Widespread dissemination of the RepA_N-type megaplasmid conferring a carbohydrate utilization advantage in clonal...

Enterococcus faecium has become a major nosocomial pathogen, primarily driven by its ability to acquire mobile genetic elements carrying antimicrobial resistance or metabolic traits. Our previous study identified a megaplasmid conferring a high-level gentamicin resistance (HLGR), which can undergo recombination between intact and IS1216V-disrupted aac(6')-Ie-aph(2″)-Ia genes, resulting in a non-HLGR phenotype. In this study, we further characterized the RepA_N-type megaplasmid in sequence type (ST)17 and ST78 E. faecium and examined its distribution among 465 isolates collected between 2011 and 2020 in Taiwan. Both sublineages carried gene clusters associated with carbohydrate utilization, albeit with differences in repertoires. Loss of the RepA_N megaplasmid in ST17 isolate E37 impaired growth on sucrose, confirming its contribution to carbohydrate metabolism. Screening of clinical isolates showed that repA_N and scrB (sucrose utilization) genes were highly prevalent (90.8% and 85.8%, respectively) in the 10 years, whereas IS1216V-inserted aac(6')-Ie-aph(2″)-Ia decreased over time (84.6% in 2011 and 55.7% in 2020), paralleling a decline in HLGR rates (57.7% in 2011 and 45.9% in 2020). Among the 465 E. faecium isolates, ST17 and ST78, both belonging to clonal complex 17, were the most dominant sublineages. The expansion of ST78 vancomycin-resistant E. faecium since 2015 corresponded with the emergence of type III IS1216V-inserted aac(6')-Ie-aph(2″)-Ia, while ST17, mainly carrying type II IS1216V insertion, remained stable throughout the decade. Collectively, these results indicate that the RepA_N-type megaplasmid was widely disseminated and may enhance metabolic adaptability in E. faecium, supporting its persistence in clinical environments.IMPORTANCEEnterococcus faecium is a major hospital-associated pathogen with high genomic plasticity. This study characterized a widespread RepA_N-type megaplasmid, which conferred both high-level gentamicin resistance and enhanced carbohydrate utilization. The co-selection of antimicrobial resistance and metabolic adaptability on a single mobile genetic element may underlie the evolutionary success of dominant clinical lineages such as sequence type (ST)17 and ST78 in Taiwan. These findings provide a framework for understanding how plasmids contribute to the persistence and spread of E. faecium in healthcare settings.