The healthy human gut can take it all: vancomycin-variable, linezolid-resistant strains and specific...

Enterococcus spp. are opportunistic human pathogens colonizing the human gut and a significant reservoir for the continuous adaptation of hospital clones. However, studies on the features of enterococci species co-colonizing healthy individuals are scarce. We investigated the prevalence, antibiotic resistance, and bacteriocin profiles of Enterococcus species in fecal samples from healthy adults in Portugal using culture-based methods, WGS, and bacteriocin inhibition assays. Results were compared with data from a 2001 study in the same region. Enterococcus spp. (n = 315; 24% MDR) were recovered from all volunteers. Enterococcus lactis was the prevalent species (75%), followed by Enterococcus faecalis (65%) and Enterococcus faecium (47%). E. lactis prevalence increased 2.5-fold since 2001. Linezolid resistance genes (optrA/poxtA) were detected in E. faecium and Enterococcus thailandicus isolates, while a vancomycin-variable E. faecium was also identified. Virulence and plasmid profiles were diverse across species, with evidence of exchange of virulence markers and plasmid replicons between E. faecium and E. lactis. Bacteriocin gene repertoires were extensive and species-specific. Higher numbers of bacteriocin genes were associated with stronger inhibition profiles, and 25% of E. faecium and E. lactis isolates were capable of inhibiting relevant VRE clones. This study unveils the co-occurrence and ecological dynamics of Enterococcus species in the healthy human gut, reinforcing its role as a reservoir for key antibiotic resistance genes and potentially pathogenic strains. The shift toward E. lactis prevalence and the detection of linezolid resistance genes in healthy individuals underscore the need for ongoing surveillance of the gut microbiome to guide public health strategies and antibiotic stewardship efforts.IMPORTANCEThis study highlights the role of Enterococcus species in the healthy human gut, revealing important insights into their prevalence and antibiotic resistance. It emphasizes that the human gut serves as a significant reservoir for antibiotic-resistant strains and shows a notable increase and prevalence of Enterococcus lactis, which has been underappreciated due to identification challenges. The research also underscores the bacteriocins' role in microbial competition, where commensal strains inhibit clinical VRE, potentially aiding the restoration of the gut microbiota, after antibiotic treatment. The findings accentuate the need for ongoing surveillance to track changes in gut bacteria, especially with the emergence of resistance genes to last resort antibiotics. Such monitoring is crucial for shaping public health strategies and managing the growing threat of antibiotic-resistant infections. Profiling bacteriocins at the species and strain level can identify ecological adaptation factors and inform strategies to target high-risk clones.