during biofilm for- of cells in biofilms. Our initial observation upon addition of puri- mation were more destabilized by treatment with sodium dodecyl fied DNA exogenously to planktonic cultures was that bacterial sulfate (SDS; Allesen-Holm et al., 2006), providing further evi- growth was inhibited at DNA concentrations greater than 5 mg/ml dence for a role in biofilm stabilization. Treatment of young (Mulcahy et al., 2008). Due to the highly anionic character of biofilms with DNase impaired the development of the cap struc- DNA, we hypothesized that DNA was a cation chelator and indeed tures of mushroom-shaped biofilms (Barken et al., 2008). DNase demonstrated that DNA efficiently binds divalent metal cations treatment of biofilms formed by Gram-negative or Gram-positive that including Mg2+,Ca2+,Mn2+, and Zn2+ (Mulcahy et al., bacteria reduces the biomass, which suggests that eDNA is a ubiq- 2008). In addition, DNA has a rapid antimicrobial killing activity uitous DNA polymer (Tetz et al., 2009). The exception to the rule that can be neutralized by pre-incubating DNA with excess cations is in Caulobacter crescentus where eDNA blocks biofilm forma- beforeexposuretobacteria(Mulcahy et al., 2008). As bacterial tion by binding to the polar holdfast structure, which is required surfaces are highly negatively charged and consequently have high for irreversible attachment (Berne et al., 2010). eDNA has been levels of Mg2+ and Ca2+ bound to the surface (Nicas and Hancock, shown to localize to specific regions of mushroom-shaped micro- 1980), we suspected that DNA chelated cations from surfaces and colonies formed by P. aeruginosa in flow-chamber biofilms. In disrupted membrane integrity. Using fluorescence microscopy to mature microcolonies, eDNA localizes primarily to the stalk struc- monitor membrane integrity, we demonstrated that DNA causes ture, at the boundary of the stalk and cap (Allesen-Holm et al., major perturbations to the outer and inner bacterial membranes, 2006). In unstructured peg-adhered biofilms, eDNA can be visu- leading to rapid cell lysis and death. In addition, cells treated with alized throughout thin biofilms with no particular organization antimicrobial concentrations of DNA released small outer mem- (Mulcahy et al., 2008). eDNA has also been shown to be present brane vesicles. This result indicated that DNA can strip sections as a matrix component in biofilms formed in vivo during infec- of outer membrane from the envelope, disrupting outer and inner tion with P. aeruginosa (Mulcahy et al., 2011; van Gennip et al., membrane integrity, resulting in cell lysis. The membrane destabi- 2012), Haemophilus influenzae (Jurcisek and Bakaletz, 2007), and lizing effects of DNA are similar to that of known cation chelator Bordetella (Conover et al., 2011). ethylenediaminetetraacetic acid (EDTA). DNA appears to have a broad-spectrum antimicrobial activity against Gram-positive and EXTRACELLULAR DNA TRAPS Gram-negative bacteria (Mulcahy et al., 2008). Neutrophil extracellular traps were first described in neutrophils, but have since been identified in other immune cell types includ- ANTIMICROBIAL PEPTIDE KILLING AND RESISTANCE MECHANISMS ing eosinophils and mast cells (Brinkmann and Zychlinsky, 2012). Cationic antimicrobial peptides (APs) are short, amphipathic pep- NETs can kill Gram-positive and Gram-negative bacteria, fungi, tides with broad-spectrum antimicrobial activity produced by parasites, and viruses (Brinkmann et al., 2004; Urban et al., 2006, the immune systems of most forms of life (Hancock and Sahl, 2009; Guimaraes-Costa et al., 2009; Saitoh et al., 2012). Although 2006). The mechanism of killing is primarily through membrane there are numerous antimicrobial neutrophil components embed- binding and disruption, although they also disrupt cytoplasmic dedinNETs(Urban et al., 2009), bacterial killing is largely processes (Hancock and Sahl, 2006; Kraus and Peschel, 2006). attributed to the antimicrobial activity of histones (Brinkmann Host defense peptides are another class of short peptides that may et al., 2004). NET killing can be blocked by either dissolving the not have direct antimicrobial activities, but are protective due to NET structure with DNase, or by the addition of neutralizing anti- their ability to modulate the innate immune response (Hancock histone antibodies, which block histone antimicrobial activity. The and Sahl, 2006). APs are positively charged and therefore inter- process of NETosis is a novel mechanism of trapping and killing act with the negatively charged lipopolysaccharide (LPS) in the bacteria, as well as limiting bacterial dissemination (Brinkmann Gram-negative outer membrane surface. The hydrophobic char- and Zychlinsky, 2012; McDonald et al., 2012; Yipp et al., 2012), acter permits membrane integration, disruption, and ultimately For the purpose of this review, it is important to note that NET cell lysis and death. Gram-negative and Gram-positive bacteria formation during infection is likely a major contribution of DNA alter their membrane charge to resist peptide killing by producing accumulation at the site of infection. NET formation has been modified phospholipids, LPS, or teichoic acid structures, whose observed in CF sputum and likely contributes to the accumula- negative charges are masked (Kraus and Peschel, 2006; Anaya- tion of eDNA during chronic CF lung infections (Marcos et al., Lopez et al., 2012). Surface modifications that contribute to AP 2010; Manzenreiter et al., 2012). Neutrophils are among the first resistance include alanine-modified teichoic acids, highly acylated immune cells recruited to the infection site and most of the DNA lipid A, as well as phosphoethanolamine and aminoarabinose- in the CF lung is derived from neutrophils (Lethem et al., 1990). modified lipid A species (Kraus and Peschel, 2006; Moskowitz and In plant roots, an eDNA barrier is produced that protects the root Ernst, 2010; Anaya-Lopez et al., 2012). Collectively, these modifi- from infection and is analogous to eDNA traps of human immune cations prevent or limit peptide binding or entry and disruption cells (Hawes et al., 2011). of bacterial membranes. CF isolates of P. aeruginosa are known to

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 21 |116

“fmicb-04-00021” — 2013/2/13 — 19:01 — page2—#2 Lewenza DNA-induced antibiotic resistance genes

produce highly acylated lipid A species and aminoarabinose mod- spermidine protects the outer membrane from APs polymyxin B ifications on the 1- and 4-phosphates of lipid A (Moskowitz and and CP10A,but also from treatment with other cationic antibiotics Ernst, 2010). including the aminoglycoside gentamicin (Johnson et al., 2012). Polyamines are typically found in the cytoplasm but here we have DNA-INDUCED EXPRESSION OF THE pmr OPERON identified a novel role for polyamines on the bacterial surface. In The pmr genes are required for the covalent addition of aminoara- the presence of eDNA, we proposed that P. aeruginosa produces binose to the 1- and 4-phosphates of lipid A (Moskowitz and spermidine as an organic polycation replacement for the divalent Ernst, 2010), which protects the outer membrane from AP treat- metal cation Mg2+ that functions to mask the negative surface ment (Johnson et al., 2012), and is required for peptide resistance charge and block AP binding (Figure 1). Magnesium ions are (Moskowitz et al., 2004; Lewenza et al., 2005). The pmr genes are essential to cross-bridge the core phosphates of lipid A, so it is not regulated by the PhoPQ and PmrAB systems in P. aeruginosa, and surprising that P. aeruginosa produces a replacement polycation in in many other Gram-negative organisms including Salmonella the presence of DNA or under Mg2+ limiting conditions. Surface enterica, Klebsiella pneumoniae, and Yersinia pestis (Macfarlane polyamines also act as antioxidants and quench reactive oxygen et al., 1999; Groisman, 2001; McPhee et al., 2006; Cheng et al., species, thereby protecting the outer membrane from oxidative 2010; O’Loughlin et al., 2010). The P. aeruginosa PhoQ sensor stress damage to lipids (Johnson et al., 2012). responds to Mg2+ levels and is activated under Mg2+ limiting conditions, leading to increased expression of the pmr operon. In DNA-INDUCED ANTIBIOTIC RESISTANCE IN BIOFILMS Mg2+-rich conditions, the presence of sub-lethal exposure to APs To test for a role of DNA-induced expression of the pmr genes also induces expression of the pmr operon (McPhee et al., 2003), in biofilm-specific antibiotic resistance, we determined the min- although this adaptive resistance is controlled by the CprRS and imum biofilm eradication concentration (MBEC) in wild type ParRS two-component systems (Fernandez et al., 2012). biofilms and in biofilms formed the presence or absence of exoge- Although DNA prevented growth at higher concentrations, we nous DNA (Mulcahy et al., 2008). DNA-enriched biofilms were examined the influence of sub-lethal concentrations of DNA on shown to be eightfold more tolerant to the APs polymyxin B pmr gene expression. In planktonic cultures grown in Mg2+ rich and colistin, and 64- to 128-fold more tolerant to the amino- media supplemented with DNA, we showed that DNA caused a glycosides gentamicin and tobramycin. Interestingly, planktonic concentration-dependent induction of the pmr operon (PA3552– cultures containing exogenous DNA also demonstrated DNA- PA3559)inP. aeruginosa (Mulcahy et al., 2008). DNA induction of induced resistance to aminoglycosides and APs (Mulcahy et al., this operon can be explained by cation sequestration by DNA, and 2008). Exogenous DNA did not have an effect on β-lactam or subsequent activation of the PhoPQ/PmrAB systems. Increased fluoroquinolones resistance. A mutant in the pmr cluster did amounts of DNA resulted in more Mg2+ sequestered and therefore not exhibit any DNA-induced resistance to APs, indicating that increasingly higher levels of pmr gene expression. Figure 1 depicts these genes were expressed and required for resistance in DNA- the cation chelating effects of DNA on the structure of LPS in enriched biofilms (Mulcahy et al., 2008). The pmr mutant showed P. aeruginosa. Gene induction by DNA can be prevented by the an intermediate aminoglycoside resistance phenotype, indicating addition of excess cations in combination with DNA, confirming that the pmr aminoarabinose modification also contributed par- that the cation chelating activity of DNA can be neutralized. We tially to DNA-induced aminoglycoside resistance. It is possible have recently shown that eDNA can also induce expression of the that the anionic eDNA bound positively charged aminoglycosides Salmonella enterica serovar Typhimurium pmr operon and causes and provided some protection as a matrix barrier, thus explain- increased AP resistance (Submitted), indicating that eDNA may ing the residual level of resistance in the presence of eDNA. It is play a general role in activating the PhoPQ system in DNA-rich known that DNA is capable of binding to aminoglycosides (Ram- environments. phal et al., 1988; Purdy Drew et al., 2009) and APs (Bucki et al., 2007). Therefore it is possible that DNA can induce specific resis- DNA-INDUCED EXPRESSION OF SPERMIDINE SYNTHESIS GENES tance mechanisms and also act as a protective matrix absorbing A large number of P. aeruginosa genes are regulated under Mg2+ and limiting antimicrobial exposure. limiting conditions; some exclusively by PhoPQ and others are + controlledbyasecondMg2 sensing two-component system CONCENTRATION OF eDNA IN BIOFILMS AND INFECTION SITES PmrAB (McPhee et al., 2006). While the pmr operon is directly An important question that has not been fully answered is to controlled by both PmrA and PhoP (McPhee et al., 2003, 2006), determine if sufficient DNA accumulates in biofilms or dur- we identified a three-gene cluster upstream of PmrAB with homol- ing infections, to induce the expression of these protective, ogy to spermidine synthesis genes PA4773 (speD) and PA4774 AP resistance phenotypes. In microarray studies comparing the (speE) that is controlled exclusively by PmrAB (McPhee et al., gene expression profiles of biofilm to planktonic cultures, the 2003). The addition of DNA to planktonic cultures also induced PhoPQ/PmrAB-controlled genes are not among the biofilm- the expression of PA4773–PA4775 in a concentration-dependent induced genes (Whiteley et al., 2001; Waite et al., 2005). This may manner (Johnson et al., 2012). Mutants in the PA4773–PA4775 be due to an insufficient accumulation of DNA in these particular genes were sensitive to APs, indicating a potential role in resistance biofilm model systems, and/or the presence of high Mg2+ levels to APs (Lewenza et al., 2005). We confirmed that PA4773–PA4774 in the growth media used, which can neutralize eDNA and pre- were required for spermidine synthesis, which is localized on the vent activation of the Mg2+ sensing PhoPQ and PmrAB systems. bacterial surface (Johnson et al., 2012). Surface and exogenous However, a recent paper described a novel regulator of biofilm

www.frontiersin.org February 2013 | Volume 4 | Article 21 |117

“fmicb-04-00021” — 2013/2/13 — 19:01 — page3—#3 Lewenza DNA-induced antibiotic resistance genes

FIGURE 1 | Lipopolysaccharide (LPS) modifications in the presence of to limiting Mg2+ or cation chelation, the PhoPQ/PmrAB systems are extracellular DNA that contribute to antimicrobial peptide resistance. activated leading to the production of covalently attached aminoarabinose to (A) Divalent metal cations including Mg2+ (orange) bind to the negatively the phosphates of lipid A (red) and the production of polycation spermidine charged phosphates of the lipid A moiety of LPS and act to stabilize LPS. (charge, +3) on the surface, which may bind electrostatically to negative Antimicrobial peptides (green) can displace cations and disrupt membrane charges in the core oligosaccharide (dark blue) of the O antigen. Both integrity, leading to cell lysis and death. (B) Extracellular DNA binds and modifications mask the negative charges and protect the outer membrane sequesters cations from the environment and the membrane. (C) In response from peptide damage. formation, BfmR, which is required for P. aeruginosa to transition Although the total concentration of eDNA can be quantitated to the maturation-1 biofilm developmental stage (Petrova et al., in biofilms (Wu and Xi, 2009), the localized concentration may be 2011). Biofilms formed by this mutant accumulated more eDNA, more important than the overall concentrations. The accumula- which was due to increased bacteriophage-mediated lysis in the tion of DNA at infection sites is not well documented but sputum bfmR mutant. Microarrays were performed on bfmR biofilms and from the lungs of persons challenged with CF is known to accumu- both the pmr and PA4774–PA4775 genes were induced in bfmR late DNA at concentrations ranging from <1 to 20 mg/ml (Shak biofilms relative to wild type PAO1 (Petrova et al., 2011). This is et al., 1990; Ranasinha et al., 1993). There are relatively low Mg2+ likely due to the increased eDNA accumulation, but it may be concentrations in the CF lung (0.08–2 mM; Palmer et al., 2005; possible that these genes are also controlled by BfmR. Sanders et al., 2006), not high enough to neutralize the cation Severalpapershavereportedthepmr-gfp gene expression pat- chelating potential of such high DNA concentrations. Based on tern in P. aeruginosa flow-chamber biofilms (Haagensen et al., the known concentration of DNA and Mg2+ in CF lung, it is 2007; Pamp et al., 2008). The pmr operon is required for col- probable that the PhoPQ/PmrAB-controlled genes are expressed istin resistance in flow-chamber biofilms, but in many of these in the CF lung and may contribute to long-term survival in the CF studies, there was little or no expression of the pmr operon in lung. Recently, colistin resistant mutants have been characterized untreated biofilms. This result suggested that there is not sufficient from CF patients and shown to contain gain-of-function PhoQ eDNA accumulation in flow-chamber biofilms cultivated under and PmrB sensor mutations, leading to increased expression of these conditions to influence pmr expression. Shortly after col- the pmr genes (Miller et al., 2011; Moskowitz et al., 2012). This istin treatment, pmr-gfp expression was seen in a colistin resistant result underscores the importance of these genes in the CF lung, subpopulation formed on the caps of mushroom-shaped micro- particularly in those patients treated with colistin. colonies (Haagensen et al., 2007). It is known that the presence of APs can induce the pmr genes, highlighting an adaptive resistance FUTURE WORK mechanism whereby the resistance genes are induced by exposure To date, we have shown that eDNA influences the expression of to sub-lethal concentrations of APs (McPhee et al., 2003). The several genes including a secreted DNase, and at least two operons colistin resistant subpopulation is metabolically active, motile, controlled by the PhoPQ and PmrAB two-component systems. requires various multi-drug efflux pumps, and appears shortly We are currently exploring the global effect of eDNA on bacterial after the early stages of surface attachment (Haagensen et al., 2007; gene expression using a genome-wide transcriptomic method and Pamp et al., 2008). Colistin treatment was effective at killing the screening a library of transcription lux fusions (Lewenza et al., cells within the inner stalk structures but not the resistant sub- 2005) to identify novel DNA-induced or repressed genes. While population on the surface, indicating that colistin penetration is aminoarabinose-modified LPS and surface spermidine both pro- not limited in flow-chamber biofilms, despite the accumulation tect the outer membrane and contribute to AP resistance in vitro, of eDNA and EPS in these biofilms (Haagensen et al., 2007; Pamp they may also protect P. aeruginosa from APs produced by the et al., 2008). innate immune system. It will be important to examine the role

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 21 |118

“fmicb-04-00021” — 2013/2/13 — 19:01 — page4—#4 Lewenza DNA-induced antibiotic resistance genes

of these surface modifications in protecting P. aeruginosa from antimicrobials and limiting their access to bacterial cells. Since innate immune cells known to produce APs, such as macrophages DNA accumulates in the environment, in infection sites and and neutrophils. in the biofilm matrix, the influence of DNA on gene expression may contribute to long-term survival in these DNA-rich CONCLUSION environments. We identified a new property of eDNA as a divalent metal cation chelator, which is required to induce the expression of multi- ACKNOWLEDGMENTS ple operons that contribute to decreasing the permeability of I would like to thank all members of my lab who contributed to the outer membrane to APs and aminoglycosides. P. aeruginosa this research, including Laetitia Charron-Mazenod, Heidi Mulc- EPS are also anionic polymers with calcium binding properties, ahy, Lori Johnson, and Shawn Horsman. Thanks to Joseph McPhee indicating that cation binding and sequestration may be a gen- for critical reading of the manuscript. This research was funded by eral feature of the biofilm matrix. The anionic charge of DNA Cystic Fibrosis Canada and the Westaim-ASRA Chair in Biofilm may also contribute to antibiotic resistance by binding to cationic Research.

REFERENCES Acids and Molecular Biology,edsY. Hancock, R. E., and Sahl, H. G. (2006). PhoP-PhoQ homologues in Pseu- Allesen-Holm, M., Barken, K. B., Kikuchi and E. Rykova (Heidelberg: Antimicrobial and host-defense pep- domonas aeruginosa regulate expres- Yang, L., Klausen, M., Webb, J. Springer), 1–14. tides as new anti-infective therapeu- sion of the outer-membrane protein S., Kjelleberg, S., et al. (2006). A Conover, M. S., Mishra, M., and tic strategies. Nat. Biotechnol. 24, OprH and polymyxin B resistance. characterization of DNA release in Deora, R. (2011). Extracellular DNA 1551–1557. Mol. Microbiol. 34, 305–316. Pseudomonas aeruginosa cultures and is essential for maintaining Bordetella Hawes, M. C., Curlango-Rivera, G., Manzenreiter, R., Kienberger, F., Mar- biofilms. Mol. Microbiol. 59, 1114– biofilm integrity on abiotic surfaces Wen, F., White, G. J., Vanetten, H. cos, V., Schilcher, K., Krautgart- 1128. and in the upper respiratory tract D., and Xiong, Z. (2011). Extracel- ner, W. D., Obermayer, A., et al. Anaya-Lopez, J. L., Lopez-Meza, J. E., of mice. PLoS ONE 6:e16861. doi: lular DNA: the tip of root defenses? (2012). Ultrastructural characteriza- and Ochoa-Zarzosa, A. (2012). Bac- 10.1371/journal.pone.0016861 Plant Sci. 180, 741–745. tion of cystic fibrosis sputum using terial resistance to cationic antimi- Dell’Anno, A., and Danovaro, R. (2005). Johnson, L., Mulcahy, H., Kan- atomic force and scanning electron crobial peptides. Crit. Rev. Microbiol. Extracellular DNA plays a key role evets, U., Shi, Y., and Lewenza, microscopy. J. Cyst. Fibros. 11, doi: 10.3109/1040841X.2012.699025 in deep-sea ecosystem functioning. S. (2012). Surface-localized spermi- 84–92. [Epub ahead of print]. Science 309, 2179. dine protects the Pseudomonas aerug- Marcos, V., Zhou, Z., Yildirim, A. Barken, K. B., Pamp, S. J., Yang, L., Gjer- Fernandez, L., Jenssen, H., Bains, inosa outer membrane from antibi- O., Bohla, A., Hector, A., Vitkov, mansen, M., Bertrand, J. J., Klausen, M., Wiegand, I., Gooderham, W. otic treatment and oxidative stress. J. L., et al. (2010). CXCR2 mediates M., et al. (2008). Roles of type IV J., and Hancock, R. E. (2012). Bacteriol. 194, 813–826. NADPH oxidase-independent neu- pili, flagellum-mediated motility and The two-component system CprRS Jones, A. P., and Wallis, C. (2010). Dor- trophil extracellular trap formation extracellular DNA in the formation senses cationic peptides and triggers nase alfa for cystic fibrosis. Cochrane in cystic fibrosis airway inflamma- of mature multicellular structures adaptive resistance in Pseudomonas Database Syst. Rev. 3, CD001127. tion. Nat. Med. 16, 1018–1023. in Pseudomonas aeruginosa biofilms. aeruginosa independently of ParRS. Jurcisek, J. A., and Bakaletz, L. O. McDonald, B., Urrutia, R., Yipp, B. G., Environ. Microbiol. 10, 2331–2343. Antimicrob. Agents Chemother. 56, (2007). Biofilms formed by non- Jenne, C. N., and Kubes, P. (2012). Berne, C., Kysela, D. T., and Brun, Y. V. 6212–6222. typeable Haemophilus influenzae in Intravascular neutrophil extracellu- (2010). A bacterial extracellular DNA Finkel, S. E., and Kolter, R. (2001). DNA vivo contain both double-stranded lar traps capture bacteria from the inhibits settling of motile progeny as a nutrient: novel role for bacte- DNA and type IV pilin protein. J. bloodstream during sepsis. Cell Host cells within a biofilm. Mol. Microbiol. rial competence gene homologs. J. Bacteriol. 189, 3868–3875. Microbe 12, 324–333. 77, 815–829. Bacteriol. 183, 6288–6293. Konstan, M. W., and Ratjen, F. (2012). McPhee, J. B., Bains, M., Winsor, G., Brinkmann, V., Reichard, U., Goos- Godeke, J., Heun, M., Bubendorfer, Effect of dornase alfa on inflamma- Lewenza, S., Kwasnicka, A., Brazas, mann, C., Fauler, B., Uhlemann, Y., S., Paul, K., and Thormann, K. M. tion and lung function: potential M. D., et al. (2006). Contribution Weiss, D. S., et al. (2004). Neutrophil (2011). Roles of two Shewanella onei- role in the early treatment of cystic of the PhoP-PhoQ and PmrA-PmrB extracellular traps kill bacteria. Sci- densis MR-1 extracellular endonucle- fibrosis. J. Cyst. Fibros. 11, 78–83. two-component regulatory systems + ence 303, 1532–1535. ases. Appl. Environ. Microbiol. 77, Kraus, D., and Peschel, A. (2006). to Mg2 -induced gene regulation in Brinkmann, V., and Zychlinsky, A. 5342–5351. Molecular mechanisms of bacterial Pseudomonas aeruginosa. J. Bacteriol. (2012). Neutrophil extracellular Groisman, E. A. (2001). The pleiotropic resistance to antimicrobial peptides. 188, 3995–4006. traps: is immunity the second func- two-component regulatory system Curr. Top. Microbiol. Immunol. 306, McPhee, J. B., Lewenza, S., and tion of chromatin? J. Cell Biol. 198, PhoP-PhoQ. J. Bacteriol. 183, 1835– 231–250. Hancock, R. E. (2003). Cationic 773–783. 1842. Lethem, M. I., James, S. L., Marriott, antimicrobial peptides activate a Bucki, R., Byfield, F. J., and Janmey, P. Guimaraes-Costa, A. B., Nascimento, C., and Burke, J. F. (1990). The origin two-component regulatory system, A. (2007). Release of the antimicro- M. T., Froment, G. S., Soares, R. of DNA associated with mucus gly- PmrA-PmrB, that regulates resistance bial peptide LL-37 from DNA/F-actin P., Morgado, F. N., Conceicao-Silva, coproteins in cystic fibrosis sputum. to polymyxin B and cationic antimi- bundles in cystic fibrosis sputum. F., et al. (2009). Leishmania ama- Eur. Respir. J. 3, 19–23. crobial peptides in Pseudomonas Eur. Respir. J. 29, 624–632. zonensis promastigotes induce and Lewenza, S., Falsafi, R. K., Winsor, aeruginosa. Mol. Microbiol. 50, Cheng, H. Y., Chen, Y. F., and Peng, H. are killed by neutrophil extracellular G., Gooderham, W. J., McPhee, 205–217. L. (2010). Molecular characterization traps. Proc. Natl. Acad. Sci. U.S.A. J. B., Brinkman, F. S., et al. Miller, A. K., Brannon, M. K., of the PhoPQ-PmrD-PmrAB medi- 106, 6748–6753. (2005). Construction of a mini- Stevens, L., Johansen, H. K., Sel- ated pathway regulating polymyxin Haagensen, J. A., Klausen, M., Ernst, R. Tn5-luxCDABE mutant library in grade, S. E., Miller, S. I., et al. B resistance in Klebsiella pneumoniae K., Miller, S. I., Folkesson, A., Tolker- Pseudomonas aeruginosa PAO1: a (2011). PhoQ mutations promote CG43. J. Biomed. Sci. 17, 60. Nielsen, T., et al. (2007). Differentia- tool for identifying differentially reg- lipid A modification and polymyxin Chiang, W., and Tolker-Nielsen, T. tion and distribution of colistin- and ulated genes. Genome Res. 15, resistance of Pseudomonas aerugi- (2010). “Extracellular DNA as matrix sodium dodecyl sulfate-tolerant cells 583–589. nosa found in colistin-treated cystic component in microbial biofilms,”in in Pseudomonas aeruginosa biofilms. Macfarlane, E. L., Kwasnicka, A., Ochs, fibrosis patients. Antimicrob. Agents Extracellular Nucleic Acids, Nucleic J. Bacteriol. 189, 28–37. M. M., and Hancock, R. E. (1999). Chemother. 55, 5761–5769.

www.frontiersin.org February 2013 | Volume 4 | Article 21 |119

“fmicb-04-00021” — 2013/2/13 — 19:01 — page5—#5 Lewenza DNA-induced antibiotic resistance genes

Moskowitz, S. M., Brannon, M. K., gene homologs are essential for com- Empey, D., et al. (1993). Efficacy and comparison of gene expression in Dasgupta, N., Pier, M., Sgambati, petitive fitness and the use of DNA safety of short-term administration planktonic cultures and developing N., Miller, A. K., et al. (2012). as a nutrient. J. Bacteriol. 188, 3902– of aerosolised recombinant human and mature biofilms. J. Bacteriol. 187, PmrB mutations promote polymyxin 3910. DNase I in adults with stable stage 6571–6576. resistance of Pseudomonas aeruginosa Palmer, K. L., Mashburn, L. M., Singh, cystic fibrosis. Lancet 342, 199–202. Whitchurch, C. B., Tolker-Nielsen, T., isolated from colistin-treated cystic P. K., and Whiteley, M. (2005). Cys- Saitoh, T., Komano, J., Saitoh, Y., Mis- Ragas, P. C., and Mattick, J. S. (2002). fibrosis patients. Antimicrob. Agents tic fibrosis sputum supports growth awa, T., Takahama, M., Kozaki, T., Extracellular DNA required for bac- Chemother. 56, 1019–1030. and cues key aspects of Pseudomonas et al. (2012). Neutrophil extracellular terial biofilm formation. Science 295, Moskowitz, S. M., and Ernst, R. K. aeruginosa physiology. J. Bacteriol. traps mediate a host defense response 1487. (2010). The role of Pseudomonas 187, 5267–5277. to human immunodeficiency virus-1. Whiteley, M., Bangera, M. G., Bum- lipopolysaccharide in cystic fibrosis Pamp, S. J., Gjermansen, M., Johansen, Cell Host Microbe 12, 109–116. garner, R. E., Parsek, M. R., Teitzel, airway infection. Subcell. Biochem. H. K., and Tolker-Nielsen, T. (2008). Sanders, N. N., Franckx, H., De Boeck, G. M., Lory, S., et al. (2001). Gene 53, 241–253. Tolerance to the antimicrobial pep- K., Haustraete, J., De Smedt, S. C., expression in Pseudomonas aerugi- Moskowitz, S. M., Ernst, R. K., and tide colistin in Pseudomonas aerugi- and Demeester, J. (2006). Role of nosa biofilms. Nature 413, 860–864. Miller, S. I. (2004). PmrAB, a nosa biofilms is linked to metaboli- magnesium in the failure of rhD- Wu, J., and Xi, C. (2009). Evaluation two-component regulatory system of cally active cells, and depends on the Nase therapy in patients with cystic of different methods for extracting Pseudomonas aeruginosa that mod- pmr and mexAB-oprM genes. Mol. fibrosis. Thorax 61, 962–968. extracellular DNA from the biofilm ulates resistance to cationic antimi- Microbiol. 68, 223–240. Shak, S., Capon, D. J., Hellmiss, R., matrix. Appl. Environ. Microbiol. 75, crobial peptides and addition of Petrova, O. E., Schurr, J. R., Schurr, Marsters, S. A., and Baker, C. L. 5390–5395. aminoarabinose to lipid A. J. Bacte- M. J., and Sauer, K. (2011). The (1990). Recombinant human DNase I Yipp, B. G., Petri, B., Salina, D., Jenne, riol. 186, 575–579. novel Pseudomonas aeruginosa two- reduces the viscosity of cystic fibrosis C. N., Scott, B. N., Zbytnuik, L. Mulcahy, H., Charron-Mazenod, L., component regulator BfmR con- sputum. Proc. Natl. Acad. Sci. U.S.A. D., et al. (2012). Infection-induced and Lewenza, S. (2008). Extra- trols bacteriophage-mediated lysis 87, 9188–9192. NETosis is a dynamic process involv- cellular DNA chelates cations and and DNA release during biofilm Tetz, G. V., Artemenko, N. K., and ing neutrophil multitasking in vivo. induces antibiotic resistance in development through PhdA. Mol. Tetz, V. V. (2009). Effect of DNase Nat. Med. 18, 1386–1393. Pseudomonas aeruginosa biofilms. Microbiol. 81, 767–783. and antibiotics on biofilm character- PLoS Pathog. 4:e1000213. doi: Pietramellar, G., Ascher, J., Borgogni, F., istics. Antimicrob. Agents Chemother. 10.1371/journal.ppat.1000213 Ceccherini, M., Guerri, G., and Nan- 53, 1204–1209. Mulcahy, H., Charron-Mazenod, L., nipieri, P. (2009). Extracellular DNA Urban, C. F., Ermert, D., Schmid, Conflict of Interest Statement: The and Lewenza, S. (2010). Pseudomonas in soil and sediment: fate and eco- M., Abu-Abed, U., Goosmann, author declares that the research was aeruginosa produces an extracellu- logical relevance. Biol. Fertil. Soils 45, C., Nacken, W., et al. (2009). conducted in the absence of any com- lar deoxyribonuclease that is required 219–235. Neutrophil extracellular traps con- mercial or financial relationships that for utilization of DNA as a nutri- Pinchuk, G. E., Ammons, C., Cul- tain calprotectin, a cytosolic pro- could be construed as a potential con- ent source. Environ. Microbiol. 12, ley, D. E., Li, S. M., McLean, J. S., tein complex involved in host flict of interest. 1621–1629. Romine, M. F., et al. (2008). Uti- defense against Candida albicans. Mulcahy, H., Sibley, C. D., Surette, lization of DNA as a sole source of PLoS Pathog. 5:e1000639. doi: Received: 24 November 2012; paper M. G., and Lewenza, S. (2011). phosphorus, carbon, and energy by 10.1371/journal.ppat.1000639 pending published: 11 December 2012; Drosophila melanogaster as an animal Shewanella spp.: ecological and phys- Urban, C. F., Reichard, U., Brinkmann, accepted: 28 January 2013; published model for the study of Pseudomonas iological implications for dissimila- V., and Zychlinsky, A. (2006). Neu- online: 14 February 2013. aeruginosa biofilm infections in tory metal reduction. Appl. Environ. trophil extracellular traps capture Citation: Lewenza S (2013) Extracellu- vivo. PLoS Pathog. 7:e1002299. doi: Microbiol. 74, 1198–1208. and kill Candida albicans yeast and lar DNA-induced antimicrobial peptide 10.1371/journal.ppat.1002299 Purdy Drew, K. R., Sanders, L. hyphal forms. Cell. Microbiol. 8, resistance mechanisms in Pseudomonas Nicas, T. I., and Hancock, R. E. K., Culumber, Z. W., Zribi, 668–676. aeruginosa. Front. Microbio. 4:21. doi: (1980). Outer membrane protein H1 O., and Wong, G. C. (2009). van Gennip, M., Christensen, L. D., 10.3389/fmicb.2013.00021 of Pseudomonas aeruginosa: involve- Cationic amphiphiles increase activ- Alhede, M., Qvortrup, K., Jensen, P. This article was submitted to Fron- ment in adaptive and mutational ity of aminoglycoside antibiotic O., Hoiby, N., et al. (2012). Inter- tiers in Antimicrobials, Resistance and resistance to ethylenediaminetetraac- tobramycin in the presence of airway actions between polymorphonuclear Chemotherapy, a specialty of Frontiers in etate, polymyxin B, and gentamicin. polyelectrolytes. J. Am. Chem. Soc. leukocytes and Pseudomonas aerugi- Microbiology. J. Bacteriol. 143, 872–878. 131, 486–493. nosa biofilms on silicone implants in Copyright © 2013 Lewenza. This is O’Loughlin, J. L., Spinner, J. L., Min- Ramphal, R., Lhermitte, M., Filliat, M., vivo. Infect. Immun. 80, 2601–2607. an open-access article distributed under nich, S. A., and Kobayashi, S. and Roussel, P. (1988). The bind- Vlassov, V. V., Laktionov, P. P., and the terms of the Creative Commons D. (2010). Yersinia pest is two- ing of anti-pseudomonal antibiotics Rykova, E. Y. (2007). Extracellular Attribution License, which permits use, component gene regulatory systems to macromolecules from cystic fibro- nucleic acids. Bioessays 29, 654–667. distribution and reproduction in other promote survival in human neu- sis sputum. J. Antimicrob. Chemother. Waite, R. D., Papakonstantinopoulou, forums, provided the original authors and trophils. Infect. Immun. 78, 773–782. 22, 483–490. A., Littler, E., and Curtis, M. source are credited and subject to any Palchevskiy, V., and Finkel, S. E. Ranasinha, C., Assoufi, B., Shak, A. (2005). Transcriptome analysis copyright notices concerning any third- (2006). Escherichia coli competence S., Christiansen, D., Fuchs, H., of Pseudomonas aeruginosa growth: party graphics etc.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 21 | 120

“fmicb-04-00021” — 2013/2/13 — 19:01 — page6—#6 REVIEW ARTICLE published: 13 February 2013 doi: 10.3389/fmicb.2013.00020 Concentration-dependent activity of antibiotics in natural environments

Steve P.Bernier1 and Michael G. Surette1,2* 1 Farncombe Family Digestive Health Research Institute, Department of Medicine, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada 2 Department of Biochemistry and Biomedical Sciences, Faculty of Health Sciences, McMaster University, Hamilton, ON, Canada

Edited by: Bacterial responses to antibiotics are concentration-dependent. At high concentrations, Fiona Walsh, Agroscope Changins antibiotics exhibit antimicrobial activities on susceptible cells, while subinhibitory con- Wädenswil, Switzerland centrations induce diverse biological responses in bacteria. At non-lethal concentrations, Reviewed by: bacteria may sense antibiotics as extracellular chemicals to trigger different cellular Charles W. Knapp, University of Strathclyde, UK responses, which may include an altered antibiotic resistance/tolerance proﬁle. In natural Carlos F.Amábile-Cuevas, Fundación settings, microbes are typically in polymicrobial communities and antibiotic-mediated Lusara, Mexico interactions between species may play a signiﬁcant role in bacterial community structure *Correspondence: and function. However, these aspects have not yet fully been explored at the community Michael G. Surette, Farncombe Family level. Here we discuss the different types of interactions mediated by antibiotics and Digestive Health Research Institute, Departments of Medicine and non-antibiotic metabolites as a function of their concentrations and speculate on how Biochemistry and Biomedical Sciences, these may amplify the overall antibiotic resistance/tolerance and the spread of antibiotic Faculty of Health Sciences, McMaster resistance determinants in a context of polymicrobial community. University, 1280 Main Street, HSC 3N 8F, Hamilton, ON, Canada L8S 4K1. Keywords: antibiotic, resistance, tolerance, interaction, signal, stress, cue, community e-mail: [email protected]

INTRODUCTION major driver in the selection for antibiotic-resistant bacteria (Levy, Antibiotics are bioactive small molecules naturally produced by 2001; Marshall and Levy, 2011; Andersson and Hughes, 2012) secondary metabolism of microorganisms such as bacteria and and the selection occurs over a large spectrum of concentrations fungi (Davies, 2006; Aminov, 2010; Davies and Davies, 2010). (Andersson and Hughes, 2012). Lethal concentrations of antibi- Their discovery as antimicrobial drugs has revolutionized the otics rarely occur outside of therapeutic applications, but bacteria management and treatment of infectious diseases. Many easily constantly face subinhibitory antibiotics in the environment and treated infectious diseases today had high mortality rates in the the host (e.g., human and other animals) following therapies. In pre-antibiotic era. However, due to the increasing prevalence of fact, the release of antibiotics in the environment from medical resistance, the in vivo efﬁcacy of antibiotics is reduced or abolished, or non-medical (e.g., agricultural) use artiﬁcially creates concen- and the spread of antibiotic-resistant microorganisms is now tration gradients that are rarely encountered by environmental threatening the treatment of otherwise manageable infections. bacteria located in areas that are normally free of human-derived Antibiotic resistance in microbes is widespread and it has been antibiotic activities (Aminov, 2009; Martinez, 2009a). The rapid demonstrated that soil bacteria are rich in resistance determi- appearance of drug-resistant bacteria upon antibiotic exposure nants to both natural and synthetic antibiotics commonly used in implies that resistance and resistance mechanisms have co-evolved clinics (D’Costa et al., 2006). The collective genes that contribute with antibiotic-derived products. The latter point raises the ques- to antibiotic resistance are referred to as the antibiotic resistome tion as to whether antibiotic resistance was already a bacterial trait (D’Costa et al., 2007; Wright, 2007). Interestingly, antibiotic resis- before the modern use of antibiotics. To address this question, ele- tance genes extracted from the soil resistome were shown to be gant metagenomic and functional studies showed the existence of identical or highly similar to those found in clinically relevant resistance determinants in pristine areas of the world (D’Costa drug-resistant human pathogens (Forsberg et al., 2012) demon- et al., 2011; Bhullar et al., 2012) demonstrating that antibiotic strating that lateral gene transfer likely plays a role in the rise of resistance predates the clinical use of antibiotics. Therefore, antibi- multidrug-resistant pathogens. In addition to the environment, otic resistance is a common bacterial feature. The medical and the human microbiome is also a niche rich in antibiotic resistance non-medical use of antibiotics may accelerate the spread of resis- determinants where exchange of resistance genes can lead to the tance through positive selection in both the environment and generation of drug-resistant bacteria with potential pathogenic the host. traits (Sommer et al., 2009, 2010; Sommer and Dantas, 2011). The focus on the medical use of antibiotics has limited fun- Altogether, these recent studies showed that the spread of antibi- damental research regarding the other potential activities of these otic resistance from non-pathogenic environmental bacterial is compounds in their natural settings, including the environment an ongoing threat to the clinical use of antibiotics, even for new (e.g., soil) and hosts such as humans, animals, and plants. In synthetic compounds, and the emergence of new drug-resistant complex communities containing antibiotic-producing microor- pathogens is a constant threat. ganisms, bacteria are naturally exposed to lethal and non-lethal Despite a better understanding of the different mechanisms antibiotics making them trained at responding to these com- leading to resistance, exposure to antibiotics is still considered the pounds. Non-lethal levels of antibiotics can alter the expression of

www.frontiersin.org February 2013 | Volume 4 | Article 20 | 121

“fmicb-04-00020” — 2013/2/15 — 18:43 — page1—#1 Bernier and Surette Antibiotics in natural communities

genes involved in a variety of bacterial functions like metabolism, beyond the scope of this review; therefore we refer the reader regulation, virulence, DNA repair, and stress response (Goh to recent reviews that have thoroughly discussed the topic (Keller et al., 2002; Tsui et al., 2004; Davies et al., 2006; Yim et al., 2006, and Surette, 2006; Diggle et al., 2007; Stacy et al., 2012). A chemi- 2007, 2011; Blazquez et al., 2012). Subinhibitory antibiotics can cal mediating intra- or interspecies interactions can be defined as also modify cellular behaviors in bacteria with the formation of a signal, cue, or coercion (chemical manipulation). For a chem- biofilms (Hoffman et al., 2005; Frank et al., 2007; Haddadin et al., ical interaction to occur, emitting bacteria must first produce a 2010; Mirani and Jamil,2011; Subrt et al.,2011; Kaplan et al.,2012) molecule that can be perceived by other individuals, and second, and persister cells (Dorr et al., 2010). Altogether, these observa- the receiver must alter its behavior in response to the signal. tions strongly suggested that antibiotics induce responses other To determine whether an interaction is mediated by a signal, than those associated to their antimicrobial activities and it is a cue, or coercion the overall benefit of the reaction is used as now accepted that they might be used as “signaling” molecules primary criteria. As shown in Table 1, a signal is defined when with regulatory functions (Yim et al., 2007; Aminov, 2009; Allen both partners take advantage of the interaction (bidirectional), et al., 2010). while cues or coercions have unidirectional benefits for receivers Antibiotics are, like other bioactive small molecules, low molec- or emitters, respectively. In true signaling interactions, the pro- ular weight metabolites produced by secondary metabolism of duction and detection of the signal have co-evolved specifically microorganisms, i.e., are not considered essential for growth and for that purpose and from an evolutionary perspective these events viability. Microorganisms such as bacteria and fungi produce will only be maintained when both partners benefit from the infor- a wealth of small molecules that has been called the parvome mation conveyed by the signal for which they evolved (Maynard (Davies, 2009; Davies and Ryan, 2012). Secondary metabolites Smith and Harper, 2003; Keller and Surette, 2006). On the other are responsible for most of the interactions taking place in nat- hand, a cue provides information to a receiver for which a response ural microbial communities (Lyon and Muir, 2003; Keller and is triggered (Keller and Surette,2006; Diggle et al.,2007; Stacy et al., Surette, 2006; Duan et al., 2009; Rath and Dorrestein, 2012) and as 2012). Although not mediated by single molecules, environmen- extracellular metabolites antibiotics have the potential to exhibit tal conditions can also be considered as cues by bacteria and they similar functions. Bacteria in natural environments are mostly include pH, osmolarity, temperature, oxidative stress/oxygen, and part of complex polymicrobial communities in which all members nutrient limitation. The main distinction with a signal is that the share nutrient resources as well as chemicals including primary biological response did not evolve for that purpose, which benefits metabolic end products and secondary metabolites, which have the only the receiver (Keller and Surette, 2006; Diggle et al., 2007; Stacy potential to induce antibiotic resistance/tolerance mechanisms. et al., 2012). Conversely, a coercion scenario is a strategy used by Natural communities also include host-associated communities the emitter, via the release of a molecule, to chemically manipulate such as the human microbiome. The role(s) of antibiotics in the receiver for its own benefit (Keller and Surette, 2006; Diggle mediating non-lethal interactions between bacterial cells, may et al., 2007; Stacy et al., 2012). in fact, play a much bigger role than previously anticipated in Figure 1 illustrates the different ways by which a single chem- the global antibiotic resistance threat that we are currently fac- ical can be perceived by bacterial cells of different species within ing. Naturally occurring antibiotics, and other related secondary a polymicrobial community. Although interactions mediated by metabolites in bacterial communities, may dictate the spread of signals, cues, or coercions can all occur in these communities, the antibiotic resistance in a concentration-dependent manner. There- majority of these dynamic interactions fall in the category of cue, fore improving our current knowledge of antibiotic-mediated because they do not require stability over time, for which only interactions in bacteria may facilitate the development of new ther- receiver cells evolve. The modulation of Pseudomonas aeruginosa apeutic strategies for the treatment of drug-resistant pathogens. virulence factors by autoinducer-2 (AI-2) from the oropharyn- Here we review the current knowledge of antibiotic responsive geal microbiota (Duan et al., 2003) and alteration of the antibiotic activities as a function of their concentrations and speculate on tolerance profile by volatile ammonia (Bernier et al., 2011)are how these antibiotic-mediated interactions may overall influence examples of interspecies interactions mediated by cues. antibiotic resistance/tolerance and community composition in Interestingly, intraspecies diversity may in some cases challenge heterogeneous polymicrobial communities. the concept of true communication by the rise of cheaters through

CHEMICAL INTERACTIONS 1,2 The explosion of research in cell–cell interactions mediated by Table 1 | Simplified description of chemical-mediated interactions . bioactive small molecules in microbiology has led to the general Benefits the emitter Benefits the receiver assumption that most interspecies cell–cell interactions could be labeled as “communication” or “signaling.” However, it is impor- Signal ++ + tant to note that the demonstration of a biological response Cue −+ upon exposure to a chemical does not necessarily imply com- +− munication. The improper use of terms like signal, signaling, Coercion or communication in microbiology has created confusion since 1 most interspecies metabolite-mediated interactions labeled as“sig- The overall benefit is used as the main determinant for the classification of the different types of bacteria–bacteria interactions and are either beneficial (+)or naling/communication” are often in conflict with evolutionary costly (−). theories. A detailed analysis of the appropriate terminology is 2Adapted from Diggle et al. (2007) and Stacy et al. (2012).

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 20 | 122

“fmicb-04-00020” — 2013/2/15 — 18:43 — page2—#2 Bernier and Surette Antibiotics in natural communities

bacterium’s perspective on a large concentration spectrum (Figure 2). From high to low concentrations, antibiotics act as either toxins, stress inducers, or as cues/coercions, respectively. Among interactions mediated by subinhibitory antibiotics, receiver bacteria can interestingly induce mechanisms leading to antibiotic resistance or tolerance (Figure 2). How antibiotics become stress inducers or cues/coercions will further be discussed in the following sections, but before we will briefly describe the toxin-like behavior of antibiotics and the impact of antibiotic resistance on the biological response exhibited by bacteria upon antibiotic exposure. At concentrations superior to or near the minimal inhibitory concentration (MIC), antibiotics behave like toxins on susceptible bacterial cells. However, although the understanding of resistance mechanisms is well-characterized, molecular mechanism(s) induced by lethal concentrations of antibiotics is an FIGURE 1 | A single chemical mediates different bacteria–bacteria area of research where our fundamental understanding is still interactions in a context of a polymicrobial community. The schematic very limited (Kohanski et al., 2010). Recent studies suggested that represents how a single chemical released by one bacterial species can be antibiotic-induced cell death was associated with increased pro- perceived differently in a multispecies community. Microorganisms that benefit positively (blue) or negatively (brown) from the different interaction duction of radical oxygen species (ROS) such as hydroxyl radicals, are represented. Arrows indicate direction of evolved pathway, e.g., an superoxide, and hydrogen peroxide for bactericidal antibiotics organism evolves to “sense” a cue or in some case can use an antibiotic as belonging to the families of quinolones, β-lactams, and amino- a nutritional source (Dantas et al., 2008). In the case of signaling and glycosides (Dwyer et al., 2007, 2009; Kohanski et al., 2007, 2008, interspecies signaling, pathways from both producer and receiver cells have co-evolved to the benefit of both microorganisms. In the case of 2010). Although antibiotic ROS-mediated killing is highly possible coercion or toxic interactions such as those exhibited by antibiotics, only in aerobic conditions, the proposed model is however, oxygen- the producer benefits from the interaction. When the producer itself dependent (Hassett and Imlay, 2007) and other mechanisms must detects the signal, this would be a case of quorum sensing or cell–cell signaling. In the case of antibiotics, the producer usually co-expresses operate in oxygen-poor environments such as those found in resistance pathways and these can be co-expressed without a sensing biofilm populations (Stewart and Franklin, 2008). circuit but often, such as with many lantibiotics, they are detected by the Antibiotic resistance mechanisms and the antimicrobial nature cell and respond in a classical quorum sensing feedback loop. of antibiotics are often associated as a cause and effect phenomenon. The presence of resistance genes in bacteria with antibiotic biosynthesis genetic loci (Benveniste and Davies, 1973; genetic mutations. The inability of cheaters to either produce Walker and Skorvaga, 1973; Davies and Benveniste, 1974)isan and/or perceive a signal may abolish the bidirectional coopera- obvious self-protective strategy (Martinez, 2009a,b; Davies and tive interaction. In a situation where the response toward a signal Ryan, 2012; Wright and Poinar, 2012). It has therefore been widely leads to the production of a protease allowing the degradation of a accepted that antibiotic resistance determinants have specifically particular substrate for nutritional purposes, cheaters impaired in evolved to tolerate the lethal activity of antibiotics (Martinez, either the production or the reception of a signal will differentially 2009a,b), but experimental data to fully support this thesis are impact the cooperative interaction. In fact, cheaters get a direct still lacking (Davies and Ryan, 2012). Antibiotics are mainly competitive advantage by avoiding the metabolic cost of produc- present at non-lethal concentrations in the environment (Mar- ing a signal or responding to it and their selfish behavior allows tinez, 2009a,b), therefore antibiotic resistance determinants are them to benefit without being cooperative. However, some sys- likely involved in response mechanisms other than those required tems may have co-regulated pathways that help to control cheaters when receiver bacteria are exposed to lethal concentrations. Simi- (Dandekar et al., 2012). lar to the antibiotic concentration-dependent response (Figure 2), antibiotic resistance would also impact receiver bacteria in an BIOACTIVITY AND ANTIBIOTIC RESISTANCE ARE DRIVEN BY antibiotic dose-dependent manner. The presence of an antibi- ANTIBIOTIC CONCENTRATIONS otic resistance mechanism would shift the spectrum of responses Antibiotics are generally known for their antimicrobial proper- to an antibiotic to higher concentrations. The resistance would ties by which they either kill (bactericidal) or inhibit bacterial lower the effective concentration of antibiotics at the target site growth (bacteriostatic). Their concentrations are highly variable (Figure 3A). Exceptions to this would occur if there were sec- in natural communities and bacteria have evolved mechanisms ondary target sites for the antibiotics that mediate other responses. to respond accordingly. Although their antimicrobial properties At toxic concentrations, resistance would function in the con- have been demonstrated in both in vitro and in vivo settings, the ventional protective role and allow receiver bacteria to avoid the biological roles of antibiotics and antibiotic resistance in natural antibiotic toxicity by blocking death or growth arrest. At subin- environments are still undefined. hibitory concentrations, antibiotic resistance genes would shift the To first differentiate the biological responses induced by effective antibiotic concentration required for inducing the bio- antibiotics, here we represent their bioactivities from a receiver logical responses (stress inducers, coercion, and cues) of receiver

www.frontiersin.org February 2013 | Volume 4 | Article 20 |123

“fmicb-04-00020” — 2013/2/15 — 18:43 — page3—#3 Bernier and Surette Antibiotics in natural communities

FIGURE 2 | Biological responses toward antibiotics are concentration- concentrations, subinhibitory, antibiotics can act as stress inducers, coercions dependent. Bacterial interactions mediated by antibiotics induce biological or be sensed as cues. Biological responses induced in receiver bacteria when responses in receiver bacteria in a dose-dependent manner. The antimicrobial antibiotics are at subinhibitory concentrations can affect various cellular behavior (toxin) of antibiotics occurs when their concentrations is high leading responses or alter gene expression leading to different adaptive responses to bacterial death or growth arrest in susceptible receiver cells. At lower impacting antibiotic resistance/tolerance. bacteria (Goh et al., 2002; Yim et al., 2007; Mesak et al., 2008; concentrations for the receiver cells would not be considered as Mesak and Davies, 2009). The displacement of the response curve a signaling event, but rather coercion at the extreme. Coercion can to subinhibitory concentrations of antibiotics due to the presence occur at subinhibitory concentrations and one bacterial cell may of resistance determinants (Figure 3A) could therefore establish use chemicals to manipulate another. If the receiver cell possesses a chemical “arms race” between producer and receiver bacteria the corresponding antibiotic resistance determinant, receiver bac- independent of lethal effect. In the context of bacterial communi- teria will be less susceptible to chemical manipulation and the ties, the displacement of the response curve for a single strain will overall benefit or harm of the interaction will therefore be reduced. modify its own behavior, which may in return shift the community Outside of the well-mixed homogeneous environments of lab- composition or activity. Stress induction, coercion, and detection oratory cultures, antibiotic effects on bacterial populations will of cues are selectable phenotypes that can be tuned to meet the be heterogeneous. For any bacteria, the response to a specific conditions of natural environments. Selections that reduce stress antibiotic will be concentration-dependent (Figure 2) and the induction and coercion could be contributing factors in the evo- active concentration for a particular response will be shifted lution of the “cryptic resistome” (Wright, 2007); genes that are higher by resistance mechanisms (Figure 3A). In natural environ- normally expressed at low levels or have low specific activity that ments, a population will be expected to exhibit a heterogeneous do not confer resistance to the toxic effects of antibiotics at higher response because of gradients of antibiotic concentration and concentrations. These may be resistance genes tuned to environ- heterogeneity in the responsiveness of different cells in the pop- ments where there are lower concentrations of these bioactive ulation (Figure 3B). These differences in cellular responsiveness molecules. may be due to genetic heterogeneity and/or through differences in In natural environments, antibiotics are likely present in a physiological states of different cells in the population. gradient of concentrations while receiver organisms may also be heterogeneous. Even in a homogeneous clonal population, cells ANTIBIOTICS AS STRESS INDUCERS may be present in different physiological states and the pres- Antibiotics at subinhibitory concentrations can act as stress induc- ence of antibiotic tolerance and resistance mechanisms will shift ers or cues/coercion on receiver bacteria (Figure 2). When the response curves for individual cells within the population behaving as stress inducers, antibiotics often induce the SOS (Figure 3B). In the context of chemical interactions within natural stress response, which is also associated with various antibi- communities, antibiotics released by emitter bacteria at lethal otic resistance mechanisms. The following section will mainly

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 20 |124

“fmicb-04-00020” — 2013/2/15 — 18:43 — page4—#4 Bernier and Surette Antibiotics in natural communities

breaks and consequently induction of the SOS stress response (Urios et al., 1991; Dwyer et al., 2007; Yim et al., 2011). This induction usually occurs within a particular window of antibiotic concentrations (Piddock and Wise, 1987). In addition to fluoroquinolones or quinolones, bactericidal β-lactam and aminoglycoside antibiotics mediate bacterial killing by stimulating the production of ROS (Dwyer et al., 2007, 2009; Kohanski et al., 2007, 2008, 2010), which are themselves potent DNA damaging molecules (Farr and Kogoma, 1991). Consequently, all antibiotics mediating the production of ROS would therefore have the potential to induce the SOS stress response. Interestingly, fluoroquinolone and β-lactam antibiotics were shown in multiple studies to induce the SOS stress response in Escherichia coli while aminoglycosides failed (Ysern et al., 1990; Miller et al., 2004; Baharoglu and Mazel, 2011; Poole, 2012b). The intimate relationship between ROS-mediated killing and SOS-induction by bactericidal antibiotics requires more investigations to explain these discrepancies, but it does suggest that the stimulation of ROS production upon antibiotic exposure may be an indirect effect. Parallel mechanisms to the SOS response may exist for aminoglycosides to kill bacteria in a ROS-dependent manner or concentrations must be lethal to induce the SOS response and not FIGURE 3 | Biological role(s) of antibiotic resistance in natural subinhibitory. Other antibiotic classes represented by trimetho- communities. (A) The response of bacterial cells to antibiotic whether it is bactericidal/bacteriostatic or subinhibitory, the biological response (as a cue prim, ceftazidime, and sulfamethoxazole are also strong inducers or coercion) will be shifted by antibiotic resistance. (B) In natural of the SOS stress response in E. coli (Blazquez et al., 2012). environments, differences in physiological states can impact the expression The SOS stress response is widespread among bacteria (Erill of antibiotic resistance or tolerance mechanisms. Antibiotic concentrations et al., 2007), but differences in the antibiotic SOS-induction in these environments may also be distributed as gradients, meaning that the population will not necessarily respond uniformly. profiles have been observed between species. For example, subinhibitory concentrations of tetracycline, chloramphenicol, and aminoglycosides induce the SOS response of Vibrio cholerae, while highlight some of the main resistance mechanisms impacted by these antibiotics have no impact in E. coli (Baharoglu and Mazel, the induction of the SOS response in bacteria upon antibiotic 2011). Despite the strong similarities between SOS systems across exposure. Gram-negative bacteria and the genetic relatedness of E. coli and Bacteria possess multiple survival mechanisms to cope with V. cholerae, these disparities suggest that antibiotics may not nec- exogenous stresses and the SOS response is the main general stress essarily induce the SOS response directly from DNA damage, response induced by bacteria in these situations (Galhardo et al., but rather via upstream pathways or targets of the SOS response 2007; Blazquez et al., 2012; Poole, 2012b). The SOS stress response (Aertsen and Michiels, 2006) that could potentially differ between is typically induced upon DNA damage caused by extracellular bacterial species. These differences between bacterial species may stresses such as bacterial cell exposure to UV light or antibiotics reflect the evolutionary selective pressure on the different bac- (Erill et al., 2007; Janion, 2008; Butala et al., 2009). It is charac- teria with specific features reflecting conditions of their natural terized by a well-coordinated global response initiating inhibition environments. of cell division and induction of DNA repair, recombination, and The induction of the SOS response is often essential for mutation (Erill et al., 2007; Janion, 2008; Butala et al., 2009). In bacterial survival in stressful environments and associated with most bacterial species, the RecA and LexA proteins govern the genetic responses that indirectly alter antibiotic resistance by response, which is conserved across bacterial phyla with a few increasing mutation rate, horizontal gene transfer, and prophage exceptions where the LexA repressor protein homolog is absent, induction (Erill et al., 2007; Janion, 2008; Butala et al., 2009; such as in Streptococcus species (Erill et al., 2007). Upon DNA Dwyer et al., 2009). Several studies have shown that induction damage, RecA stimulates cleavage of the LexA repressor leading to of the SOS response by various antibiotics like fluoroquinolones, the global response involving more than 40 SOS-regulated genes β-lactams, trimethoprim, tetracycline, chloramphenicol, rifampi- (Courcelle et al., 2001; Erill et al., 2007; Janion, 2008; Butala et al., cin, and aminoglycosides increased the mutation frequency in dif- 2009). For a more comprehensive description of the SOS stress ferent bacterial species (Ysern et al., 1990; Gillespie et al., 2005; response, we refer the reader to consult some of these reviews that Henderson-Begg et al., 2006; Cortes et al., 2008; Mesak and Davies, have thoroughly discussed the topic (Erill et al., 2007; Janion, 2008; 2009; Baharoglu and Mazel, 2011; Yim et al., 2011). Induction Butala et al., 2009). of competency by fluoroquinolones leading to horizontal gene Fluoroquinolones and quinolones are broad spectrum antibi- transfer with the potential to acquire new antibiotic resistance otics, for which resistant mechanisms have quickly emerged (Ruiz, determinants has recently been reviewed (Charpentier et al.,2012). 2003). They inhibit DNA gyrase leading to double-stranded DNA Furthermore, antibiotics inducing the SOS response can promote

www.frontiersin.org February 2013 | Volume 4 | Article 20 |125

“fmicb-04-00020” — 2013/2/15 — 18:43 — page5—#5 Bernier and Surette Antibiotics in natural communities

bacterial genetic diversity via homologous recombination, phage that antibiotics have the ability to directly induce targeted and release, and transfer of integrons or conjugative elements (Matic specific resistance mechanisms. et al., 1995; Beaber et al., 2004; Cirz et al., 2007; Hocquet et al., Although subinhibitory concentrations of antibiotics like tetra- 2012) all involved in the movement of mobile DNA like antibiotic cycline and vancomycin can induce their own resistance mecha- resistance genes. nisms, other biological responses have the potential to indirectly The induction of the SOS response is critical and relevant to our impact antibiotic tolerance as well. It was recently demonstrated understanding of antibiotic-mediated interactions on the overall that subinhibitory concentrations of β-lactam antibiotics induce impact of antibiotic resistance in bacterial populations. Bacteria the autolysin-dependent release of extracellular DNA (eDNA) by in most environments including those affected during therapeutic Staphylococcus aureus. This affects biofilm formation and autoag- treatments can potentially encounter subinhibitory concentra- gregation (Kaplan et al., 2012), two growth protective mechanisms tions of antibiotics inducing the SOS stress response. Therefore, a that will be discussed in the following section. Interestingly, eDNA better understanding of SOS-associated behaviors linked to antibi- that is part of the extracellular matrix of P. aeruginosa biofilms otic resistance traits may result in alternative approaches to control induces tolerance against aminoglycosides by chelating cations their spread. (Mulcahy et al., 2008). Additionally, subinhibitory concentrations of antibiotics like vancomycin, tetracycline, azithromycin, ANTIBIOTICS AS CUES and ampicillin induce the expression of P. aeruginosa virulence- Various antibiotics at subinhibitory concentrations induce bio- associated genes leading to increased secretion of phenazines and logical responses on receiver bacteria that are non-stress-related rhamnolipids (Shen et al., 2008). Pyocyanin, one of the four P. and frequently affect pathways of primary metabolism (Goh et al., aeruginosa phenazines (Price-Whelan et al., 2006) was recently 2002; Tsui et al., 2004; Davies et al., 2006; Yim et al., 2006, 2007, showed to induce eDNA release in P. aeruginosa biofilms through 2011; Blazquez et al., 2012). Here we highlight examples demon- hydrogen peroxide (H2O2) mediating cell lysis (Das and Mane- strating bacterial interactions mediated by antibiotics sensed as field, 2012). These examples provide evidence that in multispecies cues, which can subsequently impact the antibiotic resistance pro- communities, subinhibitory antibiotics can lead to a series of file of receiver bacteria. These inducible responses can directly sequential responses leading to antibiotic tolerance (Figure 4). target mechanisms leading to specific antibiotic resistance or These are a few examples demonstrating that sensing of subin- indirectly impact tolerance toward various antibiotics. hibitory concentrations of antibiotics as cues can trigger direct or Many bacteria can sense specific antibiotics in their envi- indirect mechanisms, for which receiver bacteria will subsequently ronment and subsequently induce the corresponding resistance have the ability to resist or tolerate lethal concentrations of antibi- mechanisms. Tetracycline and vancomycin are the two best- otics. In microbial communities, the response of one organism studied examples. Tetracycline resistance has been attributed to may lead to induction of antibiotic resistance/tolerance in other classical resistance mechanisms including efflux strategies, target bacteria. site access (TetM and TetO), and chemical inactivation (TetX; Nel- son and Levy, 2011). The regulation of some tetracycline resistance determinants is under the control of tetracycline repressor protein (TetR), which has been thoroughly reviewed elsewhere (Hillen and Berens, 1994; Berens and Hillen, 2003; Ramos et al., 2005; Nelson and Levy, 2011). Briefly, TetR has DNA binding domains targeting operators of tetracycline resistance genes. Once bound to the operator region of a target gene, TetR acts as a transcriptional repressor for which the repression can be relieved by the interaction of tetracycline with TetR (Hillen and Berens, 1994; Berens and Hillen, 2003; Ramos et al., 2005). Therefore, when tetracycline is present, the repressive function of TetR is abolished and transcription of the tetracycline resistance gene can occur normally. In the case of vancomycin, modification of the target (peptidoglycan – D-Ala-D-Ala C-terminus) is the primary mechanism of resistance (Courvalin, 2006). Six types of resistance to vancomycin (VanA, B, C, D, E, and G) were reported in Enterococcus FIGURE 4 | Antibiotic induction of extracellular DNA (eDNA) release in species (Courvalin, 2006). Interestingly, four of these operons a multispecies community and antibiotic tolerance. The schematic involved in vancomycin resistance (VanA, B, E, and G) are directly represents how two bacterial species, P.aeruginosa (green) and S. aureus inducible by vancomycin, while VanG and C types are constitu- (purple), respond to subinhibitory antibiotics to release eDNA. Subinhibitory antibiotics induced the production of pyocyanin (Shen et al., 2008), which is tive (Courvalin, 2006). Vancomycin is sensed by a two-component associated with increased H2O2 levels responsible for cell lysis and DNA regulatory system that positively activates expression of resistance release (Das and Manefield, 2012). In S. arureus, exposure to subinhibitory genes in response to vancomycin. Although the basic regulation β-lactam antibiotics induces release of eDNA via an autolysis-dependent mechanism (Kaplan et al., 2012). The release of eDNA by both bacteria can differs between tetracycline and vancomycin resistance genes, the induce antibiotic tolerance in communities (Mulcahy et al., 2008). inducible nature of their specific resistance pathways demonstrate

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 20 |126

“fmicb-04-00020” — 2013/2/15 — 18:43 — page6—#6 Bernier and Surette Antibiotics in natural communities

ANTIBIOTICS AS INDUCERS OF GROWTH PROTECTIVE demonstrated to display some level of protein translation (Gefen MECHANISMS et al., 2008). A significant portion of the antibiotic resistome is made of mobile Active mechanisms leading to the induction of persisters in resistance genes that are horizontally transferable between bacte- bacterial populations differ in terms of target. After the iden- rial cells (D’Costa et al., 2007; Wright, 2007; Allen et al., 2010). tification of hipA, as the first persistence gene and coding for Mobile resistance determinants as well as efflux pumps account the toxin of the hipAB toxin–antitoxin module (TA; Moyed for the majority of antibiotic resistance mechanisms, however, and Bertrand, 1983), many studies have shown that differ- bacteria have evolved non-inherited and transient mechanisms ent TA modules were involved in bacterial persistence (Lewis, to resist otherwise lethal antibiotic concentrations (Levin and 2010, 2012; Gerdes and Maisonneuve, 2012; Kint et al., 2012). Rozen, 2006). Environmental conditions can trigger various stress The induction of toxin genes in persister cells (Keren et al., responses making bacterial cells transiently refractory to antibi- 2004; Shah et al., 2006) or the overexpression of toxins lead- otics. Bacterial stress responses as determinants of antibiotic ing to increased persistence (Keren et al., 2004; Shah et al., resistance have become an emerging area of research. For a more 2006; Harrison et al., 2009; Maisonneuve et al., 2011)supported comprehensive description of these we refer the reader to recent the role of TA modules in bacterial persistence. Interestingly, reviews that have thoroughly addressed the topic (Poole, 2012a,b). the fluoroquinolone ciprofloxacin was shown to induce bacte- Briefly, bacterial exposures to environmental-related stresses like ria persistence via the TA module TisAB upon activation of nutrient starvation/limitation (nutrient stress), ROS and reactive the SOS response (Dorr et al., 2010). Consistently, lethal con- nitrogen species (oxidative/nitrosative stress), membrane damage centrations of ampicillin or ofloxacin induce the SOS stress (envelope stress), temperature (heat/cold stress), and ribosome response in persister cells (Kaldalu et al., 2004) while the SOS disruption (ribosomal stress) have the potential to initiate bacte- response confers persistence to fluoroquinolones (Dorr et al., rial responses leading to modified or enhanced tolerance toward 2009). Unrelated to TA modules, the extracellular chemical the lethal action of antibiotics (Poole, 2012a,b). indole was recently shown as mechanism inducing persisters in Non-inherited and transient mechanisms are mainly attributed E. coli populations (Vega et al., 2012). Interestingly, the latter to two distinct processes: persistence and drug indifference (Levin represents a non-antibiotic mediated interaction leading to antibi- and Rozen, 2006). While persistence occurs in subpopulations of otic tolerance; a topic that will be discussed in the following slow or non-growing bacteria, drug indifference can be exhibited section. by the entire population (Levin and Rozen,2006). Persister cells are Beside unicellular growth, bacteria can also adopt various generally considered to be responsible for bacterial survival follow- types of multicellular growth that exhibit phenotypes different ing antibiotic treatments although heterogeneity within bacterial than their planktonic counterparts including antibiotic tolerance. populations and reduced accessibility of the drug to some target Multicellular behaviors in bacteria include growth as biofilms, cells also contribute. A comprehensive description of persister cells aggregates, and swarming. Biofilms are sessile bacterial cells is beyond the scope of this review; therefore we refer the reader encased within an extracellular matrix that are usually attached to recent reviews that have thoroughly discussed the topic (Lewis, to a surface (Stewart and Franklin, 2008; Monds and O’Toole, 2007,2010,2012; Gerdes and Maisonneuve,2012; Kint et al.,2012). 2009). Bacterial aggregates are described as unattached biofilm- Persisters are phenotypic variants within an isogenic bacterial pop- like structures with the ability to move (Alhede et al., 2011; Haaber ulation that can tolerate high concentrations of antibiotics. In et al., 2012; Thornton et al., 2012) while swarming represents a contrast to drug-resistant cells, persisters can switch back to the type of motility exhibited by bacteria over semi-solid surfaces wild-type antibiotic sensitive phenotype when reactivated (Lewis, (Kearns, 2010). These multicellular behaviors were all associ- 2007,2010,2012; Gerdes and Maisonneuve,2012; Kint et al.,2012). ated with elevated tolerance to lethal concentrations of antibiotics This persister cell behavior has been directly observed (Balaban when compared to their planktonic counterparts (Stewart and et al., 2004). Persisters are present both in planktonic bacteria and Costerton, 2001; Kim et al., 2003; Lewis, 2007; Overhage et al., antibiotic-tolerant biofilms (Spoering and Lewis, 2001). Research 2008; Lai et al., 2009; Alhede et al., 2011; Haaber et al., 2012; emphasis in the field has largely focused on mechanisms involved Thornton et al., 2012). These studies demonstrate that multi- in persister formation and mechanisms leading to their forma- cellular assemblies in bacteria confer an advantage when facing tion have been proposed such as stochastic processes (passive) to antibiotics compared to planktonic cells. However, the growth active inducible regulation (Kint et al., 2012). Stochastic switching phase of planktonic cells (i.e., logarithmic or stationary) can has been proposed as an effective strategy for survival in unpre- have a significant impact on their antibiotic tolerance profiles. dictable environments (Kussell and Leibler, 2005; Kussell et al., For example, planktonic P. aeruginosa and E. coli cells were 2005). Dormancy is a passive mechanism involved in persister previously shown to exhibit greater levels of tolerance against formation resulting from stochastic endogenous stress leading to bactericidal antibiotics (Evans et al., 1991; Spoering and Lewis, growth arrest and the shutdown of bactericidal antibiotic tar- 2001; Bernier et al., 2013). In Salmonella typhimurium shifts gets making persisters multidrug-tolerant cells (Lewis, 2007, 2010, in primary metabolic pathways have been associated with the 2012). Experimental evidence to support the dormancy theory induced antibiotic tolerance in swarm cells (Kim and Surette, came from transcriptome analysis showing that genes involved in 2003, 2004; Turnbull and Surette, 2008, 2010) and aggregates primary metabolism and energy production were down-regulated (White et al., 2010). in persister cells (Keren et al., 2004; Shah et al., 2006). However, Antibiotic-mediated interactions impact multicellular behav- the dormancy model was challenge when persisters of E. coli were iors and indirectly the antibiotic tolerance profile of these

www.frontiersin.org February 2013 | Volume 4 | Article 20 |127

“fmicb-04-00020” — 2013/2/15 — 18:43 — page7—#7 Bernier and Surette Antibiotics in natural communities

populations. Several studies have showed that subinhibitory Briefly, under antibiotic stress, a few drug-resistant mutants arise antibiotics induce biofilm formation (Hoffman et al., 2005; Frank and then release the metabolite indole that is sensed by the entire et al., 2007; Haddadin et al., 2010; Mirani and Jamil, 2011; Subrt population allowing other less resistant isolates to survive (Lee et al., 2011; Kaplan et al., 2012) and autoaggregation (Kaplan et al., et al., 2010). In this particular case, the overall population MIC 2012). Bacterial response to extracellular stresses (Poole, 2012a,b) is totally biased by a few resistant clones since the majority of may also be an important trigger of multicellular behaviors in isolates are sensitive (Lee et al., 2010). The altruistic behavior of bacteria (Kaplan, 2011), which are better adapted as a group drug-resistant isolates comes with a fitness cost, associated with due to their physiology to tolerate lethal antibiotics (Stewart and the production of indole, that benefits the entire population (Lee Franklin, 2008). In support of this hypothesis, a recent study et al., 2010). Increased antibiotic tolerance mediated by indole is showed that non-starving planktonic cells were generally more proposed to result from induction of efflux pumps and oxida- tolerant to bactericidal antibiotics than biofilms, but when these tive stress protective mechanisms (Lee et al., 2010). In a different same bacterial cells were starved, therefore stressed, biofilm bac- study, indole is proposed to directly induce persistence through teria were significantly more resilient to antibiotics than their the generation of persister cells (Vega et al., 2012). The generation planktonic counterparts (Bernier et al., 2013). As one of the first of persisters by indole exposure is dependent on the phage-shock responses to stress, the SOS response is significantly more induced (Psp) and OxyR pathways (Vega et al., 2012). In other studies, in biofilm cells compared to their planktonic counterparts (Beloin indole was shown to promote the establishment of E. coli in dual- et al., 2004; Bernier et al., 2013). The higher intrinsic level of SOS species cultures with P. aeruginosa by inhibiting production of in biofilms may explain their increased mutation frequency com- pyocyanin and other P. aeruginosa virulence factors regulated by pared to planktonic cells (Conibear et al., 2009). Altogether, the quorum sensing (Chu et al., 2012). This example represents an increased SOS-dependent mutation rate observed in biofilms may example of coercion in which E. coli-derived indole manipulates well explain the high level of genetic variants arising in biofilm P. aeruginosa as a strategy to colonize and share a polymicro- populations in a RecA-dependent manner (Boles et al., 2004; van bial community. Conversely, over production of indole through der Veen and Abee, 2011) with the potential to impact antibiotic the induction of ROS production by subinhibitory concentra- resistance (Boles and Singh, 2008). tions of antibiotics was shown to impair E. coli biofilm formation The unique physiology of multicellular behaviors such as (Kuczynska-Wisnik et al., 2010). biofilms and swarming bacteria may render these cells better Until recently, the production of extracellular H2Sbybacteria adapted to respond and tolerate extracellular stresses such as oth- has mainly been considered as a toxic by-product of metabolism. erwise lethal antibiotic concentrations, these states may be induced However, bacterial-derived H2S is protective against the lethal directly by subinhibitory antibiotics. action of antibiotics in a ROS-dependent manner (Shatalin et al., 2011). It was also demonstrated that endogenous nitric oxide NON-ANTIBIOTIC SMALL MOLECULES AS MODULATORS OF (NO) of Gram-positive bacteria was protective against oxida- ANTIBIOTIC TOLERANCE tive stress-mediated killing by macrophages (Shatalin et al., 2008) We have highlighted previous studies demonstrating that bacteria and antibiotics (Gusarov et al., 2009). Interestingly, NO and H2S can sense antibiotics as cues to mediate bacteria–bacteria interact synergistically since the absence of one can be compensated actions with the potential to induce resistance/tolerance when for by the increased production of the other one upon antibi- lethal concentrations are subsequently reached. These can induce otic exposure (Shatalin et al., 2011). These studies demonstrate antibiotic specific mechanisms (e.g., tetracycline, vancomycin) the biological relevance of bacterial gases in mediating bacterial or more general mechanisms like biofilms induction. However, interactions and their indirect impact on antibiotic resistance. the induction of antibiotic resistance/tolerance via cues is not Ammonia, a general by-product of amino acid catabolism, was limited to antibiotics. Within natural communities, bacteria are recently shown to modulate the antibiotic tolerance of neigh- continuously exposed to a variety of small molecules other than boring bacterial cells (Bernier et al., 2011). Briefly, both Gram- antibiotics. Among these bioactive metabolites, some have been negative and Gram-positive bacterial species were able to tolerate shown to induce biological responses in bacteria leading to a otherwise lethal concentrations of ampicillin and tetracycline change in the overall antibiotic tolerance profile. upon exposure to biogenic volatile ammonia (Bernier et al., 2011). Bacterial-derived extracellular metabolites such as indole, Conversely, sensitivity toward aminoglycoside antibiotics was hydrogen sulfide (H2S),and volatile ammonia were recently shown increased in bacterial cells exposed to volatile ammonia (Bernier to impact the antibiotic tolerance profile of receiver bacteria (Lee et al., 2011). Ammonia-mediated interactions between bacterial et al., 2010; Bernier et al., 2011; Shatalin et al., 2011; Vega et al., cells were shown to induce the intracellular levels of polyamines 2012). Interestingly, all of these bioactive molecules have the abil- (Bernier et al., 2011). Consistently, addition of polyamines (sper- ity to be soluble or volatile, however, only ammonia was studied midine and putrescine) could recapitulate the ammonia-mediated under its gaseous phase (Bernier et al., 2011). phenotype demonstrating that the modified antibiotic tolerance Indole, a tryptophan-derived aromatic heterocyclic organic profile of receiver bacteria was fully dependent on the polyamine compound, was recently reported to induce antibiotic resistance modulon upon ammonia exposure (Bernier et al., 2011). Interest- in E. coli (Lee et al., 2010; Vega et al., 2012). A community-based ingly, it was demonstrated in Bacillus spp. that biofilm formation antibiotic resistance mechanism was demonstrated to occur via the could also be induced upon exposure to biogenic ammonia and release of the metabolite in continuous cultures of E. coli exposed polyamines were critical for normal biofilm development (Burrell to increasing levels of the norfloxacin quinolone (Lee et al., 2010). et al., 2010; Nijland and Burgess, 2010).

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 20 |128

“fmicb-04-00020” — 2013/2/15 — 18:43 — page8—#8 Bernier and Surette Antibiotics in natural communities

The close relationship between ammonia sensing, polyamine the lungs of cystic fibrosis (CF) patients (Sibley et al., 2008b, 2009, induction, and biofilm formation demonstrate that bacterial 2011) and dynamic interactions between these organisms have interactions mediated by non-antibiotic molecules can modu- previously been demonstrated to alter P. aeruginosa virulence late antibiotic tolerance not only locally, but also at a distance (Duan et al., 2003; Sibley et al., 2008a). In the scenario described when ammonia is under its gaseous phase. Altogether, non- below, we suggest that a single antibiotic therapy may affect antibiotic-mediated interactions clearly demonstrate the complex- the overall community structure and subsequently the antibiotic ity of dealing with antibiotic resistance/tolerance when bacteria resistance of the community by initiating a cascade of inter- are part of complex communities, making antibiotic treatments species interactions mediated by antibiotics and non-antibiotic unpredictable. metabolites (Figure 5). Following an antibiotic treatment, many of the bacterial cells METABOLITE-MEDIATED INTERACTIONS IN BACTERIAL in the community will lyse releasing their intracellular content COMMUNITIES AS MECHANISMS OF ANTIBIOTIC including eDNA and NO that could potentially induce antibiotic RESISTANCE EVOLUTION tolerance (Mulcahy et al., 2008; Gusarov et al., 2009). However, Many infections are polymicrobial and as presented above, antibi- because of the population heterogeneity in terms of susceptibility otics and non-antibiotic metabolites can mediate interactions and resistance/tolerance (persisters, stationary phase cells, aggre- between organisms that may impact their efficacy as antimicro- gates, and biofilms) a large number of cells will die while some bials. The following describes a hypothetical scenario demonstrat- will survive. Over the course of the treatment, antibiotics will ing how metabolite-mediated interactions may have the potential be present at different concentrations at different times and sites to alter the antibiotic resistance profile of an entire population and and therefore mediating different types of interactions with viable possibly leading to the spread of antibiotic resistance. bacterial cells. At subinhibitory concentrations, antibiotics will For this exercise, we chose a simple polymicrobial commu- induce different biological responses, for which antibiotic resis- nity including the pathogens P. aeruginosa and S. aureus as well tance/tolerance will subsequently be affected. In this particular as Streptococcus species belonging to the normal oropharyngeal polymicrobial community, β-lactams will induce the release of microbiota. These bacterial species can simultaneously co-infect eDNA from S. aureus in an autolysin-dependent manner and

FIGURE 5 | Metabolite-mediated interactions modulating antibiotic subsequently lyse upon antibiotic exposure, unless resistance or tolerance in polymicrobial communities. Following an cells harbor the corresponding antibiotic resistance determinant (capped antibiotic treatment, natural communities will respond, adapt, and evolve cells; red, blue, or orange corresponding to the respective antibiotic) through the establishment of dynamic interactions mediated by antibiotics or are part of multicellular structures (bioﬁlms, aggregates). Subinhibitory and non-antibiotic metabolites. A simple polymicrobial community comprising antibiotics and non-antibiotic metabolites within the community can P.aeruginosa (green), S. aureus (purple), and Streptococcus spp. (brown) is subsequently induce different antibiotic tolerance mechanisms leading used to illustrate this hypothetical scenario. A proportion of the established to an altered community in terms of composition and antibiotic community, made of multispecies bioﬁlms and planktonic cells, will die and resistance/tolerance.

www.frontiersin.org February 2013 | Volume 4 | Article 20 |129

“fmicb-04-00020” — 2013/2/15 — 18:43 — page9—#9 Bernier and Surette Antibiotics in natural communities

triggering biofilm formation as a growth protective mechanism synthetic antibiotics. Fluoroquinolones represent a good example (Kaplan et al., 2012). At the same time, subinhibitory concentra- of synthetic drugs against which bacteria have quickly evolved tions of other antibiotics will induce the production of pyocyanin resistance (Ruiz, 2003; D’Costa et al., 2006, 2007). More judicious in P. aeruginosa (Shen et al., 2008) resulting in the generation use of antibiotics clinically and restricting non-medical applica- of H2O2, cell lysis, and subsequent release of more eDNA (Das tions may slow down the spread of drug-resistant bacteria and the and Manefield, 2012). Streptococci bacteria are known to gen- emergence of new antibiotic-resistant pathogens, but the breadth erate H2O2 causing cell lysis and the release of eDNA (Shen of the antibiotic resistome and the capacity of microbes to rapidly et al., 2008). The accumulation of eDNA, through antibiotic and evolve will make this an ongoing struggle. The chemical warfare non-antibiotic interactions, will chelate cations and primarily the that has been going on in microbial communities for hundreds reduced cation concentrations that are directly sensed by the cells of millions of years is something of a double-edged sword. Most leading to antibiotic tolerance (Mulcahy et al., 2008). In addition, antibiotics in use today have a microbial origin. Microbial sec- the community would favor biofilms and aggregates as modes of ondary metabolites have also been a valuable source of drugs not growth (Kaplan et al., 2012), which are generally more tolerant to restricted to just antibiotics. At the same time these communi- antibiotics. ties have evolved complex resistance mechanisms that can rapidly The concentration of antibiotics will gradually decrease over spread from natural environments to the clinic. time allowing the remaining viable bacterial cells to grow back. Since most antibiotics are natural molecules involved in chem- The CF lung is rich in amino acids (Palmer et al., 2007) and ical interactions between bacteria in communities, it has become actively growing bacteria will release ammonia as a by-product important to expand our understanding of the effects of these of amino acid catabolism. Ammonia sensing by bacteria within interactions on bacterial behaviors including antibiotic resistance. the community will induce the synthesis of polyamines leading to Because the presence of subinhibitory antibiotics can result in increased tolerance against ampicillin and tetracycline (Bernier a phenotype, the responses are subject to evolution and natural et al., 2011) and oxidative stress (Bernier et al., 2011; Johnson selection, the same as toxic interactions. In this review, we have et al., 2012). Further, the CF lung itself is rich in extracellular discussed and presented various scenarios on how antibiotics at polyamines (Grasemann et al., 2012), which could directly affect subinhibitory concentrations have the potential to induce different the antibiotic tolerance profile of the community (El-Halfawy biological responses leading to a general modification in the antibi- and Valvano, 2012) independently of ammonia sensing. Fur- otic resistance profiles of both environmental and host-associated ther, the consequences of these responses may impact syntrophic bacteria. These non-lethal interactions can act as stress inducers or and other metabolite-mediated interactions that can further alter be sensed as cues by receiver bacteria. Activation of the SOS stress community composition. response by antibiotics appears to reduce the efficacy of antibiotic For bacterial species that are naturally competent, the abun- treatments and facilitate the evolution of resistance. Therefore, dance of eDNA may also represent an excellent source of antibi- new therapeutic strategies targeting the SOS response may in otic resistance genes. Subinhibitory antibiotics induce the SOS return increase antibiotic efficacy. Blocking the LexA cleavage, response and competence systems (Charpentier et al., 2012)in therefore the SOS response, reduces the ability of E. coli to develop Streptococci leading to horizontal gene transfer and the acqui- resistance toward ciprofloxacin and rifampicin both in vivo and sition of resistance genes. Thereafter, the new-acquired resistance in vitro, through mutations (Cirz et al., 2005). This suggests that gene will be transferred vertically through bacterial division result- suppressing the SOS response would inhibit mutation rate, which ing in a new drug-resistant Streptococci strain. Further, mutation is an important downstream SOS-associated phenotype involved rates are also increased in all bacteria upon antibiotic exposure in bacterial evolution and antibiotic resistance. leading to the generation of potential new drug-resistant mutants. Manipulating cellular physiology has the potential to enhance Resistance mechanisms that chemically inactivate antibiotics also the efficacy of antibiotics even in resistant strains. A recent study have the potential to reduce the concentration to subinhibitory explored this possibility and reported that an engineered bacte- levels for susceptible cells in the community. riophage targeting the SOS response network enhanced the killing Through various inducible mechanisms mediated by antibi- efficacy of bactericidal antibiotics and survival in mice (Lu and otics and non-antibiotic metabolites, the overall composition of Collins, 2009). The enhanced antibiotic killing by SOS-targeting the community may change over time. This complex network of phages was also effective against persister and biofilm bacteria (Lu interactions is not reflected in standard antibacterial susceptibil- and Collins, 2009). ity and these processes likely contribute to the frequent failure of The nature of the effect of volatile ammonia on antibiotic antibiotics to reduce the population of susceptible organisms in resistance is dependent on the class of antibiotic. While bac- patients. This will be more likely to occur in polymicrobial infec- terial exposure to volatile ammonia induces tolerance against tions or when pathogens are part of a normal host community β-lactam and tetracycline antibiotics it increases the efficacy of such as in upper respiratory or gastrointestinal infections. aminoglycosides (Bernier et al., 2011). Interestingly, reactivation of the metabolism of E. coli persisters by the addition of vari- CONCLUSIONS AND PERSPECTIVES ous metabolites (glucose, mannitol, fructose, pyruvate) restored The increase of antibiotic-resistant pathogens represents a very their sensitivity to aminoglycosides to a level comparable to non- significant threat and challenge in the fight against infectious persister cells (Allison et al., 2011). These two studies demonstrate diseases. The complexity of the antibiotic resistome demon- the proof of concept that bacterial interactions occurring in strates that antibiotic resistance will always be a menace even for natural communities via non-antibiotic molecules have the ability

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 20 | 130

“fmicb-04-00020” — 2013/2/15 — 18:43 — page 10 — #10 Bernier and Surette Antibiotics in natural communities

to increase antibiotic efficacy. Non-antibiotic metabolites like should include the role of antibiotics in these communities ammonia or glucose could be considered as antibiotic potentia- and mechanisms of resistance. This will provide new oppor- tors in synergistic drug combination therapies. The combinations tunities and strategies to accelerate the discovery of bioactive of antibiotics and non-antibiotic drugs were showed to enhance molecules. Finally, we have to continue our efforts toward our antimicrobial efficacy against multidrug-resistant bacteria in both global understanding of these non-classical views of antibiotic- in vivo and in vitro suggesting that synergistic drug combinations mediated resistance mechanisms as complementary and potential have therapeutic potentials (Ejim et al., 2011). strategies to new drug development programs in order to control Furthering our understanding of bacterial interactions medi- and adequately manage the spread of antibiotic resistance in the ated by antibiotics and non-antibiotic molecules will be valuable in future. the development of new strategies to combat antibiotic resistance. Although significant findings have been made in this emerging ACKNOWLEDGMENTS area of research, we need to further expand our views of these Wewould like to acknowledge Carolyn Southward for critical read- dynamic interactions in microbial communities. The conven- ing of the manuscript and helpful discussions. S. P. B is a recipient tional approach of determining in vitro susceptibilities to isolated of a postdoctoral fellowship from Cystic Fibrosis Canada. Michael organisms has many limitations clinically and in polymicrobial G. Surette is supported by grants from Cystic Fibrosis Canada and infections, many community interactions can reduce antibiotic the Canadian Institutes of Health Research. Michael G. Surette efficacy in addition to traditional resistance mechanisms. Under- is supported by a Canada Research Chair in Interdisciplinary standing chemical interactions within microbial communities Microbiome Research.

REFERENCES switch. Science 305, 1622– Blazquez, J., Couce, A., Rodriguez- the evolution of antibiotic resistance. Aertsen, A., and Michiels, C. W. (2006). 1625. Beltran, J., and Rodriguez-Rojas, A. PLoS Biol. 3:e176. doi: 10.1371/jour- Upstream of the SOS response: figure Beaber, J. W., Hochhut, B., and Waldor, (2012). Antimicrobials as promot- nal.pbio.0030176 out the trigger. Trends Microbiol. 14, M. K. (2004). SOS response promotes ers of genetic variation. Curr. Opin. Cirz, R. T., Jones, M. B., Gingles, 421–423. horizontal dissemination of antibi- Microbiol. 15, 561–569. N. A., Minogue, T. D., Jarrahi, B., Alhede, M., Kragh, K. N., Qvortrup, otic resistance genes. Nature 427, Boles, B. R., and Singh, P. K. (2008). Peterson, S. N., et al. (2007). Com- K., Allesen-Holm, M., Van Gen- 72–74. Endogenous oxidative stress pro- plete and SOS-mediated response of nip, M., Christensen, L. D., Beloin, C., Valle, J., Latour-Lambert, duces diversity and adaptability in Staphylococcus aureus to the antibi- et al. (2011). Phenotypes of non- P., Faure, P., Kzreminski, M., biofilm communities. Proc. Natl. otic ciprofloxacin. J. Bacteriol. 189, attached Pseudomonas aeruginosa Balestrino, D., et al. (2004). Global Acad. Sci. U.S.A. 105, 12503–12508. 531–539. aggregates resemble surface attached impact of mature biofilm lifestyle on Boles, B. R., Thoendel, M., and Singh, Conibear, T. C., Collins, S. L., and biofilm. PLoS ONE 6:e27943. doi: Escherichia coli K-12 gene expression. P. K. (2004). Self-generated diver- Webb, J. S. (2009). Role of mutation 10.1371/journal.pone.0027943 Mol. Microbiol. 51, 659–674. sity produces “insurance effects” in in Pseudomonas aeruginosa biofilm Allen, H. K., Donato, J., Wang, H. Benveniste, R., and Davies, biofilm communities. Proc. Natl. development. PLoS ONE 4:e6289. H., Cloud-Hansen, K. A., Davies, J., J. (1973). Aminoglycoside Acad. Sci. U.S.A. 101, 16630–16635. doi: 10.1371/journal.pone.0006289 and Handelsman, J. (2010). Call of antibiotic-inactivating enzymes Burrell, M., Hanfrey, C. C., Murray, E. Cortes, P. R., Pinas, G. E., Albarracin the wild: antibiotic resistance genes in actinomycetes similar to those J., Stanley-Wall, N. R., and Michael, Orio, A. G., and Echenique, J. R. in natural environments. Nat. Rev. present in clinical isolates of A. J. (2010). Evolution and multi- (2008). Subinhibitory concentrations Microbiol. 8, 251–259. antibiotic-resistant bacteria. Proc. plicity of arginine decarboxylases in of penicillin increase the mutation Allison, K. R., Brynildsen, M. P., and Natl. Acad. Sci. U.S.A. 70, 2276–2280. polyamine biosynthesis and essen- rate to optochin resistance in Strep- Collins, J. J. (2011). Metabolite- Berens, C., and Hillen, W. (2003). Gene tial role in Bacillus subtilis biofilm tococcus pneumoniae. J. Antimicrob. enabled eradication of bacterial per- regulation by tetracyclines. Con- formation. J. Biol. Chem. 285, 39224– Chemother. 62, 973–977. sisters by aminoglycosides. Nature straints of resistance regulation in 39238. Courcelle, J., Khodursky, A., Peter, B., 473, 216–220. bacteria shape TetR for application Butala, M., Zgur-Bertok, D., and Busby, Brown, P. O., and Hanawalt, P. C. Aminov, R. I. (2009). The role of in eukaryotes. Eur. J. Biochem. 270, S. J. (2009). The bacterial LexA tran- (2001). Comparative gene expres- antibiotics and antibiotic resistance 3109–3121. scriptional repressor. Cell.Mol.Life sion profiles following UV expo- in nature. Environ. Microbiol. 11, Bernier, S. P., Lebeaux, D., Defrancesco, Sci. 66, 82–93. sure in wild-type and SOS-deficient 2970–2988. A. S., Valomon, A., Soubigou, G., Charpentier, X., Polard, P.,and Claverys, Escherichia coli. Genetics 158, 41–64. Aminov, R. I. (2010). A brief his- Coppee, J. Y., et al. (2013). Starvation J. P. (2012). Induction of competence Courvalin, P. (2006). Vancomycin resis- tory of the antibiotic era: lessons together with the SOS response for genetic transformation by antibi- tance in gram-positive cocci. Clin. learned and challenges for the mediates high biofilm-specific otics: convergent evolution of stress Infect. Dis. 42(Suppl. 1), S25–S34. future. Front. Microbiol. 1:134. doi: tolerance to the fluoroquinolone responses in distant bacterial species D’Costa, V. M., Griffiths, E., and 10.3389/fmicb.2010.00134 ofloxacin. PLoS Genet. 9:e1003144. lacking SOS? Curr. Opin. Microbiol. Wright, G. D. (2007). Expanding the Andersson, D. I., and Hughes, D. (2012). doi: 10.1371/journal.pgen.1003144 15, 570–576. soil antibiotic resistome: exploring Evolution of antibiotic resistance at Bernier, S. P., Letoffe, S., Delepierre, Chu, W., Zere, T. R., Weber, M. M., environmental diversity. Curr. Opin. non-lethal drug concentrations. Drug M., and Ghigo, J. M. (2011). Bio- Wood, T. K., Whiteley, M., Hidalgo- Microbiol. 10, 481–489. Resist. Updat. 15, 162–172. genic ammonia modifies antibiotic Romano, B., et al. (2012). Indole D’Costa, V. M., King, C. E., Kalan, L., Baharoglu, Z., and Mazel, D. (2011). resistance at a distance in physically production promotes Escherichia coli Morar, M., Sung, W. W., Schwarz, C., Vibrio cholerae triggers SOS and separated bacteria. Mol. Microbiol. mixed-culture growth with Pseu- et al. (2011). Antibiotic resistance is mutagenesis in response to a wide 81, 705–716. domonas aeruginosa by inhibiting ancient. Nature 477, 457–461. range of antibiotics: a route towards Bhullar, K., Waglechner, N., Pawlowski, quorum signaling. Appl. Environ. D’Costa, V. M., Mcgrann, K. M., multiresistance. Antimicrob. Agents A., Koteva, K., Banks, E. D., Johnston, Microbiol. 78, 411–419. Hughes, D. W., and Wright, G. D. Chemother. 55, 2438–2441. M. D., et al. (2012). Antibiotic resis- Cirz, R. T., Chin, J. K., Andes, D. R., (2006). Sampling the antibiotic resis- Balaban, N. Q., Merrin, J., Chait, R., tance is prevalent in an isolated cave De Crecy-Lagard, V., Craig, W. A., tome. Science 311, 374–377. Kowalik, L., and Leibler, S. (2004). microbiome. PLoS ONE 7:e34953. and Romesberg, F. E. (2005). Inhi- Dandekar, A. A., Chugani, S., and Bacterial persistence as a phenotypic doi: 10.1371/journal.pone.0034953 bition of mutation and combating Greenberg, E. P. (2012). Bacterial

www.frontiersin.org February 2013 | Volume 4 | Article 20 | 131

“fmicb-04-00020” — 2013/2/15 — 18:43 — page 11 — #11 Bernier and Surette Antibiotics in natural communities

quorum sensing and metabolic Dwyer, D. J., Kohanski, M. A., Goh, E. B., Yim, G., Tsui, W., Mcclure, J., Janion, C. (2008). Inducible SOS incentivestocooperate.Science 338, Hayete, B., and Collins, J. J. (2007). Surette, M. G., and Davies, J. (2002). response system of DNA repair and 264–266. Gyrase inhibitors induce an oxida- Transcriptional modulation of bacte- mutagenesis in Escherichia coli. Int. J. Dantas, G., Sommer, M. O., Oluwase- tive damage cellular death pathway in rial gene expression by subinhibitory Biol. Sci. 4, 338–344. gun, R. D., and Church, G. M. Escherichia coli. Mol. Syst. Biol. 3, 91. concentrations of antibiotics. Proc. Johnson, L., Mulcahy, H., Kan- (2008). Bacteria subsisting on antibi- Ejim, L., Farha, M. A., Falconer, S. B., Natl. Acad. Sci. U.S.A. 99, 17025– evets, U., Shi, Y., and Lewenza, otics. Science 320, 100–103. Wildenhain, J., Coombes, B. K., Tyers, 17030. S. (2012). Surface-localized spermi- Das, T., and Manefield, M. (2012). M., et al. (2011). Combinations of Grasemann, H., Shehnaz, D., Enomoto, dine protects the Pseudomonas aerug- Pyocyanin promotes extracellular antibiotics and nonantibiotic drugs M., Leadley, M., Belik, J., and Rat- inosa outer membrane from antibi- DNA release in Pseudomonas aerug- enhance antimicrobial efficacy. Nat. jen, F. (2012). L-ornithine derived otic treatment and oxidative stress. J. inosa. PLoS ONE 7:e46718. doi: Chem. Biol. 7, 348–350. polyamines in cystic fibrosis air- Bacteriol. 194, 813–826. 10.1371/journal.pone.0046718 El-Halfawy, O. M., and Valvano, M. ways. PLoS ONE 7:e46618. doi: Kaldalu, N., Mei, R., and Lewis, Davies, J. (2006). Where have all the A. (2012). Non-genetic mechanisms 10.1371/journal.pone.0046618 K. (2004). Killing by ampicillin antibiotics gone? Can. J. Infect. Dis. communicating antibiotic resistance: Gusarov, I., Shatalin, K., Starodubtseva, and ofloxacin induces overlapping Med. Microbiol. 17, 287–290. rethinking strategies for antimicro- M., and Nudler, E. (2009). Endoge- changes in Escherichia coli tran- Davies, J. (2009). Darwin and microbial drug design. Expert Opin. Drug nous nitric oxide protects bacteria scription profile. Antimicrob. Agents biomes. EMBO Rep. 10, 805. Discov. 7, 923–933. against a wide spectrum of antibi- Chemother. 48, 890–896. Davies, J., and Davies, D. (2010). Ori- Erill, I., Campoy, S., and Barbe, J. otics. Science 325, 1380–1384. Kaplan, J. B. (2011). Antibiotic-induced gins and evolution of antibiotic resis- (2007). Aeons of distress: an evolu- Haaber, J., Cohn, M. T., Frees, biofilm formation. Int. J. Artif. tance. Microbiol. Mol. Biol. Rev. 74, tionary perspective on the bacterial D., Andersen, T. J., and Ing- Organs 34, 737–751. 417–433. SOS response. FEMS Microbiol. Rev. mer, H. (2012). Planktonic aggre- Kaplan, J. B., Izano, E. A., Gopal, Davies, J., and Ryan, K. S. (2012). 31, 637–656. gates of Staphylococcus aureus protect P., Karwacki, M. T., Kim, S., Bose, Introducing the parvome: bioactive Evans, D. J., Allison, D. G., Brown, against common antibiotics. PLoS J. L., et al. (2012). Low levels of compounds in the microbial world. M. R., and Gilbert, P. (1991). Sus- ONE 7:e41075. doi: 10.1371/jour- beta-lactam antibiotics induce extra- ACS Chem. Biol. 7, 252–259. ceptibility of Pseudomonas aerugi- nal.pone.0041075 cellular DNA release and biofilm Davies, J., Spiegelman, G. B., and nosa and Escherichia coli biofilms Haddadin, R. N., Saleh, S., Al-Adham, formation in Staphylococcus aureus. Yim, G. (2006). The world of subin- towards ciprofloxacin: effect of spe- I. S., Buultjens, T. E., and Collier, P. MBio 3, e00198–e00112. hibitory antibiotic concentrations. cific growth rate. J. Antimicrob. J. (2010). The effect of subminimal Kearns, D. B. (2010). A field guide to Curr. Opin. Microbiol. 9, 445–453. Chemother. 27, 177–184. inhibitory concentrations of antibi- bacterial swarming motility. Nat. Rev. Davies, J. E., and Benveniste, R. Farr, S. B., and Kogoma, T. (1991). otics on virulence factors expressed Microbiol. 8, 634–644. E. (1974). Enzymes that inactivate Oxidative stress responses in by Staphylococcus aureus biofilms. J. Keller, L., and Surette, M. G. (2006). antibiotics in transit to their tar- Escherichia coli and Salmonella Appl. Microbiol. 108, 1281–1291. Communication in bacteria: an eco- gets. Ann. N. Y. Acad. Sci. 235, typhimurium. Microbiol. Rev. 55, Harrison, J. J., Wade, W. D., Akierman, logical and evolutionary perspective. 130–136. 561–585. S., Vacchi-Suzzi, C., Stremick, C. A., Nat. Rev. Microbiol. 4, 249–258. Diggle, S. P.,Gardner,A.,West,S.A., and Forsberg, K. J., Reyes, A., Wang, B., Sell- Turner, R. J., et al. (2009). The chro- Keren, I., Shah, D., Spoering, A., Griffin, A. S. (2007). Evolutionary eck, E. M., Sommer, M. O., and Dan- mosomal toxin gene yafQ isadeter- Kaldalu, N., and Lewis, K. (2004). theory of bacterial quorum sensing: tas, G. (2012). The shared antibiotic minant of multidrug tolerance for Specialized persister cells and the when is a signal not a signal? Philos. resistome of soil bacteria and human Escherichia coli growing in a biofilm. mechanism of multidrug tolerance Trans. R. Soc. Lond. B Biol. Sci. 362, pathogens. Science 337, 1107–1111. Antimicrob. Agents Chemother. 53, in Escherichia coli. J. Bacteriol. 186, 1241–1249. Frank, K. L., Reichert, E. J., Piper, K. E., 2253–2258. 8172–8180. Dorr, T., Lewis, K., and Vulic, M. (2009). and Patel, R. (2007). In vitro effects Hassett, D. J., and Imlay, J. A. (2007). Kim, W., Killam, T., Sood, V., and SOS response induces persistence of antimicrobial agents on planktonic Bactericidal antibiotics and oxidative Surette, M. G. (2003). Swarm-cell to fluoroquinolones in Escherichia and biofilm forms of Staphylococcus stress: a radical proposal. ACS Chem. differentiation in Salmonella enter- coli. PLoS Genet. 5:e1000760. doi: lugdunensis clinical isolates. Antimi- Biol. 2, 708–710. ica serovar typhimurium results in 10.1371/journal.pgen.1000760 crob. Agents Chemother. 51, 888–895. Henderson-Begg, S. K., Livermore, D. elevated resistance to multiple antibi- Dorr, T., Vulic, M., and Lewis, K. Galhardo, R. S., Hastings, P. J., and M., and Hall, L. M. (2006). Effect otics. J. Bacteriol. 185, 3111–3117. (2010). Ciprofloxacin causes per- Rosenberg, S. M. (2007). Mutation as of subinhibitory concentrations of Kim, W., and Surette, M. G. (2003). sister formation by inducing the a stress response and the regulation of antibiotics on mutation frequency in Swarming populations of Salmonella TisB toxin in Escherichia coli. PLoS evolvability. Crit. Rev. Biochem. Mol. Streptococcus pneumoniae. J. Antimi- represent a unique physiological state Biol. 8:e1000317. doi: 10.1371/jour- Biol. 42, 399–435. crob. Chemother. 57, 849–854. coupled to multiple mechanisms of nal.pbio.1000317 Gefen, O., Gabay, C., Mumcuoglu, M., Hillen, W., and Berens, C. (1994). antibiotic resistance. Biol. Proced. Duan, K., Dammel, C., Stein, J., Engel, G., and Balaban, N. Q. (2008). Mechanisms underlying expression Online 5, 189–196. Rabin, H., and Surette, M. G. Single-cell protein induction dynam- of Tn10 encoded tetracycline resis- Kim, W., and Surette, M. G. (2004). (2003). Modulation of Pseudomonas ics reveals a period of vulnerability tance. Annu.Rev.Microbiol.48, Metabolic differentiation in actively aeruginosa gene expression by host to antibiotics in persister bacteria. 345–369. swarming Salmonella. Mol. Microbiol. microflora through interspecies com- Proc. Natl. Acad. Sci. U.S.A. 105, Hocquet, D., Llanes, C., Thouverez, 54, 702–714. munication. Mol. Microbiol. 50, 6145–6149. M., Kulasekara, H. D., Bertrand, Kint, C. I., Verstraeten, N., Fau- 1477–1491. Gerdes, K., and Maisonneuve, E. X., Plesiat, P., et al. (2012). Evi- vart, M., and Michiels, J. (2012). Duan, K., Sibley, C. D., Davidson, C. (2012). Bacterial persistence and dence for induction of integron- New-found fundamentals of bacte- J., and Surette, M. G. (2009). Chem- toxin–antitoxin loci. Annu. Rev. based antibiotic resistance by the rial persistence. Trends Microbiol. 20, ical interactions between organisms Microbiol. 66, 103–123. SOS response in a clinical set- 577–585. in microbial communities. Contrib. Gillespie, S. H., Basu, S., Dickens, A. ting. PLoS Pathog. 8:e1002778. doi: Kohanski, M. A., Dwyer, D. J., and Microbiol. 16, 1–17. L., O’sullivan, D. M., and Mchugh, 10.1371/journal.ppat.1002778 Collins, J. J. (2010). How antibi- Dwyer, D. J., Kohanski, M. A., and T. D. (2005). Effect of subinhibitory Hoffman, L. R., D’argenio, D. A., Mac- otics kill bacteria: from targets to Collins, J. J. (2009). Role of reactive concentrations of ciprofloxacin on coss, M. J., Zhang, Z., Jones, R. A., and networks. Nat. Rev. Microbiol. 8, oxygen species in antibiotic action Mycobacterium fortuitum mutation Miller, S. I. (2005). Aminoglycoside 423–435. and resistance. Curr. Opin. Microbiol. rates. J. Antimicrob. Chemother. 56, antibiotics induce bacterial biofilm Kohanski, M. A., Dwyer, D. J., Hayete, 12, 482–489. 344–348. formation. Nature 436, 1171–1175. B., Lawrence, C. A., and Collins, J.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 20 | 132

“fmicb-04-00020” — 2013/2/15 — 18:43 — page 12 — #12 Bernier and Surette Antibiotics in natural communities

J. (2007). A common mechanism of impacts on human health. Clin. production of virulence factors and (2008a). Discerning the complexity cellular death induced by bactericidal Microbiol. Rev. 24, 718–733. antibiotic resistance. J. Bacteriol. 190, of community interactions using a antibiotics. Cell 130, 797–810. Martinez, J. L. (2009a). Environmen- 2671–2679. Drosophila model of polymicrobial Kohanski, M. A., Dwyer, D. J., tal pollution by antibiotics and by Palmer, K. L., Aye, L. M., and Whiteley, infections. PLoS Pathog. 4:e1000184. Wierzbowski, J., Cottarel, G., and antibiotic resistance determinants. M. (2007). Nutritional cues control doi: 10.1371/journal.ppat.1000184 Collins, J. J. (2008). Mistransla- Environ. Pollut. 157, 2893–2902. Pseudomonas aeruginosa multicellu- Sibley, C. D., Parkins, M. D., Rabin, H. tion of membrane proteins and two- Martinez, J. L. (2009b). The role of nat- lar behavior in cystic fibrosis sputum. R., Duan, K., Norgaard, J. C., and component system activation trigger ural environments in the evolution of J. Bacteriol. 189, 8079–8087. Surette, M. G. (2008b). A polymicro- antibiotic-mediated cell death. Cell resistance traits in pathogenic bacte- Piddock, L. J., and Wise, R. (1987). bial perspective of pulmonary infec- 135, 679–690. ria. Proc. Biol. Sci. 276, 2521–2530. Induction of the SOS response tions exposes an enigmatic pathogen Kuczynska-Wisnik, D., Matuszewska, Matic, I., Rayssiguier, C., and Rad- in Escherichia coli by 4-quinolone in cystic fibrosis patients. Proc. E., Furmanek-Blaszk, B., Leszczyn- man, M. (1995). Interspecies gene antimicrobial agents. FEMS Micro- Natl. Acad. Sci. U.S.A. 105, 15070– ska, D., Grudowska, A., Szczepaniak, exchange in bacteria: the role of SOS biol. Lett. 41, 289–294. 15075. P., et al. (2010). Antibiotics promot- and mismatch repair systems in evo- Poole, K. (2012a). Bacterial stress Sibley, C. D., Grinwis, M. E., Field, ing oxidative stress inhibit forma- lution of species. Cell 80, 507–515. responses as determinants of antimi- T. R., Eshaghurshan, C. S., Faria, tion of Escherichia coli biofilm via Maynard-Smith, J., and Harper, D. crobial resistance. J. Antimicrob. M. M., Dowd, S. E., et al. (2011). indole signalling. Res. Microbiol. 161, (2003). Animal Signals.NewYork, Chemother. 67, 2069–2089. Culture enriched molecular profiling 847–853. NY: Oxford University Press. Poole, K. (2012b). Stress responses as of the cystic fibrosis airway micro- Kussell, E., Kishony, R., Balaban, N. Mesak, L. R., and Davies, J. (2009). determinants of antimicrobial resis- biome. PLoS ONE 6:e22702. doi: Q., and Leibler, S. (2005). Bacte- Phenotypic changes in ciprofloxacin- tance in Gram-negative bacteria. 10.1371/journal.pone.0022702 rial persistence: a model of survival resistant Staphylococcus aureus. Res. Trends Microbiol. 20, 227–234. Sibley, C. D., Parkins, M. D., Rabin, H. in changing environments. Genetics Microbiol. 160, 785–791. Price-Whelan, A., Dietrich, L. E., and R., and Surette, M. G. (2009). The 169, 1807–1814. Mesak, L. R., Miao, V., and Davies, Newman, D. K. (2006). Rethinking relevance of the polymicrobial nature Kussell, E., and Leibler, S. (2005). Phe- J. (2008). Effects of subinhibitory ‘secondary’ metabolism: physiologi- of airway infection in the acute and notypic diversity, population growth, concentrations of antibiotics on SOS cal roles for phenazine antibiotics. chronic management of patients with and information in fluctuating envi- and DNA repair gene expression Nat. Chem. Biol. 2, 71–78. cystic fibrosis. Curr. Opin. Investig. ronments. Science 309, 2075–2078. in Staphylococcus aureus. Antimicrob. Ramos, J. L., Martinez-Bueno, M., Drugs 10, 787–794. Lai, S., Tremblay, J., and Deziel, E. Agents Chemother. 52, 3394–3397. Molina-Henares, A. J., Teran, W., Sommer, M. O., Church, G. M., (2009). Swarming motility: a multi- Miller, C., Thomsen, L. E., Gaggero, C., Watanabe,K., Zhang, X., et al. (2005). and Dantas, G. (2010). The human cellular behaviour conferring antimi- Mosseri, R., Ingmer, H., and Cohen, The TetR family of transcriptional microbiome harbors a diverse reser- crobial resistance. Environ. Microbiol. S. N. (2004). SOS response induction repressors. Microbiol. Mol. Biol. Rev. voir of antibiotic resistance genes. 11, 126–136. by beta-lactams and bacterial defense 69, 326–356. Virulence 1, 299–303. Lee, H. H., Molla, M. N., Cantor, C. against antibiotic lethality. Science Rath, C. M., and Dorrestein, P. C. Sommer, M. O., and Dantas, G. (2011). R., and Collins, J. J. (2010). Bacterial 305, 1629–1631. (2012). The bacterial chemical reper- Antibiotics and the resistant micro- charity work leads to population- Mirani, Z. A., and Jamil, N. (2011). toire mediates metabolic exchange biome. Curr. Opin. Microbiol. 14, wide resistance. Nature 467, Effect of sub-lethal doses of van- within gut microbiomes. Curr. Opin. 556–563. 82–85. comycin and oxacillin on biofilm Microbiol. 15, 147–154. Sommer, M. O., Dantas, G., and Church, Levin, B. R., and Rozen, D. E. (2006). formation by vancomycin intermedi- Ruiz, J. (2003). Mechanisms of resis- G. M. (2009). Functional character- Non-inherited antibiotic resistance. ate resistant Staphylococcus aureus. J. tance to quinolones: target alter- ization of the antibiotic resistance Nat. Rev. Microbiol. 4, 556–562. Basic Microbiol. 51, 191–195. ations, decreased accumulation and reservoir in the human microflora. Levy, S. B. (2001). Antibiotic resis- Monds, R. D., and O’Toole, G. A. (2009). DNA gyrase protection. J. Antimi- Science 325, 1128–1131. tance: consequences of inaction. Clin. The developmental model of micro- crob. Chemother. 51, 1109–1117. Spoering, A. L., and Lewis, K. (2001). Infect. Dis. 33(Suppl. 3), S124–S129. bial biofilms: ten years of a paradigm Shah, D., Zhang, Z., Khodursky, A., Biofilms and planktonic cells of Lewis, K. (2007). Persister cells, dor- up for review. Trends Microbiol. 17, Kaldalu, N., Kurg, K., and Lewis, K. Pseudomonas aeruginosa have similar mancy and infectious disease. Nat. 73–87. (2006). Persisters: a distinct physio- resistance to killing by antimicrobials. Rev. Microbiol. 5, 48–56. Moyed, H. S., and Bertrand, K. P.(1983). logical state of E. coli. BMC Microbiol. J. Bacteriol. 183, 6746–6751. Lewis, K. (2010). Persister cells. Annu. hipA, a newly recognized gene of 6:53. doi: 10.1186/1471-2180-6-53 Stacy, A. R., Diggle, S. P., and Whiteley, Rev. Microbiol. 64, 357–372. Escherichia coli K-12 that affects fre- Shatalin, K., Gusarov, I., Avetissova, E., M. (2012). Rules of engagement: Lewis, K. (2012). Persister cells: molec- quency of persistence after inhibition Shatalina,Y.,Mcquade, L. E., Lippard, defining bacterial communica- ular mechanisms related to antibiotic of murein synthesis. J. Bacteriol. 155, S. J., et al. (2008). Bacillus anthracis- tion. Curr. Opin. Microbiol. 15, tolerance. Handb. Exp. Pharmacol. 768–775. derived nitric oxide is essential for 155–161. 121–133. Mulcahy, H., Charron-Mazenod, L., pathogen virulence and survival in Stewart, P. S., and Costerton, J. Lu, T. K., and Collins, J. J. (2009). Engi- and Lewenza, S. (2008). Extra- macrophages. Proc. Natl. Acad. Sci. W. (2001). Antibiotic resistance of neered bacteriophage targeting gene cellular DNA chelates cations and U.S.A. 105, 1009–1013. bacteria in biofilms. Lancet 358, networks as adjuvants for antibiotic induces antibiotic resistance in Shatalin, K., Shatalina, E., Mironov, A., 135–138. therapy. Proc. Natl. Acad. Sci. U.S.A. Pseudomonas aeruginosa biofilms. and Nudler, E. (2011). H2S: a uni- Stewart, P. S., and Franklin, M. J. 106, 4629–4634. PLoS Pathog. 4:e1000213. doi: versal defense against antibiotics in (2008). Physiological heterogeneity Lyon, G. J., and Muir, T. W. (2003). 10.1371/journal.ppat.1000213 bacteria. Science 334, 986–990. in biofilms. Nat. Rev. Microbiol. 6, Chemical signaling among bacteria Nelson, M. L., and Levy, S. B. (2011). Shen, L., Shi, Y., Zhang, D., Wei, 199–210. and its inhibition. Chem. Biol. 10, The history of the tetracyclines. Ann. J., Surette, M. G., and Duan, K. Subrt, N., Mesak, L. R., and Davies, 1007–1021. N. Y. Acad. Sci. 1241, 17–32. (2008). Modulation of secreted vir- J. (2011). Modulation of viru- Maisonneuve, E., Shakespeare, L. J., Nijland, R., and Burgess, J. G. (2010). ulence factor genes by subinhibitory lence gene expression by cell wall Jorgensen, M. G., and Gerdes, K. Bacterial olfaction. Biotechnol. J. 5, concentrations of antibiotics in Pseu- active antibiotics in Staphylococcus (2011). Bacterial persistence by RNA 974–977. domonas aeruginosa. J. Microbiol. 46, aureus. J. Antimicrob. Chemother. 66, endonucleases. Proc. Natl. Acad. Sci. Overhage, J., Bains, M., Brazas, M. D., 441–447. 979–984. U.S.A. 108, 13206–13211. and Hancock, R. E. (2008). Swarming Sibley, C. D., Duan, K., Fis- Thornton, M. M., Chung-Esaki, H. Marshall, B. M., and Levy, S. B. (2011). of Pseudomonas aeruginosa isacom- cher, C., Parkins, M. D., Storey, M., Irvin, C. B., Bortz, D. M., Food animals and antimicrobials: plex adaptation leading to increased D. G., Rabin, H. R., et al. Solomon, M. J., and Younger, J.

www.frontiersin.org February 2013 | Volume 4 | Article 20 | 133

“fmicb-04-00020” — 2013/2/15 — 18:43 — page 13 — #13 Bernier and Surette Antibiotics in natural communities

G. (2012). Multicellularity and van der Veen, S., and Abee, T. genetic diversity. Nat. Rev. Microbiol. Conflict of Interest Statement: The antibiotic resistance in Kleb- (2011). Generation of variants in Lis- 5, 175–186. authors declare that the research was siella pneumoniae grown under teria monocytogenes continuous-flow Wright, G. D., and Poinar, H. conducted in the absence of any com- bloodstream-mimicking fluid biofilms is dependent on radical- (2012). Antibiotic resistance is mercial or financial relationships that dynamic conditions. J. Infect. Dis. induced DNA damage and RecA- ancient: implications for drug discov- could be construed as a potential con- 206, 588–595. mediated repair. PLoS ONE 6:e28590. ery. Trends Microbiol. 20, 157–159. flict of interest. Tsui, W. H., Yim, G., Wang, H. H., doi: 10.1371/journal.pone.0028590 Yim, G., Mcclure, J., Surette, M. G., Mcclure, J. E., Surette, M. G., and Vega, N. M., Allison, K. R., Khalil, and Davies, J. E. (2011). Modula- Received: 04 November 2012; accepted: Davies, J. (2004). Dual effects of MLS A. S., and Collins, J. J. (2012). tion of Salmonella gene expression 28 January 2013; published online: 13 antibiotics: transcriptional modula- Signaling-mediated bacterial persis- by subinhibitory concentrations of February 2013. tion and interactions on the ribo- ter formation. Nat. Chem. Biol. 8, quinolones. J. Antibiot. (Tokyo) 64, Citation: Bernier SP and Surette MG some. Chem. Biol. 11, 1307–1316. 431–433. 73–78. (2013) Concentration-dependent activity Turnbull, A. L., and Surette, M. Walker, J. B., and Skorvaga, M. Yim, G., Wang, H. H., and Davies, of antibiotics in natural environments. G. (2008). L-Cysteine is required (1973). Phosphorylation of strepto- J. (2006). The truth about antibi- Front. Microbio. 4:20. doi: 10.3389/ for induced antibiotic resistance in mycin and dihydrostreptomycin by otics. Int. J. Med. Microbiol. 296, fmicb.2013.00020 actively swarming Salmonella enterica Streptomyces. Enzymatic synthesis of 163–170. This article was submitted to Fron- serovar Typhimurium. Microbiology different diphosphorylated deriva- Yim, G., Wang, H. H., and Davies, tiers in Antimicrobials, Resistance and 154, 3410–3419. tives. J. Biol. Chem. 248, 2435– J. (2007). Antibiotics as signalling Chemotherapy, a specialty of Frontiers in Turnbull, A. L., and Surette, M. G. 2440. molecules. Philos. Trans. R. Soc. Microbiology. (2010). Cysteine biosynthesis, oxida- White, A. P., Weljie, A. M., Apel, Lond. B Biol. Sci. 362, 1195– Copyright © 2013 Bernier and Surette. tive stress and antibiotic resistance in D., Zhang, P., Shaykhutdinov, R., 1200. This is an open-access article distributed Salmonella typhimurium. Res. Micro- Vogel, H. J., et al. (2010). A Ysern, P., Clerch, B., Castano, M., Gib- under the terms of the Creative Commons biol. 161, 643–650. global metabolic shift is linked ert, I., Barbe, J., and Llagostera, Attribution License, which permits use, Urios, A., Herrera, G., Aleixandre, V., to Salmonella multicellular develop- M. (1990). Induction of SOS genes distribution and reproduction in other and Blanco, M. (1991). Influence of ment. PLoS ONE 5:e11814. doi: in Escherichia coli and mutagene- forums, provided the original authors and recA mutations on gyrA dependent 10.1371/journal.pone.0011814 sis in Salmonella typhimurium by source are credited and subject to any quinolone resistance. Biochimie 73, Wright, G. D. (2007). The antibiotic fluoroquinolones. Mutagenesis 5, copyright notices concerning any third- 519–521. resistome: the nexus of chemical and 63–66. party graphics etc.

Frontiers in Microbiology | Antimicrobials, Resistance and Chemotherapy February 2013 | Volume 4 | Article 20 | 134

“fmicb-04-00020” — 2013/2/15 — 18:43 — page 14 — #14