Clostridium difficile is a Gram-positive, spore-forming anaerobic bacterium that has emerged as a major cause of healthcare- and antibiotic-associated diarrhoea. After antibiotic therapy, the protective intestinal microbiota is disrupted, whereupon ingested or resident C. difficile colonize the gastrointestinal tract and produce toxins and transmissible spores. C. difficile was recognized as a pathogen only three decades ago, and a number of emergent PCR ribotypes have been responsible for outbreaks worldwide, with different PCR ribotypes dominating both temporally and geographically. A major outbreak occurred in Canada in 2003, caused by a previously rare PCR ribotype 027. This 027 ribotype has spread globally and now accounts for ∼50% of isolates in United Kingdom and North American hospitals. The epidemiology of C. difficile is evolving rapidly, yet despite this continued threat, we have a poor understanding of how or why particular variants emerge.
In this paper, whole genome sequencing was used to analyze genetic variation and virulence of thirty C. difficile isolates, to determine both macro and microevolution of the species. Horizontal gene transfer and large-scale recombination of core genes has shaped the C. difficile genome over both short and long time scales. Phylogenetic analysis demonstrates C. difficile is a genetically diverse species, which has evolved within the last 1.1–85 million years. By contrast, the disease-causing isolates have arisen from multiple lineages, suggesting that virulence evolved independently in the highly epidemic lineages. The results suggest that the core C. difficile genome has been primarily shaped by purifying selection pressure, and that environmental as well as genetic effects may be responsible for its recent expansion as a major pathogen. This study also opens avenues for the development of new epidemiological tools for studying C. difficile transmission routes and for developing interventions to reduce the burden of disease.