Phenotypic and genomic features shaping the activity of a novel T4-like phage against clinical multidrug-resistant Klebsiella pneumoniae

Phenotypic and genomic features shaping the activity of a novel T4-like phage against clinical multidrug-resistant Klebsiella pneumoniae | 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 Phenotypic and genomic features shaping the activity of a novel T4-like phage against clinical multidrug-resistant Klebsiella pneumoniae Danielle Devequi Gomes Nunes, Nicole Hitchcock, Carolina de Araújo Rolo, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8388477/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background The use of bacteriophages holds a great promise in the treatment of multidrug resistant bacteria. The interaction between bacteriophages and multidrug-resistant Klebsiella pneumoniae is shaped by extensive capsular diversity, accessory genome variation and multilayered anti-phage defense systems, resulting in highly variable susceptibility profiles. Here, we describe MB01, a lytic T4-like phage isolated from wastewater in Brazil and characterize its biological properties and activity against MDR K. pneumoniae . Results MB01 showed a short replication cycle, strong bacterial suppression at low MOIs and high stability across physiological temperature and pH ranges. Among clinical isolates, MB-01 was effective for K. pneumoniae isolates. Whole genome sequencing revealed complex resistomes, heterogeneous accessory genes and marked differences in defense systems and prophage content. Defense-enriched strains were fully resistant to MB01, while permissive isolates carried fewer defenses, indicating that genomic background strongly shapes phage outcome. Conclusions These findings highlight the value of integrating phenotypic and genomic analyses for rational phage selection and support the development of targeted phage libraries to address MDR K. pneumoniae infections. Klebsiella pneumoniae bacteriophages phage therapy Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Feb, 2026 Reviews received at journal 17 Feb, 2026 Reviews received at journal 09 Feb, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 12 Jan, 2026 Reviewers invited by journal 05 Jan, 2026 Editor assigned by journal 24 Dec, 2025 Submission checks completed at journal 23 Dec, 2025 First submitted to journal 23 Dec, 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. 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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-8388477","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":569783807,"identity":"f8fd1bc9-853c-4e79-b221-e330b793ac8b","order_by":0,"name":"Danielle Devequi Gomes Nunes","email":"","orcid":"","institution":"SENAI CIMATEC University","correspondingAuthor":false,"prefix":"","firstName":"Danielle","middleName":"Devequi Gomes","lastName":"Nunes","suffix":""},{"id":569783810,"identity":"86c35555-6dc2-4b77-8c49-11ce3e831137","order_by":1,"name":"Nicole Hitchcock","email":"","orcid":"","institution":"University of California San 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pneumoniae","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella pneumoniae, bacteriophages, phage therapy","lastPublishedDoi":"10.21203/rs.3.rs-8388477/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8388477/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe use of bacteriophages holds a great promise in the treatment of multidrug resistant bacteria. The interaction between bacteriophages and multidrug-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e is shaped by extensive capsular diversity, accessory genome variation and multilayered anti-phage defense systems, resulting in highly variable susceptibility profiles. Here, we describe MB01, a lytic T4-like phage isolated from wastewater in Brazil and characterize its biological properties and activity against MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMB01 showed a short replication cycle, strong bacterial suppression at low MOIs and high stability across physiological temperature and pH ranges. Among clinical isolates, MB-01 was effective for \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates. Whole genome sequencing revealed complex resistomes, heterogeneous accessory genes and marked differences in defense systems and prophage content. Defense-enriched strains were fully resistant to MB01, while permissive isolates carried fewer defenses, indicating that genomic background strongly shapes phage outcome.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings highlight the value of integrating phenotypic and genomic analyses for rational phage selection and support the development of targeted phage libraries to address MDR \u003cem\u003eK. pneumoniae\u003c/em\u003e infections.\u003c/p\u003e","manuscriptTitle":"Phenotypic and genomic features shaping the activity of a novel T4-like phage against clinical multidrug-resistant Klebsiella pneumoniae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-07 11:27:12","doi":"10.21203/rs.3.rs-8388477/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-18T05:17:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T11:41:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-09T13:23:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171677305848063785494040018477679775520","date":"2026-01-27T09:21:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338828475910823066579052648392128626281","date":"2026-01-12T08:51:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-05T19:01:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-24T05:59:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-23T14:29:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-12-23T14:21:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e64dfe21-4b53-4c81-a4dd-035095211987","owner":[],"postedDate":"January 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-11T19:53:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-07 11:27:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8388477","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8388477","identity":"rs-8388477","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}