Synergistic Antipersister, Efflux Inhibitory & Antibiofilm Activities of Vaginal Lactobacillus-Derived Postbiotics Against UPEC: Toward a Novel Therapeutic for UTIs

Bacterial persisters, phenotypic variants that can survive high doses of antibiotics, pose a significant challenge in treating chronic infections and contribute to post-treatment relapses. In this study, we investigated the impact of cell-free supernatant (CFS) from human vaginal Lactobacillus spp on the formation of E. coli UTI89 persister cells. Among various antibiotics tested, colistin exposure resulted in the highest percentage of persister cells, followed by meropenem, with ampicillin showing the least effect. Fractionation of the CFS via column chromatography yielded seven fractions, with fractions C4 and C7 demonstrating a synergistic inhibition of E. coli persisters. Gas chromatography-mass spectrometry (GC-MS) identified itaconic anhydride and (-)-terpinen-4-ol as the key metabolites responsible for this effect. When combined with colistin and meropenem, these compounds (8 µg/ml and 5 µg/ml, respectively) significantly reduced persister cell formation. The bactericidal action was found to be ROS-dependent, as evidenced by the reduced efficacy in the presence of thiourea, a hydroxyl-radical scavenger. Additionally, these metabolites increased membrane permeability and inhibited efflux pumps, further enhancing antibiotic efficacy. Building on these findings, we developed a novel postbiotic-based vaginal wash formulation incorporating (-)-terpinen-4-ol, itaconic anhydride, and tryptamine, another postbiotic metabolite with antibiofilm properties. These bioactive compounds, isolated from indigenous human vaginal Lactobacillus spp., were incorporated into a poloxamer 407-based formulation designed to prevent urinary tract infections (UTIs) and combat antimicrobial resistance. Preclinical studies conducted on BALB/c mice models validated the formulation's efficacy and safety, highlighting its potential for clinical translation. This approach of combining the antipersister, efflux inhibitory, and antibiofilm activities of these postbiotics within a practical therapeutic formulation offers a promising strategy for improving UTI management and combating the growing challenge of antibiotic tolerance.