Comprehensive In Vitro Evaluation of Antimicrobial Combinations Against Extensively Drug- Resistant Acinetobacter baumannii: Planktonic and Biofilm Perspectives

Comprehensive In Vitro Evaluation of Antimicrobial Combinations Against Extensively Drug- Resistant Acinetobacter baumannii: Planktonic and Biofilm Perspectives | 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 Research Article Comprehensive In Vitro Evaluation of Antimicrobial Combinations Against Extensively Drug- Resistant Acinetobacter baumannii: Planktonic and Biofilm Perspectives Ana Paula Andrade, Paula Hansen Suss, Victoria Stadler, Felipe Francisco Tuon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9295991/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 Introduction: Acinetobacter baumannii is a major cause of healthcare-associated infections due to its multidrug resistance (MDR), biofilm-forming capacity, and environmental persistence. Resistance is exacerbated in biofilm-associated cells, requiring combination therapies for effective treatment. Objectives: To evaluate the in vitro antimicrobial activity and synergistic potential of clinically relevant antibiotics—doxycycline, meropenem, polymyxin B, amikacin, and gentamicin—against XDR A. baumannii isolates, with a focus on biofilm-associated resistance. Methods: Twenty clinical isolates were collected from two hospitals. Susceptibility testing was performed by broth microdilution. The presence of the blaOXA-23 gene was confirmed via qPCR. Checkerboard and time-kill assays assessed synergy. Biofilm formation on polyurethane was evaluated using crystal violet, MTT, MBEC assays, and scanning electron microscopy. Clonality was analyzed using Fourier-transform infrared spectroscopy (FTIR). Results: All isolates carried blaOXA-23 and were resistant to carbapenems. High resistance rates to gentamicin (85%) and amikacin (95%) were observed. Polymyxin B retained 100% activity. Doxycycline showed 45% susceptibility (MIC ≤1 mg/L). Synergy was detected in seven doxycycline-aminoglycoside combinations. In biofilm models, strong biomass formation persisted despite antimicrobial exposure. Doxycycline + gentamicin showed >2 log₁₀ CFU/cm² reduction in viable biofilm cells and enhanced killing in time-kill assays. FTIR identified 15 clonal clusters, indicating potential horizontal transmission. Conclusion: Doxycycline in combination with gentamicin demonstrated promising synergistic activity against XDR A. baumannii , including biofilm-associated cells. However, biofilm tolerance remains a therapeutic barrier. These findings support the consideration of combination therapies and biofilm-targeted evaluations in managing MDR infections. Further in vivo studies are warranted. Acinetobacter baumannii biofilm doxycycline gentamicin polymyxin B. Full Text Additional Declarations No competing interests reported. Table 1 and 2 are available in the Supplementary Files section. Supplementary Files Tables.docx Supplementarymaterial.docx 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-9295991","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":616578236,"identity":"f2b76a3c-387d-4374-b54a-41991ca7a36a","order_by":0,"name":"Ana Paula Andrade","email":"","orcid":"","institution":"Pontifícia Universidade Católica do Paraná","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Paula","lastName":"Andrade","suffix":""},{"id":616578237,"identity":"b931052f-6ae3-40b5-84f0-55d533180ba3","order_by":1,"name":"Paula Hansen Suss","email":"","orcid":"","institution":"Pontifícia Universidade Católica do Paraná","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"Hansen","lastName":"Suss","suffix":""},{"id":616578238,"identity":"6979bdcd-bc99-45cf-9e60-8502f16a9666","order_by":2,"name":"Victoria Stadler","email":"","orcid":"","institution":"Pontifícia Universidade Católica do Paraná","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"","lastName":"Stadler","suffix":""},{"id":616578239,"identity":"9a4ccdf4-73ee-433d-be2c-cff49a4d2bce","order_by":3,"name":"Felipe Francisco Tuon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBAC9gbmhgNQNuMDCM2DXwvPAUawFgkgZjYgWgsDVAubBHFa2BsbDxdUMNQZHO89Vl3YZpdn3sB77ANeLTwHGw7POMMgYXDmXNrtmW3JxTIH+JJn4NNiL5HYcJi3DajlRo7Zbd62A4kzGHiM8TtM/iFQyz+IlmLitEgwArU0QLQwE6eFB+iwGcckJGeeOWMsPeNccrEEM18yfi3shw9/Lqix4ec73mP4uaDMLk+CvfcwXi0gwAyJFjCDIQFMEqEFwUggQsMoGAWjYBSMMAAALapEH8YL460AAAAASUVORK5CYII=","orcid":"","institution":"Pontifícia Universidade Católica do Paraná","correspondingAuthor":true,"prefix":"","firstName":"Felipe","middleName":"Francisco","lastName":"Tuon","suffix":""}],"badges":[],"createdAt":"2026-04-01 21:08:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9295991/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9295991/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106405615,"identity":"a3e57d8d-c1f6-4de2-9ec1-c4cc38676296","added_by":"auto","created_at":"2026-04-08 09:27:45","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":838312,"visible":true,"origin":"","legend":"","description":"","filename":"JAC3500.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9295991/v1_covered_169525c9-af82-488a-a992-63aa009a00c9.pdf"},{"id":106403909,"identity":"4648e93a-39c0-437f-b60f-820f6733aab8","added_by":"auto","created_at":"2026-04-08 09:15:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23850,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9295991/v1/1c6dd07f02103f7ce94004ae.docx"},{"id":106284668,"identity":"f9ab69d8-6545-4001-a81b-16df00951f6b","added_by":"auto","created_at":"2026-04-07 06:49:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1691376,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9295991/v1/a7860ba9f0d9e085c4372187.docx"}],"financialInterests":"\u003cp\u003eNo competing interests reported.\u003c/p\u003e\n\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e","formattedTitle":"Comprehensive In Vitro Evaluation of Antimicrobial Combinations Against Extensively Drug- Resistant Acinetobacter baumannii: Planktonic and Biofilm Perspectives","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Acinetobacter baumannii, biofilm, doxycycline, gentamicin, polymyxin B.","lastPublishedDoi":"10.21203/rs.3.rs-9295991/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9295991/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction: \u003c/strong\u003e\u003cem\u003eAcinetobacter baumannii\u003c/em\u003e is a major cause of healthcare-associated infections due to its multidrug resistance (MDR), biofilm-forming capacity, and environmental persistence. Resistance is exacerbated in biofilm-associated cells, requiring combination therapies for effective treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eTo evaluate the in vitro antimicrobial activity and synergistic potential of clinically relevant antibiotics—doxycycline, meropenem, polymyxin B, amikacin, and gentamicin—against XDR \u003cem\u003eA. baumannii\u003c/em\u003e isolates, with a focus on biofilm-associated resistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eTwenty clinical isolates were collected from two hospitals. Susceptibility testing was performed by broth microdilution. The presence of the blaOXA-23 gene was confirmed via qPCR. Checkerboard and time-kill assays assessed synergy. Biofilm formation on polyurethane was evaluated using crystal violet, MTT, MBEC assays, and scanning electron microscopy. Clonality was analyzed using Fourier-transform infrared spectroscopy (FTIR).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAll isolates carried blaOXA-23 and were resistant to carbapenems. High resistance rates to gentamicin (85%) and amikacin (95%) were observed. Polymyxin B retained 100% activity. Doxycycline showed 45% susceptibility (MIC ≤1 mg/L). Synergy was detected in seven doxycycline-aminoglycoside combinations. In biofilm models, strong biomass formation persisted despite antimicrobial exposure. Doxycycline + gentamicin showed \u0026gt;2 log₁₀ CFU/cm² reduction in viable biofilm cells and enhanced killing in time-kill assays. FTIR identified 15 clonal clusters, indicating potential horizontal transmission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eDoxycycline in combination with gentamicin demonstrated promising synergistic activity against XDR \u003cem\u003eA. baumannii\u003c/em\u003e, including biofilm-associated cells. However, biofilm tolerance remains a therapeutic barrier. These findings support the consideration of combination therapies and biofilm-targeted evaluations in managing MDR infections. Further in vivo studies are warranted.\u003c/p\u003e","manuscriptTitle":"Comprehensive In Vitro Evaluation of Antimicrobial Combinations Against Extensively Drug- Resistant Acinetobacter baumannii: Planktonic and Biofilm Perspectives","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 06:49:08","doi":"10.21203/rs.3.rs-9295991/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":"45d969a5-8fd0-43df-ba96-10037bba2390","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T06:49:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 06:49:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9295991","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9295991","identity":"rs-9295991","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}