Isolation and Characterisation of Temperate Bacteriophages of the Hypervirulent Clostridium difficile 027 Strains

Clostridium difficile 027 can be divided into several subclades which vary in their disease severity. Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. Little is known about temperate bacteriophage carriage in C. difficile 027 clinical isolates. Therefore, this study was designed to induce and characterise temperate bacteriophages from C. difficile 027 subtypes’ clinical isolates as a first step to understanding their potential role in disease and diversity. Ninety-one C. difficile 027 clinical isolates were induced for prophage, and the bacteriophages present were characterised using transmission electron microscopy and pulsed-field gel electrophoresis. A correlation between phage morphology and subtype was established. Morphological and genetically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 62 and three isolates, respectively. Dual phage carriage was observed in four isolates. There were inducible phage tail-like particles in all the isolates. The capacity of the two antibiotics norfloxacin and mitomycin C to induce prophages was compared and it was found that they induced specific prophages from C. difficile isolates. PCR assays targeting the capsid, holin and portal genes of the myoviruses were designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the genes sequences from ten ribotypes showed that all sequences found in the ribotype 027 strains were identical and distinct from other C. difficile ribotypes and other bacteria species. Sporulation which can be influenced by histidine kinase gene encoded by C. difficile phages was characterised in 41 isolates. The isolates sporulated within the first 96 h producing between 10[superscript 4] and 10[superscript 12] CFU/ml spores. The variation in sporulation characteristics did not correlate to the subtypes or prophage contents of the isolates examined. This study strongly suggests that phages and sporulation contribute to diversity, evolution and success of this pathogen.