Recombination and accessory genome turnover shaped the rise and decline of vanA Enterococcus faecium ST80 in Denmark

Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause of hospital-associated infections globally. In the Capital Region of Denmark, VREfm ST80 emerged as the dominant vanA -positive clone but declined significantly over the following decade. To investigate the genomic mechanisms underlying this epidemiological shift, we assembled and performed whole-genome sequencing and phylogenomic analysis of 334 ST80 isolates collected from colonisation (83%) and infection (17%) cases across nine hospitals in the region between 2012 and 2020. Two major clusters (Clusters 1 and 2) and nine sub-clusters (1.A–G and 2.H–I) were identified. Sub-cluster-1.A (169/334) was the dominant lineage, accounting for 72% of isolates from 2012–2015. Isolates from 2016–2020 were distributed across all sub-clusters, indicating increasing diversification over time. Genetic variation was driven by accumulation of mutations, recombination and gain/loss of accessory genes and plasmids. Eight recombination hotspots were identified in core genes involved in cell wall synthesis, phosphotransferase systems, amino-acid biosynthesis, and several transporters. Accessory genome comparisons revealed sub-cluster-specific enrichment of resistance genes, including ermG (89% in 1.B), tet(S) (85% in 1.A), and tet(M) (88% in sub-cluster-1.B). Additionally, genes associated with biofilm formation, ABC transporters, and biosynthesis of polysaccharides, terpenoids, and aspartate were differentially enriched across the clusters. These genomic signatures may have contributed to niche adaptation and intra-clonal diversification. Plasmidome analysis showed increased diversity in later years, with the decline of Inc 18 family plasmids (from 80 to 38%) and the rise of two new plasmids, coinciding with reduced clonal stability. Long-read hybrid assemblies uncovered mosaic phage-like plasmids harboring the vanA operon alongside ermB , aadE , and aph(3’)-IIIa suggesting a unique mechanism for co-mobilization of antimicrobial resistance via transduction or conjugation. Despite the observed diversity, a continuous and sustained intra- and inter-hospital transmission and dissemination of this clone was evidenced by the small SNP difference (≤ 15 SNPs) between strains. This study showed the continuous hospital transmission and intra-clonal diversity of VREfm ST80. The dissemination, transmission and persistence dynamics of the vanA ST80 clone in Denmark may have been shaped by recombination events and gain/loss of genes involved in general metabolism, antibiotic resistance and virulence, and inter-clonal competition.