Comparative Genomic and Phenotypic Profiling of Commercial Probiotic Enterococcus faecium Strains: Insights into Safety and Functional Attributes

Enterococcus faecium is widely used in probiotics but can also act as an opportunistic pathogen, necessitating strain-specific safety and efficacy assessments. This study conducted a comparative genomic and phenotypic analysis of five E. faecium strains isolated from commercial probiotic products alongside five reference strains. Whole-genome sequencing revealed that the probiotic-derived strains clustered into two distinct phylogenetic clades (Clade I: ST-812; Clade II: ST-76), corroborated by Average Nucleotide Identity and Multilocus Sequence Typing analyses. These clades exhibited coherent functional genomic profiles, with Clade I enriched in genes for carbohydrate metabolism and dietary fiber degradation, while Clade II showed a different emphasis, including carotenoid biosynthesis. Crucially, all probiotic strains lacked high-risk antibiotic resistance genes ( vanA ) and major virulence determinants ( esp , hyl , gelE ) found in the clinical control strain ATCC 51559, and were phenotypically susceptible to key antibiotics. In vitro probiotic property assays further demonstrated significant inter-strain variability in gastric acid tolerance, bile salt resistance, and bile salt hydrolase activity, underscoring the strain-specific nature of functional performance. Our integrated analysis establishes a robust link between genomic identity, functional potential, and safety profile in commercial E. faecium probiotics. This study provides a comprehensive genomic framework for strain-level safety assessment and supports the informed selection of E. faecium strains for probiotic applications.