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Orally administered therapeutic drugs can interact with these bacteria in the gastrointestinal tract. Recent evidence shows modifications of multiple drugs via gut bacteria, influencing therapeutic outcomes. Trifluridine, a fluoropyrimidine drug used in colorectal cancer treatment, is susceptible to microbial metabolism and premature inactivation, yet the breadth and functional consequences of this metabolism remain incompletely defined. In this work, we further explore the potential of additional gut bacterial strains that can metabolize trifluridine and characterize how this microbial metabolism impacts cancer cell responses. Sequence-based similarity search revealed trifluridine metabolizing homologues in multiple Enterobacteriaceae species including Klebsiella oxytoca. LCMS analysis confirms that gut isolates, Escherichia coli NS-12 and K. oxytoca S-22, efficiently degraded TF within 2 h. Tipiracil, the clinical inhibitor of host thymidine phosphorylase, only partially blocked microbial TF metabolism dependent on oxygen availability and the bacterial strain. Functionally, the treatment efficacy of TF was reduced, and it lost its cytotoxic potential in cancer when pre-exposed to E. coli and K. oxytoca. Also, microbially degraded TF could not induce the oncogenic p53-dependent pathways in cancer cells compared to the intact TF drug. These findings demonstrate that microbial TF metabolism is widespread across the gut bacteria and can directly undermine its anticancer efficacy and highlight the need to consider gut microbial composition when optimizing TF-based cancer therapies.<\/jats:p>","DOI":"10.1021\/acsptsci.6c00329","type":"journal-article","created":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T16:01:41Z","timestamp":1789228901000},"page":"2842-2852","source":"Crossref","is-referenced-by-count":0,"title":["Gut Microbial Metabolism\nInactivates Trifluridine\nand Drives Loss of Anticancer Efficacy"],"prefix":"10.1021","volume":"9","author":[{"given":"Saeed","family":"Ahmad","sequence":"first","affiliation":[{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"Mills Breast Cancer Institute, Carle Foundation Hospital , , , ,","place":["Urbana, Illinois, United States, 61801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jimoh Olamilekan","family":"Igbalaye","sequence":"additional","affiliation":[{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"Mills Breast Cancer Institute, Carle Foundation Hospital , , , ,","place":["Urbana, Illinois, United States, 61801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vince","family":"Perez","sequence":"additional","affiliation":[{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"Mills Breast Cancer Institute, Carle Foundation Hospital , , , ,","place":["Urbana, Illinois, United States, 61801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth","family":"Wenning","sequence":"additional","affiliation":[{"name":"Mills Breast Cancer Institute, Carle Foundation Hospital , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophie","family":"Gauthier","sequence":"additional","affiliation":[{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"Mills Breast Cancer Institute, Carle Foundation Hospital , , , ,","place":["Urbana, Illinois, United States, 61801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason","family":"Ridlon","sequence":"additional","affiliation":[{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0630-1520","authenticated-orcid":true,"given":"Joseph","family":"Irudayaraj","sequence":"additional","affiliation":[{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"Mills Breast Cancer Institute, Carle Foundation Hospital , , , ,","place":["Urbana, Illinois, United States, 61801"]},{"name":"University of Illinois at Urbana-Champaign , , , ,","place":["Urbana, Illinois, United States, 61801"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,12]]},"reference":[{"issue":"6344","key":"2026100909101265700_cit1","doi-asserted-by":"publisher","DOI":"10.1126\/science.aag2770","article-title":"Chemical Transformation of Xenobiotics\nby the Human Gut Microbiota","volume":"356","author":"Koppel","year":"2017","journal-title":"Science"},{"issue":"7","key":"2026100909101265700_cit2","doi-asserted-by":"publisher","first-page":"1661","DOI":"10.1016\/j.cell.2020.05.001","article-title":"Personalized Mapping\nof Drug Metabolism by the Human Gut Microbiome","volume":"181","author":"Javdan","year":"2020","journal-title":"Cell"},{"issue":"3","key":"2026100909101265700_cit3","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1016\/j.chom.2022.02.004","article-title":"Gut Microbiota-Derived Bile Acids in Intestinal Immunity,\nInflammation, and Tumorigenesis","volume":"30","author":"Cai","year":"2022","journal-title":"Cell Host Microbe"},{"issue":"1","key":"2026100909101265700_cit4","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1007\/s40495-025-00429-8","article-title":"Impact of Gut Microbiota\non Drug Metabolism and Absorption","volume":"11","author":"Burke","year":"2025","journal-title":"Curr. 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