Multihospital expansion of vancomycin-resistant Enterococcus faecium ST117-CT7799 and transmission of linear plasmids co-carrying vanA and linezolid resistance genes, Comunitat Valenciana, Spain (2022-2024)

Background: Vancomycin-resistant Enterococcus faecium (VREfm) is a WHO priority pathogen. In the Comunitat Valenciana (CV), Spain, VREfm prevalence has increased since 2022. We characterized the population structure, transmission patterns and resistance determinants of VREfm across eight hospitals (2021-2024). Methods: Eight hospitals reported 870 VREfm cases during 2021-2024. We sequenced 254 VREfm isolates using WGS (Illumina) and inferred relatedness by MLST, cgMLST, and core-genome SNP analyses. Acquired antimicrobial resistance (AMR) genes, bacteriocins, plasmid replicases and putative virulence markers (PVMs) were identified in silico. Thirty-eight representatives underwent Nanopore long-read sequencing and hybrid assembly to resolve plasmids. Results: The predominant vancomycin-resistance genotype was vanB (62%; six hospitals), followed by vanA (19%; five hospitals) and vanA+vanB (17%; five hospitals). Eight sequence types (ST) and 15 clonal complexes (CT) were identified, grouped into seven main phylogenetic clades with close relatedness to publicly available genomes from other regions of Spain and Europe. A single lineage, ST117-CT7799, accounted for 198/254 (78%) isolates, persisted during 2022-2024 across seven hospitals and was enriched in the bacteriocin gene bac43 (or T8) (84%). Linezolid-resistance genes optrA and cfr(D) were present in 34% of isolates, most of which also carried vanA (32%). Hybrid assemblies revealed a diverse plasmidome including RepA_N megaplasmids with PVMs and AMR genes, and small Rep3-like plasmids harbouring bacteriocins (bac43, bac51, bacAS9); a 6 kb repA_pB82 plasmid was bac43-positive in 37/38 (97%) fully resolved carriers. Eight strains across four hospitals carried identical linear plasmids co-harbouring vanA, optrA, cfrD genes within a widespread repUS78_pZY2 background, consistent with inter-lineage plasmid transmission. Conclusions: This first comprehensive genomic analysis of VREfm in CV indicates extensive inter-hospital spread dominated by expansion of ST117-CT7799 and highlights plasmid-mediated convergence of vancomycin and linezolid resistance via linear plasmids. Strengthened infection prevention and genomic surveillance, including long-read sequencing to track linear plasmids, are warranted.