Hidden Diversity in Enterococcus faecalis Revealed by CRISPR2 Screening: Eco-evolutionary Insights into a Novel Subspecies

Enterococcus faecalis is a commensal bacterium that colonizes the gut of humans and animals, and a major opportunistic pathogen, known for causing multidrug-resistant healthcare-associated infections (HAIs). Its ability to thrive in diverse environments and disseminate antimicrobial resistance genes (ARGs) across ecological niches highlights the importance of understanding its ecological, evolutionary, and epidemiological dynamics. The CRISPR2 locus has been used as a valuable marker for assessing clonality and phylogenetic relationships in E. faecalis . In this study, we identified a group of E. faecalis strains lacking CRISPR2, forming a distinct, well-supported clade. We demonstrate that this clade meets the genomic criteria for classification as a novel subspecies, here referred to as “subspecies B”. Through a comprehensive pangenome analysis and comparative genomics, we explored the adaptive ecological traits underlying this diversification process, identifying clade-specific features and their predicted functional roles. Our findings suggest that the frequent isolation of subspecies B from meat products and processing facilities may reflect dissemination routes involving environmental contamination (e.g., water, plants, soil) from avian species. The absence of key virulence traits required for pathogenicity in mammals, particularly in humans, and the lack of clinically relevant resistance determinants indicate that subspecies B may currently pose minimal threat to public health compared to the broadly disseminated “subspecies A”. Nevertheless, the unclear potential for genetic exchange between these subspecies, and the frequent association of subspecies B with food sources, calls for continued genomic surveillance of E. faecalis from a One Health perspective to detect and mitigate the emergence of high-risk variants in advance.