Comparative analysis of an expanded Clostridium difficile reference strain collection reveals genetic diversity and evolution through six lineages.

Clostridium difficile is an anaerobic bacillus that resides in the gut and has rapidly emerged as a leading cause of antibiotic associated diarrheal disease in humans. The genetic basis of the pathogenicity of C. difficile remains poorly understood. In this study we aimed at characterizing the genetic diversity of C. difficile strains by three different methods (PCR ribotyping, multilocus sequence typing and genetic markers) to improve the typing of C. difficile. Our study was performed on a reference collection (Leeds-Leiden/ECDC) of C. difficile PCR ribotype (RT) strains (n=70) expanded with six PCR RT strains highly related to the emerging PCR RTs 027 and 078. Besides PCR ribotyping we used multilocus sequence typing (MLST) using seven housekeeping genes (MLST 7HG) that has recently been developed for characterizing C. difficile isolates as well as analysis of unique genetic markers. Evolutionary relatedness of the sequences determined by MLST 7HG was analyzed in phylogenetic analysis. In total 56 MLST 7HG sequence types (STs) were identified, nine of which were new. Phylogeny reconstruction of the reference set of strains supplemented with the online available C. difficile MLST reference database, revealed six monophyletic lineages of closely related STs. ST-122 (PCR RT131) formed a well-separated branch in the tree and was thus designated as a novel lineage. Furthermore, we confirmed that several PCR RTs are highly related to the emerging PCR RTs 027 and 078 since these types display the same STs (ST-1 and ST-11, respectively). Based on the observed results, we conclude that MLST 7HG is a valuable method to study C. difficile phylogeny.