show Abstracthide AbstractObjectives: From 2012-2015, a sudden huge increase of vancomycin-resistant (vanA) Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. Clonal relatedness of VREfm and vancomycin-susceptible E. faecium (VSEfm) was investigated, transmission events between hospitals were identified and the pan-genome and plasmids from the largest VREfm clonal group were characterized.Methods: WGS of 1058 E. faecium isolates was carried out on the Illumina platform to perform SNP-analysis and to identify the pan-genome. One isolate was also sequenced on the PacBio platform to close the genome. Epidemiological data were collected from laboratory information systems.Results: Phylogeny of 892 VREfm and 166 VSEfm revealed a polyclonal structure with a single clonal group (ST80) accounting for 40% of the VREfm isolates. VREfm and VSEfm co-occurred within many clonal groups; however, no VSEfm were related to the dominant VREfm group. A similar vanA plasmid was identified in =99% of isolates belonging to the dominant group and 69% of the remaining VREfm. Ten plasmids were identified in the completed genome and approximately 29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among all isolates and the dominant group was 13,320 and 5,905 genes, respectively.Conclusions: Most likely, VREfm emerged due to import of a successful VREfm clone which has rapidly transmitted to the majority of hospitals in the region whilst simultaneously disseminating a vanA plasmid to pre-existing VSEfm. Acquisition of a heterogeneous accessory genome may account for the success of this clone by facilitating adaption to new environmental challenges.