Conditioning chemotherapy exposure is associated with epigenetic modifications in Clostridioides difficile isolates from stem cell transplant recipients

Conditioning chemotherapy exposure is associated with epigenetic modifications in Clostridioides difficile isolates from stem cell transplant recipients | 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 Conditioning chemotherapy exposure is associated with epigenetic modifications in Clostridioides difficile isolates from stem cell transplant recipients Jacob Ng, Jennifer Trannguyen, Racheal Wilkinson, Fritz Conard, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8865869/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Clostridioides difficile is a highly methylated organism within the gut microbiome that is responsible for Clostridioides difficile infection (CDI), a common disease that is mediated by toxins production from the bacterium. C. difficile infection is ten times more common in chemotherapy patients than the average patient, but the reasons for this disparity are unclear. Conditioning chemotherapy (CC), an integral part of cancer treatments, has the ability to induce methylation changes in many cell types. We posit that CC induces methylation changes within C. difficile that may promote toxin production and consequently CDI. To test our hypothesis, we sought to identify the epigenetic changes, particularly methylation changes, within C. difficile isolates before and after chemotherapy and within isolates that express toxin and isolates that do not. After stool sampling, we isolated C. difficile by culture then sequenced and created a hybrid assembly of each isolate using nanopore long read sequencing and Illumina short read sequencing. Bioinformatics tools such as Dorado and Samtools were used to basecall and determine methylation states, while Unicycler was used for genome assembly. Methylartist was then used for data visualization. Genome-wide methylation profiling revealed distinct epigenetic signatures in Clostridioides difficile associated with toxin expression and chemotherapy exposure. Whole-genome 6mA analysis demonstrated significant differences between toxin-positive and toxin-negative isolates, with prominent methylation changes in tcdA and tcdE , while selected sporulation genes were unmethylated in toxin-negative strains. Chemotherapy was associated with a significant shift in global 6mA methylation patterns. Targeted 5mC analysis of the pathogenicity locus revealed reduced methylation around tcdB and across multiple toxin genes following chemotherapy, whereas sporulation genes remained unaffected. These findings suggest chemotherapy-associated epigenetic remodeling of toxin-associated loci in C. difficile . Biological sciences/Microbiology Biological sciences/Stem cells Full Text Additional Declarations No competing interests reported. Figures are available in the Supplementary Files section. Supplementary Files Figures.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 18 May, 2026 Reviews received at journal 13 May, 2026 Reviews received at journal 09 May, 2026 Reviewers agreed at journal 03 May, 2026 Reviewers agreed at journal 02 May, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers invited by journal 27 Apr, 2026 Editor assigned by journal 27 Apr, 2026 Editor invited by journal 10 Mar, 2026 Submission checks completed at journal 02 Mar, 2026 First submitted to journal 02 Mar, 2026 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. 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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-8865869","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":634187064,"identity":"091a5f48-2e9f-455f-b620-71cff1611cc8","order_by":0,"name":"Jacob Ng","email":"","orcid":"","institution":"Texas A\u0026M University","correspondingAuthor":false,"prefix":"","firstName":"Jacob","middleName":"","lastName":"Ng","suffix":""},{"id":634187065,"identity":"fe434fe4-6137-4d8a-9ba9-fd957ba8aafa","order_by":1,"name":"Jennifer Trannguyen","email":"","orcid":"","institution":"University of 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Conditioning chemotherapy (CC), an integral part of cancer treatments, has the ability to induce methylation changes in many cell types. We posit that CC induces methylation changes within \u003cem\u003eC. difficile\u003c/em\u003e that may promote toxin production and consequently CDI. To test our hypothesis, we sought to identify the epigenetic changes, particularly methylation changes, within \u003cem\u003eC. difficile \u003c/em\u003eisolates before and after chemotherapy and within isolates that express toxin and isolates that do not. After stool sampling, we isolated \u003cem\u003eC. difficile\u003c/em\u003e by culture then sequenced and created a hybrid assembly of each isolate using nanopore long read sequencing and Illumina short read sequencing. Bioinformatics tools such as Dorado and Samtools were used to basecall and determine methylation states, while Unicycler was used for genome assembly. Methylartist was then used for data visualization. Genome-wide methylation profiling revealed distinct epigenetic signatures in \u003cem\u003eClostridioides difficile\u003c/em\u003e associated with toxin expression and chemotherapy exposure. Whole-genome 6mA analysis demonstrated significant differences between toxin-positive and toxin-negative isolates, with prominent methylation changes in \u003cem\u003etcdA\u003c/em\u003e and \u003cem\u003etcdE\u003c/em\u003e, while selected sporulation genes were unmethylated in toxin-negative strains. Chemotherapy was associated with a significant shift in global 6mA methylation patterns. Targeted 5mC analysis of the pathogenicity locus revealed reduced methylation around \u003cem\u003etcdB\u003c/em\u003e and across multiple toxin genes following chemotherapy, whereas sporulation genes remained unaffected. 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