Comparison of Multi-Locus Variable Number Tandem Repeat Analysis to PCR-Ribotyping for the Classification of Clostridium difficile Isolates
Clostridium difficile is an important enteropathogen in humans and various domestic animal species. Concern has been expressed about the potential for interspecies transmission of C. difficile. The use of molecular typing methods is critical for elucidating the potential for interspecies transmission, however there is no consensus regarding optimal methods. PCR-ribotyping is widely used, but it may not have optimal discriminatory power and inter-laboratory comparisons remain difficult. Other molecular typing methods are labor-intensive, subjective, or lack the discriminatory power to differentiate between closely related strains. Multi-locus Variable-Number Tandem-Repeat Analysis (MLVA) is an attractive alternative, which is objective, can be standardized to have good inter-laboratory reproducibility, and is amenable to automation. The objectives of the present study were to use MLVA to assess genetic relatedness between C. difficile ribotypes of human, environmental, and animal origin.
DNA from 102 previously ribotyped isolates from humans (n=39), dogs (16), horses (15), cattle (11), pigs (8), cats (2), sheep (2), elk (1) and the environment (8) were used. PCR-MLVA was performed using 7 selected C. difficile repeat (CDR) loci. The number of copies at each of the seven CDR loci were concatenatedto generate an MLVA type for each isolate, and these were clustered according to MLVA results to establish their geneticrelationship. A minimum-spanning tree (MST) was generated using the Bionumerics 4.01 software (Applied Maths, Austin,TX).
Eight clusters of MLVA types with summed tandem repeat difference (STRD) < 10 were generated by the MST. The largest cluster consisted of 18 isolates of varying species and ribotypes. A second closely related cluster (n=11) was comprised of multiple ribotypes from a majority of human isolates, several canine isolates, 1 environmental and 1 equine isolate. The third cluster consisted of 2 closely related isolates with STRD = 3. Both of these isolates were recovered from calves and belonged to different ribotypes. Cluster 4 (n=8), was comprised of a variety of species and ribotypes. Five isolates from 4 species within this cluster had the same MLVA type. Cluster 5 (n=13) consisted of non-toxigenic isolates from different species. Cluster 6 contained 1 human and 1 equine isolate belonging to the hypervirulent 027/BI/NAP1 clone. Cluster 7 consisted of 1 canine and 1 equine isolate, and cluster 8 contained 2 porcine isolates with identical MLVA type.
The study demonstrated that PCR-MLVA might provide useful information to sole or combined molecular typing of C. difficile. The finding of isolates from multiple species within the same MLVA cluster supports the concerns for interspecies transmission of this bacterium.