Investigation of strain variation and virulence factors in clostridium difficile.

Abstract

Over the last decade Clostridium difficile has emerged as a serious issue, causing hospital-based epidemics and community-associated disease. While initially seen as a nuisance pathogen, the emergence of “hypervirulent” ribotype 027 strains has seen an increase in the morbidity and mortality attributable to C. difficile. These “hypervirulent” variants are currently spreading throughout the developed world. Other strain types are also becoming a cause for concern, with ribotype 017 and ribotype 078 strains also associated with more severe disease in human patients. Little is known about the types of C. difficile strains in Australia, and there was no information available regarding the types of C. difficile strains infecting patients within the Victorian healthcare system when this study began. The concern caused by the emergence of “hypervirulent” C. difficile overseas highlighted the need for a survey of local isolates. The work described in this thesis represents the first molecular survey of isolates from Victorian healthcare networks, illustrating the diversity of isolates found in this state. In addition, it covers the first reported isolation and characterisation of a locally-acquired ribotype 027 strain in Australia, followed by characterisation of isolates from other local outbreaks, allowing hospital laboratories to identify important strain types infecting patients and to target C. difficile as a potential issue in our healthcare facilities. The genetic diversity of C. difficile may also have implications beyond the hospital setting and may influence the potential of variant strain types to cause disease. The other major aim of this project was to investigate the role of the global regulator Spo0A in the regulation of putative virulence factors. A key finding of the study outlined in this PhD thesis is that Spo0A appears to regulate cell phenotypes differently in the various strain backgrounds tested. Through targeted gene disruption and complementation studies, it was found that Spo0A differentially regulates toxin production in divergent strain types. In ribotype 027 strains, Spo0A was found to negatively regulate toxin A and toxin B production. In the ribotype 078 strain, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin levels during late stationary phase. This contrasts with the finding for 630∆erm, where Spo0A does not appear to influence toxin production. In addition to toxin production, growth and motility phenotypes were examined. Spo0A may be involved in regulating some aspects of C. difficile motility, with an altered growth phenotype on agar plates observed in the spo0A mutants. This change was most distinct in the ribotype 078 strain tested. Prior to this study, the genetic manipulation of a ribotype 078 strain had not been reported. Other studies have shown that ribotype 078 strains are genetically diverse compared to other strain types. The work outlined in this study is a clear demonstration that differences at the genomic level reflect phenotypic differences. This thesis highlights the genetic diversity of C. difficile and illustrates the need to examine a broad range of strains when examining potential virulence factors.