Culture-enriched metagenomic sequencing reveals within-patient diversity and transmission of vancomycin-resistant Enterococcus faecium.

Colonization of the gastrointestinal (GI) tract by vancomycin-resistant Enterococcus faecium (VREfm) often precedes bloodstream infection and serves as a reservoir for onward patient transmission in healthcare settings. Routine clonal isolate-based sequencing often underestimates within-patient diversity and can miss transmission involving low-abundance and co-colonizing strains. Here, we applied culture-enriched metagenomic sequencing to matched GI tract and blood VREfm populations collected ≤14 days apart from 35 patients with positive VREfm blood cultures obtained between 2020 and 2025 at a single hospital. GI tract populations exhibited greater within-patient diversity than bloodstream populations, including multi-strain colonization in five patients. Among single-strain populations, variant analysis suggested distinct environment-specific pressures between the GI tract and bloodstream environments. To assess transmission using culture-enriched metagenomic sequencing, we compared all 70 VREfm populations against 470 contemporary clinical VREfm isolate genomes collected from the same hospital and identified 19 putative transmission clusters including 6 clusters involving multi-strain populations. Together, these results demonstrate how culture-enriched metagenomic sequencing improves resolution for assessing within-patient VREfm diversity and enhances the detection of transmission events that could be missed by clonal isolate-based surveillance.