Author ORCID Identifier

https://orcid.org/0009-0002-3008-0526

Date of Award

8-1-2025

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.N.Saisubramanian

Keywords

Urinary Tract Infections, Human Vaginal Lactobacillus, Postbiotics, Biofilm, Efflux Inhibition, Antipersister

Abstract

Probiotics, which support host health by maintaining normal microbial flora, offer a promising approach for preventing recurrent infections. To evaluate the prophylactic potential of vaginal probiotics against urinary tract infections (UTIs), we conducted a study on 54 healthy South Indian women aged 18–40 who visited the Obstetrics and Gynaecology outpatient clinic at Trichy SRM Medical College and Research Centre between June and July 2022. Vaginal swabs were collected following ethical guidelines, and commensal microbiota were isolated for probiotic characterization, with the broader objective of developing a customized feminine hygiene wash enriched with vaginal postbiotics.

Lactobacilli cell-free supernatant (CFS) demonstrated potent antibiofilm activity against Uropathogenic Escherichia coli (UPEC). Tryptamine, a major bioactive compound identified in the CFS, significantly inhibited UPEC biofilm formation by disrupting bacterial motility, reducing cell surface hydrophobicity, and impairing extracellular matrix production. Structural analyses confirmed alterations in biofilm morphology, along with reduced bacterial viability and increased reactive oxygen species (ROS) generation. Gene expression studies further supported these findings by showing downregulation of key biofilm-associated genes, including fimA, fimH, papG, and csgA.

In addition to antibiofilm effects, the study explored the efflux pump inhibitory potential of metabolites produced by Lactobacillus jensenii. Seven compounds were isolated from the CFS, among which Compound 7 (C7) exhibited strong fluorescence under UV light. MIC reversal assays demonstrated that C7 significantly enhanced erythromycin susceptibility in Klebsiella pneumoniae, reducing the MIC from 512 μg/mL to 8 μg/mL. GC–MS analysis identified C7 as (–)-Terpinen-4-ol, an isomer of terpineol.

The effectiveness of (–)-Terpinen-4-ol was further validated in vivo using zebrafish infection models, where its combination with erythromycin substantially lowered bacterial bioburden in resistant K. pneumoniae strains. The study also investigated how Lactobacillus-derived metabolites influence antibiotic persistence in UPEC. Antibiotics such as colistin and meropenem increased persister cell formation, but metabolites including itaconic anhydride and (–)-Terpinen-4-ol significantly reduced persistence by enhancing membrane permeability and elevating ROS levels, thereby improving antimicrobial penetration.

Overall, the findings highlight the strong antibiofilm, efflux inhibitory, and antipersister properties of Lactobacillus-derived metabolites, underscoring their potential role in preventing UTIs. In vivo validation using BALB/c mice models demonstrated that the formulated feminine hygiene wash significantly reduced urinary tract colonization and biofilm formation. These results support the development of a postbiotic-based vaginal wash as an effective preventative strategy against UTIs.

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