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Journal of Clinical Microbiology, November 2008, p. 3703-3713, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00866-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic Relatedness of Clostridium difficile Isolates from Various Origins Determined by Triple-Locus Sequence Analysis Based on Toxin Regulatory Genes tcdC, tcdR, and cdtR{triangledown}

Philippe J. M. Bouvet* and Michel R. Popoff

Centre National de Référence des Bactéries Anaérobies et du Botulisme, Institut Pasteur, Paris, France

Received 6 May 2008/ Returned for modification 14 July 2008/ Accepted 19 September 2008

A triple-locus nucleotide sequence analysis based on toxin regulatory genes tcdC, tcdR and cdtR was initiated to assess the sequence variability of these genes among Clostridium difficile isolates and to study the genetic relatedness between isolates. A preliminary investigation of the variability of the tcdC gene was done with 57 clinical and veterinary isolates. Twenty-three isolates representing nine main clusters were selected for tcdC, tcdR, and cdtR analysis. The numbers of alleles found for tcdC, tcdR and cdtR were nine, six, and five, respectively. All strains possessed the cdtR gene except toxin A-negative toxin B-positive variants. All but one binary toxin CDT-positive isolate harbored a deletion (>1 bp) in the tcdC gene. The combined analyses of the three genes allowed us to distinguish five lineages correlated with the different types of deletion in tcdC, i.e., 18 bp (associated or not with a deletion at position 117), 36 bp, 39 bp, and 54 bp, and with the wild-type tcdC (no deletion). The tcdR and tcdC genes, though located within the same pathogenicity locus, were found to have evolved separately. Coevolution of the three genes was noted only with strains harboring a 39-bp or a 54-bp deletion in tcdC that formed two homogeneous, separate divergent clusters. Our study supported the existence of the known clones (PCR ribotype 027 isolates and toxin A-negative toxin B-positive C. difficile variants) and evidence for clonality of isolates with a 39-bp deletion (toxinotype V, PCR ribotype 078) that are frequently isolated worldwide from human infections and from food animals.

* Corresponding author. Mailing address: Institut Pasteur, Unité Bactéries anaérobies et toxines, 25-28 rue du Docteur Roux, F-75724 Paris cedex 15, France. Phone: 33 (0)1 40613509. Fax: 33 (0)1 40613123. E-mail: philippe.bouvet{at}pasteur.fr

{triangledown} Published ahead of print on 1 October 2008.

Journal of Clinical Microbiology, November 2008, p. 3703-3713, Vol. 46, No. 11
0095-1137/08/$08.00+0     doi:10.1128/JCM.00866-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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