A novel mechanism of unstable heteroresistance via reversible ompP2 mutations in Haemophilus influenzae

A novel mechanism of unstable heteroresistance via reversible ompP2 mutations in Haemophilus influenzae | 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 A novel mechanism of unstable heteroresistance via reversible ompP2 mutations in Haemophilus influenzae Matthias Merker, Sabine Petersen, Margo Diricks, Christian Utpatel, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7027370/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 Heteroresistance, the presence of a resistant subpopulation within a predominantly susceptible bacterial isolate, is usually difficult to detect and may cause treatment failure. Mechanisms driving unstable heteroresistant phenotypes, which rapidly revert in absence of antibiotics, remain poorly understood. To investigate the mechanisms underlying heteroresistance in H. influenzae , we isolated four heteroresistant clones and characterized them through experimental evolution, gradient diffusion assays, population analysis profiling, and whole genome sequencing. All clones initially exhibited increased ceftriaxone minimum inhibitory concentrations but rapidly reverted to wild-type susceptibility within four days in drug-free conditions. The main mechanism was reversible structural changes of the ompP2 resistance gene. Three clones exhibiting a 168 bp genomic inversion affecting ompP2 reverted to wild-type sequence. A fourth clone with an ompP2 frameshift insertion reproducibly acquired compensatory deletions restoring the open reading frame. PAP analysis still detected minority subpopulations with 4x-8x increased MICs at day 4. We identified site-specific DNA breakpoints with a palindrome and a homologous sequence facilitating rapid structural genome changes and phenotypic switching. Overall, we discovered novel mechanisms of unstable heteroresistance in H. influenzae , mediated by reversible structural changes in the gene ompP2. Phenotypic switching was induced by ceftriaxone exposure and likely reflects the mutational plasticity of ompP2. Biological sciences/Evolution/Experimental evolution Biological sciences/Microbiology/Microbial genetics/Bacterial genes Biological sciences/Microbiology/Bacteria/Bacterial evolution Haemophilus influenzae ceftriaxone heteroresistance mutational reversion antibiotic resistance Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplTablesv120250415.xlsx Supplementary Tables SupplFigureS1ReplicatesFigure1.pdf Supplementary Figure S1 SupplFigureS4HAE605BWAAlignment.pdf Supplementary Figure S4 SupplFigureS2Gradientdiffusiontestsymbols.pdf Supplementary Figure S2 SupplFigureS5MechanismDeletion.pdf Supplementary Figure S5 SupplFigureS3Gradientdiffusiontestphotos.pdf Supplementary Figure S3 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. 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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-7027370","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":484806560,"identity":"7abb6d28-fdaf-462e-ab50-ab14d9dc2285","order_by":0,"name":"Matthias 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Mechanisms driving unstable heteroresistant phenotypes, which rapidly revert in absence of antibiotics, remain poorly understood. To investigate the mechanisms underlying heteroresistance in \u003cem\u003eH. influenzae\u003c/em\u003e, we isolated four heteroresistant clones and characterized them through experimental evolution, gradient diffusion assays, population analysis profiling, and whole genome sequencing. All clones initially exhibited increased ceftriaxone minimum inhibitory concentrations but rapidly reverted to wild-type susceptibility within four days in drug-free conditions. The main mechanism was reversible structural changes of the \u003cem\u003eompP2\u003c/em\u003e resistance gene. Three clones exhibiting a 168 bp genomic inversion affecting \u003cem\u003eompP2\u003c/em\u003e reverted to wild-type sequence. A fourth clone with an \u003cem\u003eompP2\u003c/em\u003e frameshift insertion reproducibly acquired compensatory deletions restoring the open reading frame. 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