The Human Gut Microbiota As a Reservoir for Antimicrobial Resistance Genes

The human gut microbiota as a reservoir for antimicrobial resistance genes Elena Buelow The human microbiota as a reservoir for antimicrobial resistance genes PhD Thesis, University of Utrecht, The Netherlands ISBN/EAN: 978-94-6295-117-4 Cover art: Elena Buelow, Ivo Jelinek. Layout and design: Elena Buelow, Ivo Jelinek Printed by: Uitgeverij BOXPress | Proefschriftmaken.nl © Elena Buelow, Utrecht, The Netherlands. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system or transmitted by any means without permission of the author. The copyright of the articles that have been published or accepted for publication has been transferred to the respective journals. This work was supported by The Netherlands Organisation for Health Research and Development ZonMw (Priority Medicine Antimicrobial Resistance; grant 205100015) and by the European Union Seventh Framework Programme (FP7-HEALTH-2011-single-stage) ‘Evolution and Transfer of Antibiotic Resistance’ (EvoTAR) under grant agreement number 282004 The printing of this thesis was kindly supported by: University Medical Center Utrecht; Infection and Immunity Center Utrecht; and the Netherlands Society of Medical Microbiology (NVMM) and the Royal Netherlands Society for Microbiology (KNVM). The human gut microbiota as a reservoir for antimicrobial resistance genes De darm microbiota van de mens als een reservoir van antimicrobiële resistentiegenen (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 24 maart 2015 des middags te 12.45 uur door Elena Bülow geboren op 7 augustus 1982 te Kevelaer, Duitsland Promotor: Prof. dr. M.J.M. Bonten Copromotoren: Dr. ir. W. van Schaik Dr. R.J.L. Willems Es kann die Ehre dieser Welt Dir keine Ehre geben, Was dich in Wahrheit hebt und hält, Muß in dir selber leben. Wenn’s deinem Innersten gebricht An echten Stolzes Stütze, Ob dann die Welt dir Beifall spricht, Ist dir all wenig nütze. Das flücht’ge Lob des Tages Ruhm Magst du dem Eitlen gönnen; Das aber sei dein Heiligtum: Vor dir bestehen können. Theodor Fontane Commisse: Prof. dr. P.I.W. de Bakker Prof. dr. M. Kleerebezem Prof. dr. M.O.A. Sommer Prof. dr. J.A.J.W. Kluytmans Dr. B.E. Dutilh Paranimfen: Nuno Rodrigues Faria Fernanda Paganelli Content Chapter 1 General introduction 9 Chapter 2 Effects of selective decontamination (SDD) 28 on the gut resistome J Antimicrob Chemother, 2014, 69 (8): 2215-2223. doi:10.1093/jac/dku092 Chapter 3 Dynamics of the gut microbiota and resistome 51 during ICU hospitalization Manuscript in preparation Chapter 4 Limited dispersal of antibiotic resistance 84 genes through the sewerage system Manuscript in preparation Chapter 5 Genes conferring resistance to the disinfectant 112 benzalkonium chloride in the gut microbiota of hospitalized patients Manuscript in preparation Chapter 6 General discussion 132 English summary 148 Dutch summary 152 Acknowledgements 158 Curriculum Vitae 164 Chapter 1 General introduction Chapter 1 The human microbiota The human body is densely populated with trillions of microbes which are referred to as the human microbiota. The human microbiota mostly consists of bacterial cells, and is estimated to outnumber the cells of the human body by a factor of ten. The number of bacterial genes in the human microbiota outnumbers the number of genes in the human genome by several orders of magnitude [1, 2]. The collection of genes contained by the human microbiota is referred to as the human microbiome. Historically, the human microbiota was studied by laboratory culture, but culture techniques are limited in their ability to capture the full diversity of the bacteria that inhabit the human body [3]. The introduction of microbial DNA-based culture-independent approaches to study microbial communities (termed ‘metagenomics’) has revolutionized our understanding of microbiology and substantially contributed to the advances in human microbiome research in the last decade [4, 5]. Metagenomic approaches are commonly used in studies of the human microbiota, either by means of 16S rRNA sequencing for taxonomic profiling, or by means of metagenomic shotgun-sequencing, which allows both taxonomic profiling and the identification of functionally associated features of sequences [1, 5] (Figure 1). A complementary approach to assign biological functions to the genes that are identified by high-throughput sequencing approaches is termed functional metagenomics. In functional metagenomics, DNA isolated from a microbial ecosystem is randomly cloned into a vector and transformed to a microbial host (most commonly E. coli), resulting in large clone libraries. These clone libraries can then be used to screen for novel enzymatic activities, resistance to antimicrobial compounds or other relevant phenotypes [6-8]. 10 Chapter 1 Functional metagenomics Microbial community Extract DNA Library in E. coli Screen for phenotypes sample 11 Chapter 1 Figure 1: Methods for DNA-based analyses of microbial communities Different methods for the analysis of the composition and function of microbial communities are outlined in this figure. All these methods start with the isolation of microbial-DNA from an environmental sample (e.g. soil, feces, water). To characterize the phylogenetic profile of an environmental community, the 16S rRNA gene is amplified along defined variable regions and subsequently sequenced. Analysis is performed by grouping highly similar sequences (usually at least 97% identical sequences on the nucleotide level) into operational taxonomic units (OTUs), which are then compared to 16S rRNA databases such as Silva [9], Green Genes [10], and RDP [11], to identify the OTUs as precisely as possible. Communities then can be described in terms of presence, relative abundance and phylogenetic relatedness of the OTUs. A different approach is to directly sequence metagenomic DNA, in a technique is called shotgun metagenomic sequencing, in the figure referred to as “shotgun metagenomic approach”. The assembled sequences or sequence reads are compared to reference genomes or gene catalogs. Compared to 16S rRNA analysis, this is a more costly approach but generally provides higher taxonomic resolution, and allows the analysis of single nucleotide polymorphisms (SNPs) and other variant sequences. Furthermore, metagenomic sequencing allows the in silico assessment of functional characteristics of a microbial community through an analysis pipeline, such as MG-RAST [12]. In functional metagenomics, DNA isolated from a microbial ecosystem is randomly cloned into a vector and transformed to a microbial host (most commonly E. coli), resulting in large clone libraries. These clone libraries can then be used to screen for novel enzymatic activities, resistance to antimicrobial compounds or other relevant phenotypes [6-8]. This figure was adapted from Morgen et al. [1]. The human gut microbiota The human gut harbors the most densely colonized microbial ecosystem of the human body. The gut microbiota has an important role in human health since it is involved in numerous metabolic, physiological and immunological processes and is responsible for maintaining gut homeostasis [4, 13-16]. The phyla Firmicutes and Bacteroidetes are the most dominant phyla in the human gut microbiota, with Proteobacteria, Verrucomicrobia, Actinobacteria, Fusobacteria, and Cyanobacteria, also being ubiquitously present, but generally at lower levels than the Firmicutes and Bacteroidetes [15]. The gut microbiota has high inter- individual variability and can be influenced by several factors including age, ethnicity, host immune response, nutritional variations and the administration of antibiotics [15-18]. The majority of gut microbes are harmless gut commensals that determine the beneficial symbiotic relationship of the gut microbiota and its host. Dysbiosis of the human gut microbiota, which means an imbalanced gut microbiota that can be caused by shifts in microbial composition, has been associated with increased disease susceptibility of the host, and, consequently, with various diseases, including metabolic and immunological disorders and cancer [13, 15, 16]. Current research focuses on identifying and understanding 12 Chapter 1 environmental, ecological and host factors that lead to the distortion of the function and composition of the gut microbiota, which may subsequently lead to disease. The exposure of the gut microbiota to antimicrobial agents has substantial short-term and long-term effects on the community structure of the gut microbiota and may be one of the main causes by which the bacterial communities of the gut are currently affected [17-19]. One of the consequences of antibiotic use is a reduction of microbial diversity [19] which can have a direct impact on the host’s health. The healthy gut microbiota provides colonization resistance against opportunistic pathogens by the natural abundance of gut commensals. The term colonization resistance defines the ability of the healthy gut microbiota to prevent colonization with ingested bacteria or to inhibit the overgrowth of bacteria that are normally present in the gut at low abundance [20]. In this process gut microbe-microbe and host-microbiota interactions are thought to play equally important roles. During microbe- microbe interactions the competition for nutrients and available

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