Multilocus variable-number tandem repeat analysis: a helpful tool for subtyping French Clostridium difficile PCR ribotype 027 isolates.

The objective of this study was to evaluate the usefulness of multilocus variable-number tandem repeat analysis (MLVA) for typing and subtyping of Clostridium difficile. Sixty-eight strains were studied, including strains from PCR ribotypes 027, 078/126, 014/020/077, 017 and 023. The stability of variable-number tandem repeat (VNTR) loci was tested by comparing the MLVA results of two strains subcultured 11 times. After DNA extraction, seven tandem repeat loci (A6, B7, C6, E7, F3, G8, H9) from published MLVA schemes were amplified by PCR and sequenced. The distance between two strains was determined by calculating the summed tandem repeat difference. Genomic diversity was evaluated by using the minimum spanning tree (Bionumerics 5.1 software program; Applied Maths). Among the 68 C. difficile isolates examined, 65 unique MLVA types were identified, suggesting a high discriminatory power. An overall good agreement was observed between MLVA types and PCR ribotypes. The stability of VNTR loci was good. MLVA could separate isolates of the hypervirulent PCR ribotype 027 clone in several clusters; all 027 strains isolated within a hospital were grouped in a specific cluster or were placed very close to each other. Results of MLVA confirmed that strains from PCR ribotypes 078 and 126 were closely related although some were located in different branches of the tree. Similar results were observed for most strains from PCR ribotypes 014, 020 and 077. This highly discriminatory method is time-consuming and expensive, but is a valuable tool for subtyping of C. difficile, especially of 027 strains.