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Current FDA-approved therapies for\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      infection (CDI) are limited and are frequently associated with treatment failure and high rates of disease recurrence. To address this unmet clinical need, we employed a drug repurposing strategy focused on FDA-approved compounds with established safety profiles and limited systemic absorption characteristics, which are desirable for localized gastrointestinal infections. Through this approach, we identified the antifungal azoles miconazole, econazole, and tioconazole as potent inhibitors of\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      . These compounds exhibited robust\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      activity against a diverse panel of clinical\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      isolates, with MIC\n                      <jats:sub>50<\/jats:sub>\n                      values of 1, 2, and 2 \u00b5g\/mL, respectively. Time-kill analyses demonstrated that all tested azoles exerted rapid bactericidal activity within 2 h, exceeding the killing kinetics of the current standard-of-care agents vancomycin and fidaxomicin, likely attributable to their membrane-disruptive mechanism that compromises cytoplasmic membrane integrity and leads to rapid cellular collapse. Additionally, the azoles displayed prolonged post-antibiotic effects (&gt;12 h). Furthermore, the azoles exhibited limited activity against two representative members of the commensal intestinal microbiota (\n                      <jats:italic toggle=\"yes\">Bifidobacterium<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Lactobacillus<\/jats:italic>\n                      spp.) and retained antibacterial efficacy under physiologically relevant conditions, including high inoculum, variable pH, and simulated gastrointestinal fluids. Mechanistic studies supported membrane targeting as the primary mode of action. Azole exposure significantly disrupted bacterial membrane integrity, and scanning electron microscopy revealed marked morphological damage, including membrane distortion and cellular collapse, consistent with structural membrane damage. Notably, cholesterol supplementation increased\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      tolerance to azoles and other membrane-disrupting agents, suggesting that host cholesterol levels or dietary factors may influence CDI outcomes. Finally, in a\n                      <jats:italic toggle=\"yes\">Caenorhabditis elegans<\/jats:italic>\n                      CDI infection model, all tested azoles significantly improved host survival following lethal\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      challenge. Collectively, our findings highlight the potential of azole antifungals as promising platforms that merit further investigation and modification in the pursuit of safer, more effective therapeutics for CDI treatment.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      <jats:italic toggle=\"yes\">Clostridioides difficile<\/jats:italic>\n                      infection (CDI) is a serious and recurrent intestinal disease for which current treatment options remain limited and often disrupt the protective gut microbiota, increasing the risk of relapse. In this study, we show that the antifungal drugs miconazole, econazole, and tioconazole exhibit rapid and sustained activity against\n                      <jats:italic toggle=\"yes\">C. difficile<\/jats:italic>\n                      under conditions that mimic the gastrointestinal environment. Unlike existing therapies, these agents display minimal activity against commensal gut bacteria and provide prolonged post-antibiotic effects. We also demonstrate that cholesterol, a common host-derived molecule, modulates bacterial susceptibility to these drugs, revealing a previously underappreciated link between host factors and therapeutic efficacy. Finally, these compounds confer significant protection in an\n                      <jats:italic toggle=\"yes\">in vivo Caenorhabditis elegans<\/jats:italic>\n                      model of CDI, highlighting their potential as candidates for repurposing and further development.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.02900-25","type":"journal-article","created":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T12:00:47Z","timestamp":1779105647000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Repurposing azole antifungals as rapid bactericidal, membrane-disruptive therapeutics against\n                    <i>Clostridioides difficile<\/i>"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9937-742X","authenticated-orcid":true,"given":"Ahmed A.","family":"Abouelkhair","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]},{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for One Health Research, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1730-4150","authenticated-orcid":true,"given":"Nader S.","family":"Abutaleb","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]},{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for One Health Research, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brice J.","family":"Stolz","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]},{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for One Health Research, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0939-0458","authenticated-orcid":true,"given":"Mohamed N.","family":"Seleem","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]},{"id":[{"id":"https:\/\/ror.org\/02smfhw86","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for One Health Research, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","first-page":"319","article-title":"Clostridioides difficile infection: landscape and microbiome therapeutics","volume":"19","author":"Gawey BJ","year":"2023","unstructured":"Gawey BJ, Khanna S. 2023. 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