Vaginal Probiotic Potential of Lactobacillus acidophilus: Population Genomic and Phenotypic Analysis

Background/Objectives: Certain strains of Lactobacillus acidophilus are widely used as probiotics. However, their functional potential for female reproductive tract health remains insufficiently characterized. While most studies have focused on individual strains, the distribution of putative probiotic-associated genes across the species remains unclear. This study aimed to evaluate the vaginal probiotic potential of Lb. acidophilus using population genomic analysis and comparative phenotypic characterization. Methods: Pan-genomic analysis was performed on 109 Lb. acidophilus genomes (107 public genomes and 2 vaginal isolates). Putative probiotic-associated gene clusters were identified by functional annotation, categorized into functional modules, and compared among ecological-origin groups. Two vaginal isolates (strains A2 and A3) were characterized in vitro for growth under different pH conditions, cell surface hydrophobicity, lactic acid and hydrogen peroxide production, antimicrobial activity, hemolysis, and antimicrobial susceptibility. Results: The Lb. acidophilus pan-genome was closed, with 1782 of 1902 gene clusters (93.69%) classified as core. Thirty-six putative probiotic-associated gene clusters were identified and grouped into four modules: environmental tolerance, adhesion/colonization, exopolysaccharide/biofilm synthesis, and nutrient metabolism/microbial competition. Thirty-four of the 36 gene clusters were present in all 109 genomes, and no general ecological origin-specific distribution pattern was observed. Three bacteriocin-related gene clusters were conserved across all genomes. A2 and A3 exhibited similar lactic acid production and growth patterns at pH 4–6. Both produced relatively low amounts of hydrogen peroxide compared with the reference strains. A3 showed higher cell surface hydrophobicity and moderate inhibition against Gardnerella vaginalis, while A2 showed no inhibition of this organism. Both isolates were non-hemolytic, and were susceptible to vancomycin and linezolid, resistant to clindamycin, and non-susceptible to daptomycin. No acquired antibiotic resistance genes were detected. Conclusions: Most putative probiotic-associated gene clusters were conserved across the Lb. acidophilus population, whereas A2 and A3 showed strain-dependent phenotypic differences. These findings support combining population genomic analysis with strain-level phenotypic testing when selecting Lb. acidophilus candidates for bacterial vaginal infections.