One panel consisted of CDR4, CDR5, CDR9, CDR48, CDR49, CDR59, and CDR60, and the other panel consisted of C6cd, H9cd, F3cd, CDR4, CDR9, CDR48, and CDR49 [13, 14]. However, our study indicated that MLVA4, which consisted of C6cd, CDR4, CDR49, and CDR60, was able to discriminate all 142 test strains (Table 3), as previously observed for MLVA of Salmonella typhimurium . Furthermore, all of these VNTR loci exhibited higher allelic number and copy number variation than previously reported (Table 1) . Our results may be explained by two reasons: 1) among these loci, CDR60 loci was found exhibit incomplete RO4929097 purchase copy number and was assigned by repeat array
size, as this could increase the allelic number; and 2) we validated these loci in a more random population than previous studies [13, 14], which would increase the value of allelic diversity. In addition, we used a categorical coefficient instead of STRD to analyze the MLVA data and to analyze the loci represented by the repeat array size. Although this may find more reduce the sensitivity to differentiate the outbreak strains, analyses using the STRD coefficient were found to be too variable and may obscure the epidemiological links between C. difficile outbreak strains when several
repeats at a locus are deleted or duplicated simultaneously . All clusters detected by MLVA4 and MLVA10 combined can be explained by epidemiological information. Apart from the two patients from cluster D were C. difficile infection cases, other patients from other clusters were assumed to be C. difficile carriers (GSK2126458 Figure 4; Additional file 3). The major limitation of this validation for the study of outbreak strains was the sample population we used; the 142 test strains used in the current study were a randomly sampled population that did not contain mafosfamide outbreak strains, and the genetic relationship between these was distant. For these reasons, this may have overestimated the discriminatory power of the MLVA 4. Therefore, the MLVA4 panel requires further validation using closely related strains, such as outbreak strains from hospitals, before any conclusions as to its discriminatory power can be made. Five imperfect
VNTR loci (cd5, cd6, cd7, CDR59, and CDR60) were used in this study, except for CDR59, the other four loci were long-repeat VNTR loci with incomplete repeats (Additional file 1). The incomplete repeats may be caused by insertions and deletions, which often result in horizontal gene transfer between bacteria strains and obscured the phylogenic relationship in the bacteria population . However, the long-repeat regions exhibited a higher frequency of recombinations, and were considered attractive candidate regions that could be used for determining phylogenetic relatedness between species and strains . The long-repeat VNTR loci have been known to be responsible for adaptive evolution, as for antigenic variation , and were also used to differentiate the C. botulinum and N. meningitides[36, 37].