Genetic diversity of the low-level vancomycin resistance gene vanC-2/vanC-3 and identification of a novel vanC subtype (vanC-4) in Enterococcus casseliflavus.

An intrinsic low-level vancomycin resistance (VanC phenotype) in Enterococcus casseliflavus is conferred by either of two subtypes of vanC genes, that is, vanC-2 or vanC-3, which are genetically closely related. To know genetic diversity of vanC-2/C-3 genes among E. casseliflavus, nucleotide sequences of vanC-2/C-3 and other genetic components in vanC gene cluster (vanXYc, vanTc, vanRc, and vanSc) were analyzed for nine clinical isolates and four standard strains that showed low-level vancomycin resistance. While the vanC-2/C-3 gene sequences showed 93-100% identities among the strains examined, two genetic groups were discriminated by phylogenetic analysis: one closely related to the previously reported vanC-2 or vanC-3 genes (vanC-2/C-3 genotype) with 98-100% identity, and the other distinct from the vanC-2/C-3 genotype (93-95% identity). The latter group found in three clinical isolates was considered as a new subtype of vanC and tentatively designated as vanC-4. Between strains with the vanC-2/C-3 genotype and those with vanC-4, vanXYc genes were also genetically discriminated with 92-93% identity. Similar sequence diversity was observed for vanTc, vanRc, and vanSc (88-93% identity). Clonal relatedness among the E. casseliflavus strains was investigated by phylogenetic analysis of atpA gene. While among E. casseliflavus strains with vanC-2/C-3 genotype, extremely high sequence identities of atpA were found (98.7% or higher), these strains showed slightly lower identity to those with vanC-4 (94-96%). These two groups of E. casseliflavus strains were also discriminated by genotyping with arbitrarily primed PCR. These findings indicated that among E. casseliflavus there are at least two genetic lineages with the distinct vanC genes, that is, a single subtype including previously known vanC-2/C-3, and a novel subtype vanC-4.