First report of regional spreading and long-term inhabitation of VanD-type vancomycin-resistant Enterococcus faecalis clinical strains: A proposal of new classification of vanD gene subtypes.

OBJECTIVE: VanD-type vancomycin resistance is rarely reported in Enterococcus faecalis. This study aimed to characterise five VanD-type vancomycin-resistant E. faecalis strains isolated over 8 years from three hospitals in a local city in Japan, with a focus on resistance mechanisms, genetic background, and phylogenetic classification.

METHODS: Five E. faecalis strains (SVR2085, SVR2281, SVR2330, SVR2331, and SVR2332) were analysed using antimicrobial susceptibility testing, plasmid profiling, and whole-genome sequencing. Genomic islands (GIs) containing vanD gene clusters were characterised. Core GI gene and core genome phylogenies were compared, and vanD homologues were classified using public database sequences.

RESULTS: All strains exhibited high-level resistance to vancomycin (MIC >1024-64 mg/L), multiple drug resistance, and carried pheromone-responsive plasmids encoding bacteriocin 41 as a colonisation factor. Four strains shared nearly identical GIs (126-185 kbp), while SVR2330 had a structurally distinct GI. Phylogenetic analysis showed that the four similar strains formed a single cluster, suggesting a common ancestor, whereas SVR2330 was divergent. Comparative analysis of 37 vanD homologues revealed high genetic diversity, allowing classification into three subgroups - vanD(I), vanD(II), and vanD(III). The vanD of SVR2330 was assigned as vanD(III)-4, and the others as vanD(II)-6, -11, -15, and -17.

CONCLUSIONS: This study proposes a new classification scheme for diverse vanD genes and provides the first evidence of the regional spread and long-term persistence of VanD-type vancomycin-resistant E. faecalis in Japan.