Clostridium difficile is the major underlying cause of antibiotic-associated diarrhoea and poses a risk for healthcare systems worldwide. Endospores produced during sporulation are widely regarded to be the infectious agent of C. difficile associated diarrhoea. These spores are able to withstand a variety of antimicrobial agents and industrial cleaning products and are therefore able to reside on surfaces in healthcare settings for prolonged periods of lime. In order to cause disease in susceptible individuals, spores need to abjure dormancy and return to vegetative cell growth through germination. Sporulation and germination have been studied extensively in Bacillus spp. Knowledge about the sporulation and germination pathways in C. difficile, however, remains incomplete. Here, forward and reverse genetics methods were employed to analyse sporulation and germination phenotypes of C. dfficile. Using forward genetics, 19 mutants with potential sporulation and/or germination phenotypes were isolated, three of which were completely deficient for sporulation. In an attempt to explore the use of transposon suicide vectors, a protocol for the successful transformation of C. difficile was developed. A reverse genetic mutant in the germination specific lytic transglycosylase Slee created by ClosTron mutagenesis was used to study spore germination in vivo. This study is the first report of the use of a germination mutant in vivo. The sporulation characteristics of 52 clinical C. difficile isolates have been analysed indicating that a variation in the rate of sporulation is not associated with molecular type. The germination characteristics of 37 clinical C. difficile isolates were examined, indicating that different isolates exhibit varying germination characteristics in response to bile salts.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594825 |
| Date | January 2013 |
| Creators | Heeg, Daniela |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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