Practical considerations in developing Clostridium difficile bacteriophages as therapeutics: characterisation, evolutionary dynamics and model systems

Clostridium difficile is a major nosocomial pathogen responsible for epidemics associated with significant mortality and morbidity. The occurrence of Clostridium difficile infections is associated with the use of antibiotics to treat primary infections. Consequently, alternative treatment methods are being considered. Bacteriophages are bacterial viruses that have long been used as therapeutics, are a plausible alternative. This thesis investigates the potential of bacteriophages of C. difficile to be used a therapeutic. Eight novel bacteriophages were isolated, all of which were characterised in terms of their morphology, host range, adsorption and growth dynamics. One of these bacteriophages was sequenced on the basis of its broad host range, which included strains of the clinically relevant ribotype 027. Individual and combinations of phages were shown to significantly reduce the amount of bacterial counts in vitro, with combinations being more effective than individual bacteriophages. The therapeutic potential of bacteriophages was also tested in vivo in a hamster model. Treatment in vivo was shown to be able to significantly reduce the bacterial counts in a carriage model where the hamsters were infected with an avirulent strain of C. difficile and delay the onset of C. difficile infection and death in a hamster model infected with a highly virulent strain of C. difficile. The way that C. difficile and its bacteriophages co-evolve was tested in vitro. The results showed that time progresses bacterial become more resistant to bacteriophages and bacteriophages become more infectious, creating an evolutionary arms race. Genomic analysis of bacteriophage resistant bacteria showed the presence of SNPs mainly associated with cell surface proteins. In conclusion, this study has provided evidence that bacteriophages of C. difficile do indeed have the potential of being used as therapeutic agents. However this is not a straightforward process and more research needs to be invested in this area.