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Use of different molecular typing methods for the study of heterogeneity within Clostridium difficile toxinotypes V and III.

Clostridium difficile strains of toxinotypes III (n = 13) and V (n = 45) were typed by agarose gel-based PCR ribotyping, capillary gel electrophoresis-based PCR ribotyping and PFGE using two different restriction enzymes, SmaI and SacII. With conventional agarose gel-based PCR ribotyping, toxinotype III strains were distributed among six different PCR ribotypes and toxinotype V strains into three different PCR ribotypes. Capillary gel electrophoresis-based ribotyping was more discriminatory for toxinotype V strains, with six different ribotypes found. With PFGE using SmaI, all toxinotype III strains grouped together into a single pulsotype. Using SacII, ribotype 027 strains grouped together with >90 % similarity and were <83 % similar to other ribotypes of toxinotype III strains. Within ribotype 078, seven (SmaI) and eight (SacII) different pulsotypes were found, whilst ribotype 126 strains belonged to one (SmaI) and two (SacII) pulsotypes. Within ribotype 066, it was possible to distinguish between pig and human isolates. Using SacII, a further distinction could also be made between pig isolates from two different farms. PFGE (SmaI and SacII) clustered strains according to their toxinotype; however, correlation of PFGE and ribotyping was better with SacII. These data suggest that toxinotype III strains are a more heterogeneous group than toxinotype V strains and that SacII is more discriminatory than SmaI. Alternatively, the use of both enzymes simultaneously could improve PFGE typing of C. difficile.