{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:22:50Z","timestamp":1785410570131,"version":"3.56.0"},"posted":{"date-parts":[[2026,4,23]]},"group-title":"Microbiology","reference-count":111,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T00:00:00Z","timestamp":1776902400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["R21AI171634"],"award-info":[{"award-number":["R21AI171634"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"crossref","award":["R03AI180614"],"award-info":[{"award-number":["R03AI180614"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"crossref"}]},{"award":["R35GM142924"],"award-info":[{"award-number":["R35GM142924"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Clostridioides difficile<\/jats:italic>\n                  is a healthcare-associated infection that arises when broad-spectrum antibiotic treatment disrupts the gut microbiota and is transmitted by highly resistant spores. Vancomycin-resistant\n                  <jats:italic>Enterococcus faecium<\/jats:italic>\n                  (VRE) is an opportunistic pathogen frequently co-isolated from\n                  <jats:italic>C. difficile<\/jats:italic>\n                  patients. We found that\n                  <jats:italic>C. difficile<\/jats:italic>\n                  sporulation is significantly reduced in VRE-\n                  <jats:italic>C. difficile<\/jats:italic>\n                  co-culture. Physical separation of\n                  <jats:italic>C. difficile<\/jats:italic>\n                  and VRE in transwell co-culture restored sporulation. Mixed macrocolony culture assays on solid agar confirmed physical contact is necessary for sporulation inhibition. We screened a panel of enterococci and found that most strains reduce sporulation, except\n                  <jats:italic>Enterococcus saccharolyticus<\/jats:italic>\n                  , which lacks predicted surface displayed virulence factors in its genome. We performed a candidate gene screen using an\n                  <jats:italic>Enterococcus faecalis<\/jats:italic>\n                  OG1RF transposon library and found that an insertion in the major pilin\n                  <jats:italic>ebpC<\/jats:italic>\n                  partially restored\n                  <jats:italic>C. difficile<\/jats:italic>\n                  sporulation in co-culture. These data were confirmed with in-frame deletions in the\n                  <jats:italic>ebpABC<\/jats:italic>\n                  pilus operon and a clinical isolate of\n                  <jats:italic>E. feacalis<\/jats:italic>\n                  lacking\n                  <jats:italic>ebpABC<\/jats:italic>\n                  . These findings suggest enterococci modulate\n                  <jats:italic>C. difficile<\/jats:italic>\n                  sporulation through a contact-dependent mechanism involving the Ebp pilus.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    A characteristic of\n                    <jats:italic>C. difficile<\/jats:italic>\n                    infection is multiple episodes of acute disease. Spores are the transmission vector of\n                    <jats:italic>C. difficile<\/jats:italic>\n                    and are necessary for recurrence in models of disease. Our research demonstrates that\n                    <jats:italic>C. difficile<\/jats:italic>\n                    spore production is significantly reduced in the presence of enterococci, a common group of beneficial and pathogenic bacteria present in the gut microbiota. Physical contact with enterococci reduces\n                    <jats:italic>C. difficile<\/jats:italic>\n                    spore production. We attribute this effect to a protein structure on the surface of enterococci. This finding suggests a potential role for enterococci and the gut microbiota in general to uncover regulators of\n                    <jats:italic>C. difficile<\/jats:italic>\n                    spore formation. This may provide an avenue for innovative treatment strategies that reduce spore formation.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.04.22.718763","type":"posted-content","created":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T03:40:13Z","timestamp":1777002013000},"source":"Crossref","is-referenced-by-count":0,"title":["Contact dependent suppression of\n                  <i>Clostridioides difficile<\/i>\n                  sporulation by enterococci requires the endocarditis and biofilm associated pilus"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-8101-5456","authenticated-orcid":false,"given":"Alicia K.","family":"Wood","sequence":"first","affiliation":[{"name":"Binghamton Biofilm Research Center, Department of Biological Sciences, Binghamton University SUNY, Binghamton, New York 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USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-0311-9563","authenticated-orcid":false,"given":"Bryce","family":"Chong","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, University at Albany, State University of New York, Albany, New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7079-9570","authenticated-orcid":false,"given":"Laura C.","family":"Cook","sequence":"additional","affiliation":[{"name":"Binghamton Biofilm Research Center, Department of Biological Sciences, Binghamton University SUNY, Binghamton, New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6353-294X","authenticated-orcid":false,"given":"Adam","family":"Session","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Binghamton University SUNY, Binghamton, New York 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