Antimicrobial and Anti-inflammatory Activities of Bacteriocin-like Inhibitory Substance-producing Lactiplantibacillus plantarum against Vaginitis-associated Pathogens.

Vaginal dysbiosis is characterized by disruption of the vaginal microbial community, pathogen overgrowth, and mucosal inflammation. Here, we integrated in vitro screening, whole-genome sequencing, LC-MS/MS profiling, machine learning, and a murine bacterial vaginosis (BV) model to characterize potentially beneficial Lactiplantibacillus plantarum strains. Among 88 Lactobacillaceae isolates, 53 exhibited bacteriocin-like inhibitory substance activity, and 7 demonstrated broad antimicrobial effects against Gardnerella vaginalis, Neisseria gonorrhoeae, Streptococcus agalactiae, and Candida spp. Two L. plantarum strains, CYLB2146 and CYLB2189, were selected based on their antimicrobial activity, adhesion capacity (15.0% and 13.5%, respectively), and significant anti-inflammatory effects, including reductions in IL-6, IL-8, and TNF-α levels (p < 0.05). Genomic analysis revealed plantaricin biosynthetic gene clusters and secondary metabolite pathways, while LC-MS/MS identified predominantly low-molecular-weight and cyclic peptides. Machine-learning models achieved ROC-AUC > 0.92 and prioritized candidate peptides. Cyclo(Tyr-Pro) inhibited multiple pathogens and reduced biofilm formation at sub-inhibitory concentrations without membrane disruption, supporting a potential anti-virulence role. In a murine BV model, probiotic treatment markedly reduced G. vaginalis compared with PBS and MRS controls (51.2 and 41.6-fold). After 5 days, levels decreased to 3.2 and 11.1-fold in live CYLB2146 and CYLB2189 groups and to 3.2 and 0.7-fold in CFS groups, with further reductions by day 10, while controls remained high (34.0 and 50.6-fold). Probiotic treatment also attenuated infection-associated inflammation, particularly TNF-α, and improved vaginal tissue morphology and histological scores. Collectively, CYLB2146 and CYLB2189 exhibited complementary antimicrobial, antibiofilm, and anti-inflammatory activities, supporting their potential as probiotic candidates for BV management.