From Lab to Life: Validating and Studying P. mirabilis’ Crystalline Biofilms in an in-vitro Bladder Model

From Lab to Life: Validating and Studying P. mirabilis’ Crystalline Biofilms in an in-vitro Bladder Model | 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 From Lab to Life: Validating and Studying P. mirabilis’ Crystalline Biofilms in an in-vitro Bladder Model Malshani Chathuranika Nissanka, Ayomi Dilhari, Gayan Priyadarshana, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8463469/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose: Proteus mirabilis forms crystalline biofilms that cause catheter encrustation and blockage. This study aimed to develop and validate an in vitro bladder model to study crystalline biofilms of P. mirabilis on urinary catheters. Methods: Double-walled glass vessel was used as a bladder model (BM), maintained at 37°C by circulating water. Artificial urine was supplied at 0.7 mL/min via a peristaltic pump, and a Foley catheter connected to a drainage bag formed a sterile closed system. Four catheterized BMs were connected in series. Model validation included calcium and magnesium leaching over 5 days and reproducibility based on catheter encrustation after 2 weeks. Artificial urine medium(AUM) was validated by comparing uropathogen growth with human urine. BM1 contained P. mirabilis–inoculated AUM (positive control), BM2 uninoculated AUM (negative control), and BM3–BM4 AUM inoculated with clinical P. mirabilis strains (n=6). Models were evaluated at 2,7, 14 days and until catheter blockage. Biofilm quantification and visualization were done using MTT, CV assays, scanning electron microscopy-energy dispersive X-ray analysis, atomic absorption spectroscopy. A quantitative PCR was performed to detect expression of biofilm-associated virulence genes. Results: All P. mirabilis strains formed strong biofilms after 14 days. The increase in catheter encrustation with calcium and magnesium over time was significant (p<0.05). SEM-EDX revealed biofilm architecture, microcolonies, calcium, and magnesium deposits. UreC, SpeA, MrpA, and RsbA genes showed significant expression after biofilm formation compared to initial stage. Conclusion:The developed bladder model provides a valid platform to study CAUTIs, biofilm formation, and develop novel approaches for controlling infections. Biological sciences/Microbiology Health sciences/Urology Proteus mirabilis Biofilms Encrustation Urinary Tract Infections Bladder model In vitro Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-8463469","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":573508505,"identity":"057f147b-289d-4eac-a4e0-b842b1390987","order_by":0,"name":"Malshani Chathuranika Nissanka","email":"","orcid":"","institution":"University of Sri Jayewardenepura","correspondingAuthor":false,"prefix":"","firstName":"Malshani","middleName":"Chathuranika","lastName":"Nissanka","suffix":""},{"id":573508507,"identity":"92825988-974f-4dae-8b8e-d5e3b64ce661","order_by":1,"name":"Ayomi 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Model","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Proteus mirabilis, Biofilms, Encrustation, Urinary Tract Infections, Bladder model, In vitro","lastPublishedDoi":"10.21203/rs.3.rs-8463469/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8463469/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePurpose: Proteus mirabilis forms crystalline biofilms that cause catheter encrustation and blockage. This study aimed to develop and validate an in vitro bladder model to study crystalline biofilms of P. mirabilis on urinary catheters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethods: Double-walled glass vessel was used as a bladder model (BM), maintained at 37°C by circulating water. Artificial urine was supplied at 0.7 mL/min via a peristaltic pump, and a Foley catheter connected to a drainage bag formed a sterile closed system. Four catheterized BMs were connected in series. Model validation included calcium and magnesium leaching over 5 days and reproducibility based on catheter encrustation after 2 weeks. Artificial urine medium(AUM) was validated by comparing uropathogen growth with human urine. BM1 contained P. mirabilis–inoculated AUM (positive control), BM2 uninoculated AUM (negative control), and BM3–BM4 AUM inoculated with clinical P. mirabilis strains (n=6). Models were evaluated at 2,7, 14 days and until catheter blockage. Biofilm quantification and visualization were done using MTT, CV assays, scanning electron microscopy-energy dispersive X-ray analysis, atomic absorption spectroscopy. A quantitative PCR was performed to detect expression of biofilm-associated virulence genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults: All P. mirabilis strains formed strong biofilms after 14 days. The increase in catheter encrustation with calcium and magnesium over time was significant (p\u0026lt;0.05). SEM-EDX revealed biofilm architecture, microcolonies, calcium, and magnesium deposits. UreC, SpeA, MrpA, and RsbA genes showed significant expression after biofilm formation compared to initial stage.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusion:The developed bladder model provides a valid platform to study CAUTIs, biofilm formation, and develop novel approaches for controlling infections.\u003c/p\u003e","manuscriptTitle":"From Lab to Life: Validating and Studying P. mirabilis’ Crystalline Biofilms in an in-vitro Bladder Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-13 15:04:52","doi":"10.21203/rs.3.rs-8463469/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8fbf1c6e-d2fb-423d-a6eb-d0272324e568","owner":[],"postedDate":"January 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61023358,"name":"Biological sciences/Microbiology"},{"id":61023359,"name":"Health sciences/Urology"}],"tags":[],"updatedAt":"2026-02-05T05:25:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-13 15:04:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8463469","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8463469","identity":"rs-8463469","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}