A NOVEL TOXINOTYPING SCHEME AND CORRELATION OF TOXINOTYPES WITH SEROGROUPS OF CLOSTRIDIUM-DIFFICILE ISOLATES

Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR f ragments were checked for length polymorphisms and cut with several re striction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were fo und compared to the toxin genes of reference strain VPI 10463. Polymor phisms were usually observed in both toxin genes. RFLPs were more comm only found in the tcdB gene, in which a single restriction enzyme coul d give up to five different patterns. Restriction sites seemed to be l ess heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortened tcdA genes are described. Accord ing to the changes in their toxin genes, variant strains could be divi ded into 10 groups (toxinotypes I to X). A toxinotype was characterize d by similar patterns of changes in the toxin genes and in other regio ns of the pathogenicity locus and also similar pulsed-field gel electr ophoresis patterns. Variant toxinotypes were found in 9 of the 22 sero groups studied, and some toxinotypes were clearly associated with spec ific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were common ly found are A1, A15, E, and X. Testing of variability in C. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.