Comprehensive Genomic Characterization and Probiotic Properties Evaluation of Limosilactobacillus fermentum JAC 231 Isolated from Vaginal Microbiota.

Limosilactobacillus fermentum JAC 231, a strain isolated from the vaginal microbiota of an asymptomatic woman, was investigated for its genomic features, safety, probiotic potential, and antimicrobial properties in this study. Whole-genome sequencing revealed a 2.06 Mbp genome with 51.4% GC content and 1,993 predicted coding sequences (CDS). Functional annotation indicated that the genes were related to carbohydrate metabolism, stress tolerance, adhesion, and bacteriocin biosynthesis. Comparative analysis revealed that L. fermentum JAC 231 shared 1,587 orthologous groups with the other five probiotic strains of L. fermentum. No virulence factors or transferable antibiotic resistance genes were detected, and only an incomplete prophage region was identified, which supports the safety of this strain. Phenotypically, JAC 231 exhibited tolerance to acidic pH and bile salts and demonstrated high adhesion capacity to porcine gastric mucin (5.97 log₁₀ CFU/mL) and HeLa epithelial cells (5.79 log₁₀ CFU/mL). Antimicrobial susceptibility testing indicated resistance to vancomycin and moderate susceptibility to ciprofloxacin, features considered intrinsic and nontransferable. The strain exhibited broad-spectrum antimicrobial activity, with inhibition zones ranging from 16.60 ± 2.08 mm to 28.30 ± 2.88 mm against various Gram-positive and Gram-negative pathogens. Agar diffusion assays indicated that the antimicrobial activity of the cell-free supernatant was primarily due to the production of organic acids. Furthermore, JAC 231 significantly reduced pathogen adherence to epithelial cells in competitive, exclusion, and displacement assays. Collectively, these genomic and functional attributes support the safety and probiotic potential of L. fermentum JAC 231 and underscore its promise as a candidate for therapeutic applications targeting the urogenital and gastrointestinal microbiota. The online version contains supplementary material available at 10.1007/s12602-026-11025-7.