Characterization of mobile genetic elements from clostridium difficile and development of molecular tools for genetic manipulations

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Clostridium difficile is now considered as one of the most important pathogen responsible for nosocomial infections in North America and Europe, especially among the elderly. Virulence of C. difficile relies mainly on two toxins (TcdA and TcdB) and on the ability to sporulate and persist in the environment. The recent genome sequencing from a virulent strain of C. difficile revealed several mobile genetic elements, including a plasmid and two complete prophages. Thus, horizontal gene transfer is most likely an important mechanism for the evolution of this pathogen. However, prophages and plasmids of C. difficile have only been poorly studied so far and according to their role in virulence of other pathogens, there is a need to better characterize them and assess their impact on the biology and evolution of C. difficile. In addition, efficient molecular tools for the genetic manipulation of C. difficile remain very limited, which blocks further fundamental studies. Our long-term objective is to characterize prophages and plasmids from C. difficile and to develop molecular tools for its genetic manipulation in order to get insight into the ecology, diversity, virulence and evolution of this bacterium. A collection of ~1,000 strains will be classified by molecular techniques, and prophages within the genome of these strains will be identified and characterized at the microbiological and molecular level, including whole genome sequencing. Plasmids will also be isolated, sequenced and fully characterized. The distribution of prophages and plasmids across our C. difficile collection will be determined, which will hopefully give us clues about their role and impact on C. difficile ecology and evolution. Finally, these plasmids will be used to develop molecular tools (e.g. vectors) to manipulate the C. difficile genome. For the first time, this study will provide data about prophage diversity in a large collection of C. difficile strains. We will also gain fundamental data regarding C. difficile plasmids and their potential function(s). New virulence or fitness factors are expected to be found within these mobile genetic elements, which could help us better understand the microbial diversity of C. difficile.