How to Evaluate Non-Growing Cells—Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria
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antibiotics Review How to Evaluate Non-Growing Cells—Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria Susanne Fleischmann 1,†, Christian Robben 2,†, Thomas Alter 1, Peter Rossmanith 3,4 and Patrick Mester 4,* 1 Institute of Food Safety and Food Hygiene, Department of Veterinary Medicine, Freie Universitaet Berlin, 14163 Berlin, Germany; [email protected] (S.F.); [email protected] (T.A.) 2 Christian-Doppler Laboratory for Monitoring of Microbial Contaminants, University of Veterinary Medicine Vienna, 1210 Wien, Austria; [email protected] 3 Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA; [email protected] 4 Unit of Food Microbiology, Institute of Food Safety, Food Technology and Veterinary Public Health, University of Veterinary Medicine Vienna, 1210 Wien, Austria * Correspondence: [email protected]; Tel.: +43-12507-3529 † These authors contributed equally to this work. Abstract: Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our in- creasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or Citation: Fleischmann, S.; through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a Robben, C.; Alter, T.; Rossmanith, P.; drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize Mester, P. How to Evaluate traditional culture-based methods represents a considerable experimental hurdle to investigate their Non-Growing Cells—Current increased antimicrobial resistance and impedes the development and evaluation of effective treat- Strategies for Determining ments or interventions against bacteria in the VBNC state. Although experimental approaches were Antimicrobial Resistance of VBNC developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance Bacteria. Antibiotics 2021, 10, 115. screening and this review aims to provide an overview of possible methodological approaches. https://doi.org/10.3390/antibiotics 10020115 Keywords: viable but non-culturable; VBNC; antimicrobial resistance; antibiotic resistance; tolerance; Academic Editors: Anthony Clarke bacterial viability; live/dead; viability PCR and Alexandro Rodríguez-Rojas Received: 18 December 2020 Accepted: 22 January 2021 Published: 26 January 2021 1. Introduction Humanity0s history is a continuous battle between us and microbial pathogens and for Publisher’s Note: MDPI stays neutral the most part, we were on the losing side with bacterial and viral infections being among with regard to jurisdictional claims in the major causes of morbidity and mortality worldwide. Thanks to the development and published maps and institutional affil- improvement of sanitation, hygiene practices, and especially the discovery of antibiotics iations. and vaccinations since the early 20th century, deaths from infectious diseases have declined markedly which can be considered one of the biggest success stories in human history [1]. However, although the burden of disease has been drastically reduced, public health threats posed by microbes are still present and new challenges demand new solutions. Over the Copyright: © 2021 by the authors. last years, an increase in infectious diseases has been observed in almost all countries, Licensee MDPI, Basel, Switzerland. regardless of their economic development, with a rising incidence of foodborne illness This article is an open access article and nosocomial infections [2–5]. For effective prevention and intervention measures, the distributed under the terms and assessment of microbial viability is crucial in determining the safety of food and drinking conditions of the Creative Commons water, as well as the environmental and medical sector. Together with the rising drug Attribution (CC BY) license (https:// resistance of pathogens against traditional antibiotic therapies, the WHO has defined this creativecommons.org/licenses/by/ problem as one of the most serious public health challenges [6–8]. Figure1 highlights the 4.0/). Antibiotics 2021, 10, 115. https://doi.org/10.3390/antibiotics10020115 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, x 2 of 25 Antibiotics 2021, 10, 115 2 of 24 has defined this problem as one of the most serious public health challenges [6–8]. Figure 1 highlights theuse use of of antibiotics antibiotics in in human human medicine, medicine, animal animal husbandry, husbandry, agriculture, agriculture, and and the subsequent the subsequentcirculation circulation between between these these applications applications and and the the environment environment leading leading to to an an increase in the increase in theprevalence prevalence of of antibiotic antibiotic resistant resistant bacteria bacteria in in different different ecological ecological niches. niches. Figure 1. A schematicFigure 1. A overview schematic of overview the ecology of the of antibiotics, ecology of showingantibiotics, how showing these drugshow these are circulated drugs are betweencircu- different environments,lated such between as the medicaldifferent environment, environments, agricultural such as the settings, medical the environment, aquacultural agricultural environment, settings, the pharmaceutical the aquacultural environment, the pharmaceutical industry and the wider environment, adapted from industry and the wider environment, adapted from [9]. [9]. During the last decades, a plethora of antibiotic resistant bacterial genotypes have During thebeen last reported decades, and a asplethora a result, of research antibiotic has resistant intensified bacterial to identify genotypes novel antibacterialhave agents been reportedand and antibiotics as a result, [9–11 research]. While cominghas intensified to the fore to recently,identify the novel impact antibacterial of phenotypic plasticity agents and antibioticson antimicrobial [9–11]. While and antibiotic coming to resistance the fore recently, has been the far impact less studied. of phenotypic Phenotypic plasticity plasticity on antimicrobialdescribes the and ability antibiotic of bacteria resi tostance change has theirbeen phenotype far less studied. (in this Phenotypic case their antimicrobial plasticity describesresistance the ability profile) of withoutbacteria underlyingto change thei geneticr phenotype changes. (in Whilethis case phenotypic their an- plasticity in timicrobial resistancebacteria hasprofile) been without known forunderlying a long time genetic already, changes. the understanding While phenotypic of the underlying plasticity in bacteriaadaptive has and been regulatory known processes,for a long astime well already, as their the role understanding in the infectious of processthe has only underlying adaptiverecently and been regulatory significantly processes, expanded as well [6,12 as]. their role in the infectious pro- cess has only recentlyIn order been tosignificantly preserve viability expanded and [6,12]. adaptability in constantly changing environmental In order toconditions, preserve bacteriaviability haveand adaptability developed various in constantly mechanisms changing for environmental switching from a vegetative conditions, bacteriato a metabolically have developed inactive, various but highlymechanisms resistant for and switching persistent from state. a vegetative The most studied type to a metabolicallyof such inactive, cellular but dormancy highly resistant is sporulation and persistent of sporogenous state. The most bacteria, studied while type in recent years, of such cellularthe dormancy viable but is non-culturable sporulation of (VBNC) sporogenous state for bacteria, many bacterial while in pathogens recent years, has gained more the viable but attentionnon-culturable [12–16 (VBNC)]. The VBNC state statefor many is a condition bacterial pathogens of reduced has metabolic gained activity,more induced in attention [12–16].response The VBNC to conditions state is a of conditio stress. Entryn of reduced into the metabolic VBNC state activity, can be induced facilitated in by a number response to conditionsof environmental of stress. factors,Entry into such the as VBNC a lack state of nutrients, can be facilitated temperature by a shifts, number or the presence of environmentalof antibiotics, factors, such disinfectants, as a lack of and nutrients, sanitizers temperature [12,17–19]. shifts, When or a the microorganism presence enters the of antibiotics,VBNC disinfectants, state, both and the sanitizers morphology [12,17–19]. and physiology When a microorganism of the cell changes enters to allowthe its survival VBNC state, bothunder the adverse morphology conditions. and physiology Then, once of the the environmentalcell changes to conditionsallow its survival return to normality, under adversethe conditions. cell can get Then, back once to its the culturable environmental state, through conditions a reactivation return to process, normality, which is referred the cell can getto back as “resuscitation”. to its culturable state, through a reactivation