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Journal of Clinical Microbiology, August 1998, p. 2240-2247, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Novel Toxinotyping Scheme and Correlation of Toxinotypes with Serogroups of Clostridium difficile Isolates

Maja Rupnik,1 Véronique Avesani,1 Miha Janc,2 Christoph von Eichel-Streiber,3 and Michel Delmée1,*

Microbiology Unit, Catholic University of Louvain, 1200 Brussels, Belgium1; Department of Biology, University of Ljubljana, 1000 Ljubljana, Slovenia2; and Institute of Medical Microbiology, Johanes-Gutenberg-University, 55101 Mainz, Germany3

Received 17 November 1997/Returned for modification 23 February 1998/Accepted 14 April 1998

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 fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less 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. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly 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.

* Corresponding author. Mailing address: Microbiology Unit, Catholic University of Louvain, Avenue Hippocrate 54.90, B1200 Brussels, Belgium. Phone: 32 2 764 94 41. Fax: 32 2 764 94 40. E-mail: delmee{at}mblg.ucl.ac.be.

Journal of Clinical Microbiology, August 1998, p. 2240-2247, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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