Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution

Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution | 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 Biological Sciences - Article Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution Nitin Baliga, Evan Pepper-Tunick, Vivek Srinivas, Fred Mast, Song Li, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8167376/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global TB control, yet the mechanisms enabling rapid evolution of drug resistance in Mtb remain poorly understood. Here we reveal that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INH R ) without fitness costs, challenging the paradigm that resistance mutations always precede their fitness compensatory adaptations. Using M. smegmatis mc 2 155 (Msm) as a model, we show that brief exposure to sublethal INH (2× IC 50 ) enriches for "low-level resistance and tolerance" (LLRT) mutants in a single step. These LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (> 500× IC 50 ) at 6-fold higher rates than wildtype, primarily through otherwise-deleterious mycothiol biosynthesis mutations that become tolerable in the oxidative stress-buffered background. Crucially, we demonstrate that sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH resistance evolution in Msm. Bayesian analysis of 1,578 clinical Mtb isolates from Vietnam confirmed that mutations in oxidative stress response genes were significantly associated with the emergence of INH R strains ( p-value = 1.09×10 -7 ). Independently, reanalysis of genome-wide CRISPRi screens revealed that the OSR network and high Bayes probability genes are functionally associated with treatment escape and survival with multiple antibiotics, including isoniazid, rifampicin, ethambutol, bedaquiline, vancomycin, clarithromycin, linezolid, and streptomycin. Our findings that host-imposed oxidative stress and inadequate drug penetration may synergistically prime Mtb populations for rapid resistance evolution suggest that targeting pre-resistance mechanisms, such as oxidative stress defenses, could help slow the emergence of antibiotic resistance in tuberculosis. Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance Biological sciences/Microbiology/Pathogens Biological sciences/Evolution/Experimental evolution Biological sciences/Computational biology and bioinformatics/High-throughput screening Biological sciences/Microbiology/Bacteria/Bacterial evolution Full Text Additional Declarations There is NO Competing Interest. Supplementary Files S1isolatemutations.csv Supplemental Table S1 S2OSRnetworkSigBPlociannotated.xlsx Supplemental Table S2 SupplementPepperTunicketal20251119.pdf Supplemental Text and Figures Cite Share Download PDF Status: Published Journal Publication published 07 May, 2026 Read the published version in Nature Communications → 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. 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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-8167376","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":551304083,"identity":"c73449e8-ff59-4e6a-9a9b-c6996b28e991","order_by":0,"name":"Nitin 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Here we reveal that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INH\u003csup\u003eR\u003c/sup\u003e) without fitness costs, challenging the paradigm that resistance mutations always precede their fitness compensatory adaptations. Using \u003ci\u003eM. smegmatis\u003c/i\u003e mc\u003csup\u003e2\u003c/sup\u003e155 (Msm) as a model, we show that brief exposure to sublethal INH (2× IC\u003csub\u003e50\u003c/sub\u003e) enriches for \"low-level resistance and tolerance\" (LLRT) mutants in a single step. These LLRT mutants, particularly those with \u003ci\u003eohrR\u003c/i\u003e loss-of-function mutations, acquire high-level resistance (\u003e 500× IC\u003csub\u003e50\u003c/sub\u003e) at 6-fold higher rates than wildtype, primarily through otherwise-deleterious mycothiol biosynthesis mutations that become tolerable in the oxidative stress-buffered background. Crucially, we demonstrate that sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH resistance evolution in Msm. Bayesian analysis of 1,578 clinical Mtb isolates from Vietnam confirmed that mutations in oxidative stress response genes were significantly associated with the emergence of INH\u003csup\u003eR\u003c/sup\u003e strains (\u003ci\u003ep-value\u003c/i\u003e = 1.09×10\u003csup\u003e-7\u003c/sup\u003e). Independently, reanalysis of genome-wide CRISPRi screens revealed that the OSR network and high Bayes probability genes are functionally associated with treatment escape and survival with multiple antibiotics, including isoniazid, rifampicin, ethambutol, bedaquiline, vancomycin, clarithromycin, linezolid, and streptomycin. Our findings that host-imposed oxidative stress and inadequate drug penetration may synergistically prime Mtb populations for rapid resistance evolution suggest that targeting pre-resistance mechanisms, such as oxidative stress defenses, could help slow the emergence of antibiotic resistance in tuberculosis.","manuscriptTitle":"Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 06:51:07","doi":"10.21203/rs.3.rs-8167376/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"92824b77-d260-4427-8b2d-0f2a5b3fd96a","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":58664868,"name":"Biological sciences/Microbiology/Antimicrobials/Antimicrobial resistance"},{"id":58664869,"name":"Biological sciences/Microbiology/Pathogens"},{"id":58664870,"name":"Biological sciences/Evolution/Experimental evolution"},{"id":58664871,"name":"Biological sciences/Computational biology and bioinformatics/High-throughput screening"},{"id":58664872,"name":"Biological sciences/Microbiology/Bacteria/Bacterial evolution"}],"tags":[],"updatedAt":"2026-05-08T07:06:20+00:00","versionOfRecord":{"articleIdentity":"rs-8167376","link":"https://doi.org/10.1038/s41467-026-72496-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-05-07 04:00:00","publishedOnDateReadable":"May 7th, 2026"},"versionCreatedAt":"2025-12-04 06:51:07","video":"","vorDoi":"10.1038/s41467-026-72496-4","vorDoiUrl":"https://doi.org/10.1038/s41467-026-72496-4","workflowStages":[]},"version":"v1","identity":"rs-8167376","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8167376","identity":"rs-8167376","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}