Antagonistic Activity, Genomic Characterization, and Probiotic Potential of Clonal Enterococcus faecalis Strains Isolated from Human Breast Milk: An In Vitro and In Silico Study

The objective of this study was to characterize four clonal Enterococcus faecalis strains isolated from human breast milk through in vitro and in silico analyses and to evaluate their microbiological, antagonistic, molecular, genomic, and potential probiotic properties. The four isolates were Gram-positive cocci, negative for catalase, oxidase, and hemolytic activity, and showed phenotypic resistance to cephalothin, gentamicin, and tetracycline. The strains showed antagonistic and lytic activity against Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. Molecular identification by 16S rRNA sequencing and average nucleotide identity (ANI) analysis confirmed species assignment and genomic similarity among the isolates, with ANI values exceeding 99%. Whole-genome analysis identified bacteriocin-associated genes, including Enterocin_96 and Enterolysin_A, as well as ireK, efrA, and dfrE, with no evidence of vancomycin resistance genes. The analysis also revealed two virulence-associated genes (asa1 and gelE) and few mobile genetic elements. The strains showed tolerance under simulated gastrointestinal conditions, maintaining counts of ≥7 log10 CFU/mL after 4 h from an initial inoculum of approximately 9 log10 CFU/mL, in addition to exhibiting mucin adherence and biofilm-forming capacity. This study provides further evidence for the presence of E. faecalis in the human breast milk microbiota. In addition, the results provide phenotypic, functional, and genomic information relevant to the evaluation of human breast milk-derived E. faecalis strains in the context of probiotic research.