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.
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