Successful Fecal Microbiota Transplants in Post-antibiotic Treated Recurrent Clostridioides difficile Patients Induce Acylcarnitine and Sphingolipid Lipidomic Shifts

Successful Fecal Microbiota Transplants in Post-antibiotic Treated Recurrent Clostridioides difficile Patients Induce Acylcarnitine and Sphingolipid Lipidomic Shifts | 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 Successful Fecal Microbiota Transplants in Post-antibiotic Treated Recurrent Clostridioides difficile Patients Induce Acylcarnitine and Sphingolipid Lipidomic Shifts Abigail Gancz, Guozhi Zhang, Arthur McMillan, Michael Dougherty, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7888346/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 Clostridioides difficile infection (CDI) is an urgent public health threat in the United States, resulting on an annual basis in over half a million cases, more than 29,000 deaths, and $4.8 billion in healthcare costs. While fecal microbiota transplants (FMTs) have proven more effective than standard-of-care antibiotics in resolving recurrent CDI (rCDI), their inherent risks underscore the need for advancements in regulated alternative therapies such as live biotherapeutic products (LBPs). The development of effective LBPs, however, is contingent upon better understanding the biological mechanisms underlying FMT efficacy. Building on our previously published untargeted metabolomic study which identified lipids as major explanatory factors associated with successful FMTs, we assessed additional lipid species using an instrumental platform coupling liquid chromatography, ion mobility spectrometry, collision induced dissociation, and mass spectrometry (LC-IMS-CID-MS) techniques. This platform and data analysis workflow enable the evaluation of >850 unique lipid species across 26 classes. Here, we confidently identified 397 lipids in the stools of 15 rCDI patients at pre- and post-FMT (2 week, 2 month, and 6 month) time points. Statistical evaluations of the lipidomic data illustrated that FMT-administration drastically reshapes the lipidome (adonis test, R 2 =0.11999, Pr(>F) <0.001), including 96 specific lipid species across 18 lipid classes (mixed effects modeling, BH correction, p < 0.05). In particular, we noted that medium and long-chain acylcarnitines decreased following FMT administration, while very long-chain acylcarnitines were elevated in post-FMT samples. Additionally, we observed assayed sphingolipids to be elevated pre-FMT with the exception of trihydroxy ceramides, which were highly upregulated post-FMT. These lipidomic alterations suggest that FMT administration may influence intestinal barrier integrity, inflammatory signaling, or apoptosis pathways. Interestingly, there was a strong co-occurrence of medium and long-chain acylcarnitines with Enterobacteriaceae, a bacterial family that has been demonstrated to utilize carnitine for growth. These findings highlight the critical role of the lipidome in patient susceptibility to rCDI and suggest the interactions between microbiota and lipids pre- and post-FMT as targets for developing next-generation LBPs. Full Text Additional Declarations No competing interests reported. Supplementary Files SupplementalFigure1.tif Figure S1: Boxplots showing changes over time in (A) significant fatty acyls and (B) significant fatty acyl esters of hydroxyl fatty acids. Statistical analysis was done on log₂-transformed, 1 × 10⁶-scaled, minimum-imputed lipid data using a MEM model. SupplementalFigure2.tif Figure S2: Boxplots showing changes over time in (A) significant gangliosides, (B) significant hexosylceramides, and (C) significant sphingomyelins. Statistical analysis was done on log₂-transformed, 1 × 10⁶-scaled, minimum-imputed lipid data using a MEM model. SupplementalFigure3.tif Figure S3: Correlations between acylcarnitines and bacterial families. Analyses used repeated measures correlation of MEM-significant lipids and bacterial genera. SupplementalFigure4.tif Figure S4: Stacked bar plots showing the bacterial genera encoding lipid associated biosynthesis and degradation pathways, with each column representing an individual patient. SupplementalTablesFMTLipidsAbigailGancz8212025.xlsx 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-7888346","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":538085312,"identity":"70eabd6e-fd14-4788-a4ba-1b2b98924fd7","order_by":0,"name":"Abigail Gancz","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Abigail","middleName":"","lastName":"Gancz","suffix":""},{"id":538085314,"identity":"eded5188-ffd4-4d1e-b40a-fdf5ee27ded6","order_by":1,"name":"Guozhi Zhang","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Guozhi","middleName":"","lastName":"Zhang","suffix":""},{"id":538085316,"identity":"63f7a088-15fb-47a2-bb25-91d781f36073","order_by":2,"name":"Arthur McMillan","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Arthur","middleName":"","lastName":"McMillan","suffix":""},{"id":538085318,"identity":"1830ef82-77b0-48f0-8cb2-ffb96dbacda5","order_by":3,"name":"Michael Dougherty","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Dougherty","suffix":""},{"id":538085320,"identity":"958523bb-7e30-4fab-bbe0-b5108efbe161","order_by":4,"name":"Sarah McGill","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"McGill","suffix":""},{"id":538085321,"identity":"c319299d-40ec-4baa-bcb0-8da49fbd0682","order_by":5,"name":"Ajay Gulati","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Ajay","middleName":"","lastName":"Gulati","suffix":""},{"id":538085323,"identity":"e5050013-ec11-4f1b-a822-42c1ccfd9b1d","order_by":6,"name":"Erin Baker","email":"","orcid":"","institution":"University of North Carolina at Chapel Hill","correspondingAuthor":false,"prefix":"","firstName":"Erin","middleName":"","lastName":"Baker","suffix":""},{"id":538085325,"identity":"f4f222ba-a2a3-43c7-8344-6b14dc9d4704","order_by":7,"name":"Casey Theriot","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYHACNiCWYOCH8hgbCOtghmiRbCBRCwODwQFitfBPO3/swcc9FnLGt5sff+ZhsJHdcICAFonbyeyGM55JGJvdOWYmzcOQZkxQi4F0Mps0zwGJxG03ctiYeRgOJxKn5Q9Qy+YZOcxAh/0nUgsDUMsGiRwGoMMOENYC9IuZZM8BCWOJG2lmknMMko1nEtLCPzvxmcSPA3Vy/DOSH394U2En20dIC7o7SVM+CkbBKBgFowAHAADDKTydP0bsNwAAAABJRU5ErkJggg==","orcid":"","institution":"North Carolina State University","correspondingAuthor":true,"prefix":"","firstName":"Casey","middleName":"","lastName":"Theriot","suffix":""}],"badges":[],"createdAt":"2025-10-17 16:08:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7888346/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7888346/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95229836,"identity":"93a1df91-afc5-4ff2-8fb7-e20a48ec27cd","added_by":"auto","created_at":"2025-11-05 16:36:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1733787,"visible":true,"origin":"","legend":"","description":"","filename":"FullPaperFMTLipidsDraftAbigailGancz10202025.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7888346/v1_covered_f2d644c6-c47d-4a7e-a199-5cf9eb4f18bc.pdf"},{"id":95014532,"identity":"a11d6137-15d7-4656-b063-b93825675f3d","added_by":"auto","created_at":"2025-11-03 10:54:05","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":816409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1:\u003c/strong\u003e Boxplots showing changes over time in (A) significant fatty acyls and (B) significant fatty acyl esters of hydroxyl fatty acids. 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