Isolation and Characterisation of Bacteriophages Infecting Environmental Strains of Clostridium Difficile

Clostridium difficile is a leading cause of nosocomial infections associated with antibiotic treatment. Despite its’ pathogenic role, the bacterium can be carried asymptomatically in the GI tract and has an ubiquitous presence in the environment. The ecology of C. difficile outside clinical settings is not fully understood, but the evolution of pathogenic strains may occur in its zoonotic and environmental reservoirs. A major driver of bacterial genome evolution is bacteriophages. This project has investigated the prevalence and diversity of phages associated with strains from these reservoirs. A large and diverse collection of C. difficile isolates was established in order to isolate and characterise the phages associated with them. 27 phages were isolated. These were characterised according to their particle morphology, genome size and plaque morphology which showed they could be sub-grouped according to morphology. Host range analyses showed there is a complex network of phage-host interactions within this species. To characterise to a genetic level, the genomes of seven phages were sequenced and annotated. Their genomes show they are related to known C. difficile phages. However, genes which are unusual or novel to phage genomes were also identified. Comparative genomics identified distinct lineages within C. difficile phages, which correspond to their morphological sub-grouping. Also the bioinformatic analyses provide evidence of genetic exchange occurring between them, presumably during co-infection as multiple prophage carriage is common in this species. Interestingly, analysis of the C. difficile CRISPR system showed that in addition to the bacterial arrays, several prophages encode CRISPR arrays that target phage sequences. Evidence of their co-evolution suggests phage infection impacts both genome evolution of their host and other C. difficile phages. In summary, a diverse C. difficile phage collection has been established which has the potential for development to allow their exploitation.