Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes

University of Ljubljana, Department of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia 1 Anaerobe Reference Unit, Public Health Laboratory, University Hospital of Wales, Cardiff, UK 2 Author for correspondence: Maja Rupnik. Tel: +386 61 256 5584. Fax: +386 61 257 3390. e-mail: maja.rupnik{at}mf.uni-lj.si Toxinotyping and PCR ribotyping are two methods that have been used to type Clostridium difficile isolates. Toxinotyping is based on PCR-RFLP analysis of a 19 kb region encompassing the C. difficile pathogenicity locus. PCR ribotyping is based on comparison of patterns of PCR products of the 16S–23S rRNA intergenic spacer region. Representative strains (101) from a C. difficile PCR ribotype library and 22 strains from previously described toxinotypes were analysed to compare ribotyping with toxinotyping. Within this panel of strains all 11 toxinotypes (0–X) described previously and an additional 5 novel toxinotypes (XI–XV) were observed. PCR ribotyping and toxinotyping correlated well and usually all strains within a given ribotype had similar changes in toxin genes. The new toxinotype XI comprises strains that did not express toxins TcdA or TcdB at detectable levels, but contained part of the tcdA gene. Strains of toxinotype XII exhibit changes only in the 5' end of the tcdB gene. Toxinotype XIV is composed of strains that have a large insertion at the beginning of the tcdA gene. A total of 25 of the 89 tested PCR ribotypes of C. difficile contained variant strains. It was estimated that they represent 7·7% of the total number of strains in the Anaerobe Reference Unit collection. Keywords: Clostridium difficile , toxinotyping, PCR ribotypes, tcdA + B - strains, variant toxin genes Abbreviations: UPGMA, unweighted pair group method with arithmetic averages