Cairns, Michelle Dawn; (2018) Evolution of Clostridium difficile. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Clostridium difficile continues to be a leading cause of healthcare-associated infections in the developed world. Increased detection of C. difficile infection (CDI) and development of typing schemes to differentiate between strains is primarily due to the recognition of global outbreaks of a single strain, BI/NAP1/027 which is characterised by three common typing techniques; restriction endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE) and PCR ribotyping. Phylogenetic analysis using multilocus sequence typing (MLST) divides C. difficile into five phylogenetic lineages which align the well-known PCR ribotypes; 027, 023, 017, 078 and a lineage containing diverse PCR ribotypes. MLST data in this thesis confirmed the five phylogenetic lineages were maintained after testing a larger collection of isolates from varied sources with further micro-diversity within the individual lineages. MLST investigation did not identify a lineage exclusive to nonhuman strains or any correlation between sequence type and geographical location. Data in this thesis also supports the notion that PCR ribotyping and REA do not correspond as well as previously considered. This may result in phylogenetically similar strains being designated as a different type or variant. The toxin A-B+ PCR ribotype 017 strain that forms a predominant lineage is little investigated. Through whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis, a historical clone of PCR ribotype 017 was identified from a London hospital ward. Although no phenotype exclusive to the clonal strain was characterised, this is the first report in the UK investigating the phylohistory of isolates from hospitalised patients with CDI due to PCR ribotype 017. Further investigation of PCR ribotype 017 with a larger and global collection of strains revealed two distinct sub-lineages containing multiple independent clonal expansions, antimicrobial resistant SNP determinants, deletions and insertions which were well distributed geographically and temporally. The data suggests transmission between humans and animals and findings support a USA origin with multiple, global transmission events. The key findings of this thesis are that C. difficile as a species is continually evolving with the appearance of divergent sub-lineages. WGS is superior to routine typing methodologies for tracking this evolution and will have significant impacts for outbreak investigation, understanding the phylohistory and phylogeography of C. difficile and other pathogens that are a threat to human health.

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