Saisubramanian Nagarajan

Background: Recurrent urinary tract infections(rUTI) represent a major clinical challenge due to persistent clinical symptoms, repeated antibiotic exposure, and increased risk of multidrug resistance. Further clinical management of rUTI remains challenging, as existing diagnostic and treatment guidelines are largely designed for uncomplicated, acute infections. Though uropathogenic Escherichia coli (UPEC) is the predominant cause of community-acquired UTIs, pathogen-derived genomic features that may predispose certain E. coli strains to repeatedly establish infection are not fully understood. Methods: To comprehensively dissect distinct genetic signals across genomic compartments that distinguish rUTI-associated isolates from those causing sporadic infection, the pangenome analysis in three different frameworks (i) Combined genomes (chromosome + plasmid), (ii) bacterial chromosomes only and (iii) plasmid-only was conducted. A comprehensive evaluation of population structure was performed using Gubbins, recombination-aware phylogeny IQTree, phylogroup distribution, pan-genome openness using Heap’s law, and plasmidome architecture using MOBSUITE. Findings: Supervised machine learning models showed that the highest discriminatory performance was achieved using the combined genomic dataset (accuracy ∼0.98), and integration of feature-selected genes with PanGWAS (Pyseer and Scoary) identified a robust set of recurrence-associated genes, namely cbtA, cbeA , and ldrD , which were consistently detected across machine learning and association frameworks. Subsequent association rule mining further revealed cooperative gene networks enriched in rUTI isolates, particularly involving toxin-antitoxin modules and metabolic regulators. Interpretation: Overall, this integrated ML-PanGWAS approach demonstrates that rUTI is a lineage-independent, polygenic phenotype encoded within a combined chromosomal-plasmid genomic context, providing new insights into the bacterial genomic architecture underlying recurrent disease and offering candidate biomarkers for future diagnostic and therapeutic development. Funding: The Department of Biotechnology (DBT), Government of India (grant numbers: BT/PR40150/BTIS/137/81/2023 and the SASTRA TRR grant (SASTRA TRR SCBT OCT-23). Evidence before this study: We searched PubMed, Scopus, and Web of Science for studies published from database inception to March 2026, without language restrictions, using combinations of the terms “recurrent urinary tract infection”, “uropathogenic Escherichia coli ”, “pan-genome”, “genome-wide association”, “plasmid”, and “machine learning”. We included studies investigating genomic, phylogenetic, or functional differences between recurrent and sporadic UTI isolates. Previous studies have primarily used phylogenetic and single nucleotide polymorphism (SNP)-based approaches and reported limited genomic differentiation, with no consistent clustering or robust gene-level associations. Overall, existing evidence is heterogeneous and largely limited to single-layer genomic analyses. Added value of this study: This study integrates pan-genome analysis across chromosomal, plasmid, and combined datasets with machine learning, genome-wide association analysis, and association rule mining. In contrast to previous SNP-based studies, we show that recurrent UTI isolates can be robustly discriminated only when chromosomal and plasmid features are analysed jointly. We identify reproducible recurrence-associated genes, including cbtA, cbeA , and klcA , and demonstrate that these genes form cooperative networks involving persistence, plasmid-mediated transfer, and metabolic adaptation, supporting a polygenic basis of recurrence. Implications of all the available evidence: Our findings indicate that recurrent UTI is not driven by lineage alone but by coordinated accessory gene networks spanning chromosomal and plasmid compartments. Although certain sequence types are more frequently associated with recurrence, they are not exclusive and likely serve as backgrounds for enriched gene modules. These results highlight the importance of integrated genomic profiling for predicting recurrence risk and identifying persistence and adaptation pathways as potential targets for future diagnostic and therapeutic strategies.

Introduction: Acinetobacter baumannii is a highly problematic nosocomial pathogen, designated a “red alert” organism by the Infectious Diseases Society of America. Multidrug-resistant (MDR) strains are associated with mortality rates of 25–68%, particularly in critically ill patients. Efflux-mediated resistance, along with serum-induced upregulation of efflux pump genes, highlights the role of efflux systems in driving MDR phenotypes. Targeting these systems represents a promising therapeutic strategy. Methods: The efflux inhibitory potential of 13 plant-derived polyphenols was evaluated to restore antibiotic susceptibility in MDR/XDR clinical isolates of A. baumannii . Approaches included in silico molecular docking and molecular dynamics simulations, in vitro assays (MIC reversal, real-time efflux, checkerboard synergy, intracellular accumulation, membrane permeability, and ROS generation), and in vivo bioburden analysis using a zebrafish infection model. Results and Discussion: Gallotannin exhibited the strongest activity, achieving a 64-fold reduction in erythromycin MIC in an XDR strain. Efflux inhibition was confirmed through real-time efflux and accumulation assays, showing 32–64-fold MIC reductions across multiple antibiotic classes in five MDR/XDR isolates. Molecular simulations revealed stable binding of gallotannin to the AdeB efflux pump via persistent hydrogen bonding. Combination therapy resulted in a ∼5-log CFU reduction in vitro and ∼4-log reduction in zebrafish bioburden. Mechanistically, gallotannin enhanced membrane permeability and intracellular antibiotic accumulation, without affecting membrane potential. The combination also induced significantly elevated ROS levels.Gallotannin potentiates erythromycin activity by inhibiting efflux, increasing permeability, and promoting ROS-mediated killing, significantly reducing A. baumannii burden in vitro and in vivo .

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 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.

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.

Probiotics present a promising preventive strategy for addressing urinary tract infections (UTIs) and serve as a viable alternative to conventional antimicrobial treatments. The integration of Lactobacillus-derived efflux pump inhibitors (EPIs) with conventional antibiotics presents a promising strategy to enhance the efficacy of treatments against antibiotic-resistant superbugs. In this study, we isolated eight vaginal Lactobacillus spp. from 54 healthy Indian women to explore their probiotic properties, specifically the ability of cell-free supernatant (CFS) from Lactobacillus jensenii. to inhibit efflux in Multi Drug Resistant (MDR) E. coli and K. pneumoniae. From the CFS, we obtained seven fractions, among which compound 7 (C7) markedly reduced the MIC of erythromycin against the K. pneumoniae MTCC 432 strain by 32-fold from 64 to 2 µg and restored its sensitivity, indicating potent efflux inhibitory activity. Gas Chromatography-Mass Spectrometry (GC-MS) analysis identified one major compound from the C7 fraction as Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (R)-, also known as (-)-Terpinen-4-ol. In vivo studies using zebrafish infected with clinical strain of K. pneumoniae 1845 followed by treatment with (-)-Terpinen-4-ol and erythromycin caused a significant 7-log reduction in bacterial bioburden. Additionally, time-kill assay demonstrated that the combination of erythromycin with (-)-Terpinen-4-ol caused a remarkable 7-log reduction in both K. pneumoniae, and E. coli cell counts. Furthermore, (-)-Terpinen-4-ol -erythromycin combination exhibited a six-log reduction in bacterial burden in infected T24 bladder cell lines and was found to be non-toxic. This study underscores the efflux inhibitory potential of (-)-Terpinen-4-ol extracted from the CFS of human vaginal probiotics, paving the way for future pharmacological research and therapeutic applications.

Probiotics present a promising preventive strategy for addressing urinary tract infections (UTIs) and serve as a viable alternative to conventional antimicrobial treatments. The integration of Lactobacillus-derived efflux pump inhibitors (EPIs) with conventional antibiotics presents a promising strategy to enhance the efficacy of treatments against antibiotic-resistant superbugs. In this study, we isolated eight vaginal Lactobacillus spp. from 54 healthy Indian women to explore their probiotic properties, specifically the ability of cell-free supernatant (CFS) from Lactobacillus jensenii. to inhibit efflux in MDR E. coli and K. pneumoniae. From the CFS, we obtained seven fractions, among which compound 7 (C7) markedly reduced the MIC of erythromycin against the K. pneumoniae MTCC 432 strain by 32-fold from 64 µg to 2 µg and restored its sensitivity, indicating potent efflux inhibitory activity. Gas Chromatography-Mass Spectrometry (GC-MS) analysis identified one major compound from the C7 fraction as Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-, (R)-, also known as (-)-Terpinen-4-ol. In-vivo studies using zebrafish infected with clinical strain of K. pneumoniae 1845 followed by treatment with (-)-Terpinen-4-ol and erythromycin caused a significant 7-log reduction in bacterial bioburden.. Additionally, time-kill assay demonstrated that the combination of erythromycin with (-)-Terpinen-4-ol caused a remarkable 7-log reduction in both K. pneumoniae, and E. coli cell counts. Furthermore, (-)-Terpinen-4-ol -erythromycin combination exhibited a six-log reduction in bacterial burden in infected T24 bladder cell lines and was found to be non-toxic. This study underscores the efflux inhibitory potential of (-)-Terpinen-4-ol extracted from the CFS of human vaginal probiotics, paving the way for future pharmacological research and therapeutic applications.

The rising prevalence of antimicrobial resistance has intensified the search for innovative therapeutic strategies, particularly in the prevention of urinary tract infections (UTIs). This study presents the development and evaluation of a novel postbiotics vaginal wash formulated to prevent UTIs by utilizing metabolites derived from indigenous vaginal Lactobacillus spp. The primary objective was to create a cost-effective, stable, and non-invasive solution targeting uropathogenic bacteria. Key metabolites, including tryptamine, (-)-terpinen-4-ol, and itaconic anhydride, were identified from cell free supernatant of vaginal Lactobacillus and incorporated into a poloxamer 407-based formulation. In vitro assays demonstrated significant bioactivity against uropathogenic bacteria, effectively inhibiting bacterial colonization and biofilm formation. Preclinical validation was conducted using BALB/c mice models to assess both the safety and efficacy of the vaginal wash. Results indicated a substantial reduction in infection rates among treated mice, with no observed adverse effects, confirming the formulation’s safety profile. In conclusion, this novel postbiotics vaginal wash represents a promising non-invasive therapeutic approach for UTI prevention. By harnessing bioactive metabolites from human vaginal Lactobacillus spp., the formulation offers a potential solution to combat antimicrobial resistance while improving women’s health outcomes. Further clinical studies are warranted to validate these findings and explore broader applications in clinical practice, paving the way for new strategies in managing UTIs and enhancing overall female health.