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

Synergistic Antipersister, Efflux Inhibitory & Antibiofilm Activities of Vaginal Lactobacillus-Derived Postbiotics Against UPEC: Toward a Novel Therapeutic for UTIs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synergistic Antipersister, Efflux Inhibitory & Antibiofilm Activities of Vaginal Lactobacillus-Derived Postbiotics Against UPEC: Toward a Novel Therapeutic for UTIs Veena G Nair, David Raj Chellappan, Ramya Devi Durai, Rajesh Y.B.R.D, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8039921/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract 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. Biological sciences/Drug discovery Biological sciences/Microbiology Uropathogenic E coli persisters Vaginal probiotics Cell-free supernatant Postbiotics Anti-persister potential Customised vaginal wash Preclinical validation Full Text Additional Declarations No competing interests reported. Supplementary Files MMATSupplementary5Nov.docx Cite Share Download PDF Status: Published Journal Publication published 10 Jan, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 05 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviews received at journal 28 Nov, 2025 Reviewers agreed at journal 27 Nov, 2025 Reviewers agreed at journal 23 Nov, 2025 Reviewers agreed at journal 20 Nov, 2025 Reviewers agreed at journal 18 Nov, 2025 Reviewers invited by journal 18 Nov, 2025 Editor assigned by journal 17 Nov, 2025 Editor invited by journal 13 Nov, 2025 Submission checks completed at journal 12 Nov, 2025 First submitted to journal 12 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8039921","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":549419053,"identity":"5294e3e9-b882-4286-8694-242d5fd76f0e","order_by":0,"name":"Veena G Nair","email":"","orcid":"","institution":"SASTRA Deemed to be University","correspondingAuthor":false,"prefix":"","firstName":"Veena","middleName":"G","lastName":"Nair","suffix":""},{"id":549419055,"identity":"235ac06a-7655-4802-9535-b0927f37e8e0","order_by":1,"name":"David Raj Chellappan","email":"","orcid":"","institution":"SASTRA Deemed to be 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UTIs","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Uropathogenic E coli persisters, Vaginal probiotics, Cell-free supernatant, Postbiotics, Anti-persister potential, Customised vaginal wash, Preclinical validation","lastPublishedDoi":"10.21203/rs.3.rs-8039921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8039921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacterial 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 \u003cem\u003eLactobacillus\u003c/em\u003e spp on the formation of \u003cem\u003eE. coli\u003c/em\u003e 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 \u003cem\u003eE. coli\u003c/em\u003e 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 \u0026micro;g/ml and 5 \u0026micro;g/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 \u003cem\u003eLactobacillus\u003c/em\u003e 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.\u003c/p\u003e","manuscriptTitle":"Synergistic Antipersister, Efflux Inhibitory \u0026amp; Antibiofilm Activities of Vaginal Lactobacillus-Derived Postbiotics Against UPEC: Toward a Novel Therapeutic for UTIs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 12:06:29","doi":"10.21203/rs.3.rs-8039921/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-05T09:33:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-03T10:30:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T05:54:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267234717803671911411666212598515273151","date":"2025-11-27T06:22:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300555891025336572446629921464878582354","date":"2025-11-23T14:42:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"40775048854701627481472544240948003873","date":"2025-11-20T05:17:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"147871160017603943053340080013246846111","date":"2025-11-18T05:22:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-18T05:10:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-18T04:58:21+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-13T14:16:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T09:21:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-12T09:16:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7cca4fd2-6362-4902-81bf-2ea9206da5e1","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58458180,"name":"Biological sciences/Drug discovery"},{"id":58458182,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-01-12T16:16:22+00:00","versionOfRecord":{"articleIdentity":"rs-8039921","link":"https://doi.org/10.1038/s41598-026-35736-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-01-10 15:59:03","publishedOnDateReadable":"January 10th, 2026"},"versionCreatedAt":"2025-11-26 12:06:29","video":"","vorDoi":"10.1038/s41598-026-35736-7","vorDoiUrl":"https://doi.org/10.1038/s41598-026-35736-7","workflowStages":[]},"version":"v1","identity":"rs-8039921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8039921","identity":"rs-8039921","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}