Exploiting Bacteriophages to Tackle Clostridium difficile Infection
Clostridium difficile infection (CDI) currently affects around 20,000 people each year, in healthcare institutions and in the community, and will often follow disruption of the gut microbiome. Current treatment strategies call for the use of further antibiotics, of which there is a limited choice. There is a need for additional remedial and prophylactic solutions with greater specificity and low levels of toxicity and resistance. This thesis describes the pathogenesis of CDI, the current treatment strategies and navigates the growing body of studies investigating the potential use of phage. The project involved extensive screening including faecal samples and environmental sources in an attempt to identify novel phages of C. difficile and documents efforts to improve the therapeutic capacity of a selected phage, ФCD27, by mutagenesis. No exclusively lytic phages were isolated or obtained following mutagenesis with ethylmethane sulphonate, hydroxylamine or sodium pyrophosphate. Batch fermentation models of CDI showed that a prophylactic approach to phage therapy of CDI offers a higher efficacy than a remedial regime. A continuous model of CDI in a colon model was successfully produced and demonstrated variable efficacy rates from no apparent decrease in the burden of C. difficile to a reduction to below the limit of detection by culture, with no detrimental effect on commensal microbiota. The lysogenic capacity of ФCD27 appeared to prevent clearance of C. difficile in the models, but some strains containing the prophage exhibited reduced toxin production phenotypically. A possible mechanism of this altered phenotype included the action of ФCD27 repressor proteins on the promoter regions of C. diffiicle toxin genes or regulatory elements, but affinity of a candidate repressor, ORF44, to PaLoc constituents was not demonstrated. Studies have also demonstrated the ability of ФCD27 to prevent outgrowth of germinating C. difficile spores, thus potential as an environmental decontaminant. iii The findings of the project and the future prospects of phage therapy as an agent against CDI are discussed.
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