Probiotic potential of Enterococcus faecium ED22: evidence from genomic analysis and in vitro characterization

Abstract Enterococcus species are ubiquitous members of the human microbiota, but their potential to act as opportunistic pathogens requires careful, strain-specific safety assessment. This study characterized Enterococcus faecium ED22 using a polyphasic approach combining whole-genome sequencing (WGS) with in vitro phenotypic assays. The strain was initially identified by MALDI-TOF/MS and subsequently confirmed by WGS. Genomic analyses included genome assembly and annotation, antimicrobial resistance (AMR) and virulence-associated gene screening, plasmid characterization. Functional characterization included antibacterial and antifungal activity, DPPH radical-scavenging activity, gastrointestinal tolerance, and aggregation assays, while hemolytic and gelatinase activities were evaluated as safety-related traits. ED22 exhibited detectable antibacterial activity against Staphylococcus aureus , Escherichia coli , and Pseudomonas aeruginosa , with proteinase K-sensitive activity suggesting a contribution of proteinaceous antimicrobial compounds. Antifungal activity was observed against Trichophyton rubrum , but not Candida albicans . Fermented milk containing ED22 showed DPPH radical-scavenging activity of 66.23 ± 0.38%, together with measurable phenolic and flavonoid contents. ED22 remained viable under simulated gastrointestinal conditions and exhibited auto-aggregation and co-aggregation properties. The strain was non-hemolytic and gelatinase-negative. Genomic screening detected no major acquired vancomycin resistance determinants, key virulence genes, or plasmid-borne AMR genes. A RiPP biosynthetic cluster displayed 66% similarity to enterocin A. These findings support ED22 as a preliminary candidate for probiotic development, though further MIC profiling, genomic validation, plasmid transferability assessment, and in vivo efficacy trials remain essential.