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PHYSIOLOGICAL and ECOLOGICAL INVESTIGATIONS of CLOSTRIDIUM DIFFICILE by Catherine D. R

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PHYSIOLOGICAL and ECOLOGICAL INVESTIGATIONS of CLOSTRIDIUM DIFFICILE by Catherine D. R PHYSIOLOGICAL AND ECOLOGICAL INVESTIGATIONS OF CLOSTRIDIUM DIFFICILE By Catherine D. Robinson A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Microbiology and Molecular Genetics- Doctor of Philosophy 2014 ABSTRACT PHYSIOLOGICAL AND ECOLOGICAL INVESTIGATIONS OF CLOSTRIDIUM DIFFICILE By Catherine D. Robinson Disease caused by Clostridium difficile is currently the most prevalent nosocomial infection and leading cause of antibiotic-associated diarrhea. It is clear that the intestinal microbiota plays a role in preventing C. difficile infection in the absence of antibiotics; however, the mechanisms involved in this protective function are poorly understood. Since antibiotic administration is an inducing factor of C. difficile infection, treatment employing antibiotics often results in recurrent disease, yet it is still the primary line of treatment. Therefore, a central goal of research in this area is to better define the role of the intestinal microbiota in suppression of disease, and ultimately develop alternative ways to prevent and treat C. difficile infection. In this thesis, I present a novel in vitro model that was developed to study complex fecal communities. This in vitro model is a continuous-culture system that utilizes arrays of small-volume reactors; it is unique in its simple set-up and high replication. We adapted this model to operate as a C. difficile infection model, where in vivo C. difficile invasion dynamics are replicated in that the fecal communities established in the reactors are resistant to C. difficile growth unless disrupted by antibiotic administration. We then go on to use this model to show that newly emerged, epidemic strains of C. difficile have a competitive fitness advantage when competed against non-epidemic strains. We also show this competitive advantage in vivo, using a mouse infection model. This result is exciting, as it suggests that physiological attributes of these strains, aside from classical virulence factors, contribute to their epidemic phenotype. Finally, the metabolic potential of C. difficile in regards to carbon source utilization is explored, and reveals that epidemic strains are able to grow more efficiently on trehalose, a disaccharide sugar. Moreover, preliminary in vivo mouse studies suggest that trehalose utilization plays a role in colonization. Therefore, the growth advantage conferred by this increased ability to utilize trehalose may contribute to the ecological fitness of these strains in vivo. The in vitro model developed and presented in this thesis could be used to study many aspects of C. difficile-microbiota interactions and has the potential to elucidate mechanisms that are important for in vivo resistance to establishment of disease. In addition, the metabolic investigations described provide insight into understanding the physiology of not only C. difficile as a whole, but also physiological attributes unique to epidemic strains. Ultimately, these types of ecological and physiological investigations will bring us closer to finding better ways to treat and prevent disease caused by C. difficile. ACKNOWLEDGMENTS First of all I would like to thank my advisor Rob Britton for helping to make my experience as a doctoral student so successful and positive. Having had one child coming into graduate school and then two babies during my PhD, I had many extra challenges and responsibilities in addition to those of a graduate student. Dr. Britton was amazingly supportive of my family obligations and understanding about the difficulties that having children brings. Above that, his encouragement, support, and mentorship from an academic standpoint was exceptional. I also want to thank the other members of my graduate committee, Dr. Gemma Reguera, Dr. Chris Waters, Dr. Terry Marsh, and Dr. Vince Young for their insight and guidance. I would also like to thank all of the Britton lab members, both past and present. It was a genuine pleasure working with all of them. I couldn’t have asked for a more collaborative and pleasant lab to be a part of. I want to especially thank Jenny Auchtung for being such a great colleague and friend. She was like a second mentor to me and always had helpful advice, drawn from her abundant experiences in different areas of research as a graduate student and postdoc. She was always willing to lend an ear for venting about both research and child-rearing frustrations and offered invaluable support and encouragement. I can’t adequately express how much the support of my parents has meant to me over the years. They deserve all of the credit for making me the successful person I am today. I am thankful that they instilled in me the work ethic and sense of self- pride and dedication required to persevere through this process. The encouraging iv and reassuring words from them really helped me along the way and they never fail to express how proud they are of me. Finally, I want to thank my family- my husband, Dan, and my three girls, Alex, Lana, and Veda. They are my real motivation. I truly don’t think a better, more supportive husband and father exists. He’s my rock and my biggest fan and always encourages me when I question my abilities. I want to thank him for believing in me and giving me the confidence to believe in myself. I want my daughter Alex to know how much having her by my side has meant to me, not only for all of her help with the little ones, but how she supports and encourages me in her own way. I hope that I am setting a good example for all of my girls and that they will see that they can accomplish anything they set their minds to. v TABLE OF CONTENTS LIST OF TABLES ............................................................................................................................................x LIST OF FIGURES ........................................................................................................................................xi CHAPTER 1: Overview of Clostridium difficile Disease and Physiology……….……………..1 Antibiotic-Associated Diarrhea…………………………………………………………………....1 C. difficile: the Bacterium and the Disease…………………..……………………………..….2 C. difficile infection cycle…………………………………………………………………...2 Epidemiology, disease, and virulence factors………………………………...…...6 Molecular typing schemes………………………………………………………………...8 Emergence of Epidemic C. difficile Strains………………………………………………...…..9 Current impact on the health care system…………………..…………………....11 Understanding Disease Development: Colonization Resistance……………………11 Bile acids and germination……………………………………………………….…...…12 Competitive exclusion………………………………………………...……………….…..13 Direct antagonism…………………………………………………………………………. 14 Understanding C. difficile Physiology: Metabolism……………………………….……...16 Early Characterization of C. difficile physiology………………………….……..18 Studies of C. difficile-microbiota nutrient competition…………….………...19 Hydrolytic enzymes……………………………………………………….………………..22 Proteolysis, amino acids, and stickland fermentation……….……………….23 “Omics” approaches to investigating metabolism….……………………….…27 Genomics……………………………………………………………………………..27 Transcriptomics and proteomics…………………………………….…….28 Metabolomics…………....………………………..……………………….……….30 Autotrophy………………………………………………………………………….….………31 Summary………………………………………………………………………………….…….……..…..31 REFERENCES………………………………………………………………………….………...……….34 CHAPTER 2: Development of Single Chamber Human Gut Microbiota Mini-bioreactor Arrays (MBRA)……………………………………………………………………………….……………………46 Abstract………………………………………………………………………………….……………...….47 Introduction…………………………………………………………………………..………………….48 Materials and Methods……………………………………………………………………………....50 Design of mini-bioreactor arrays (MBRA)………………………………………...50 Collection of fecal samples……………………………………………………..….…….51 Preparation of fecal samples……………………………………………………………51 Media……………………………………………………………………………………..………52 MBRA operating conditions and sampling………………………..………………53 HPLC of SCFA……………………………………………………………….…………………54 SCFA Analysis…………………………………………………………………………………54 DNA extraction……………………………………………….………………………………55 vi Preparation of 16S rRNA amplicons for sequencing…………………….……55 Analysis of amplicon data………………………………………………………….…….56 Comparison of cultures grown in BRMW, BRMW10 and BRMG to the fecal slurry……………………………………………………………..…..57 Analysis of variation in cultures grown in BRMW10 and comparison to the starting fecal inoculum……………………………..59 Results………………………………………………………………………………………………….…..59 Mini-bioreactor design……………………………………………………………..……..59 MBRA operation…………………………………………………………….……………….61 Short chain fatty acid profiles of cultures grown in each medium revealed that BRMW10 communities were highly stable and most similar to the fecal inoculum………………………………………………………..…..62 Comparison of MBRA microbial composition and structure demonstrates that communities cultured in BRMW10 are most similar to the fecal inoclum.………………………………………………………………...….......67 Comparison of class-level differences among reactor communities revealed the extent of reorganization of the microbial community during culture……………………………………………………………….........................70 Examining the microbial community structure in BRMW10 communities at more frequent time intervals revealed how communities diverge from day-to-day and reactor-to-reactor…………………………………...………71 Discussion……………………………………………………………………………….……………...…75 Comparison of community ecology in BRMW10 reactors to previously published models reveals similar trends among the in vitro models….78 Conclusions…………………………………………………………………………………….80
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