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Ribotype 033 (RT033) is predominantly associated with animal and environmental reservoirs, yet key traits underlying its ecology are poorly defined. Here we show that RT033 strains produce spores with enhanced germination sensitivity and expanded germinant specificity, enabling germination at low concentrations of bile\u2010salts and in response to bile\u2010salts that are typically inhibitory, including those more typical of animal hosts. Genetic analysis identified a single amino acid substitution (R1036I) in CspA, a component of the CspBAC germination apparatus, as the determinant of this phenotype. Expression of the RT033\n                    <jats:italic>cspBAC<\/jats:italic>\n                    operon in a laboratory strain was sufficient to confer increased sensitivity and expanded bile\u2010salt responsiveness. The R1036I substitution disrupts a conserved salt bridge at the CspA:CspC interface while not causing major destabilisation of the complex in the absence or presence of germinants and co\u2010germinants. Together, our findings reveal a naturally occurring rewiring of bile\u2010salt sensing in spores of RT033 strains that likely reflects adaptation to animal\u2010associated bile\u2010acid environments and may influence persistence, transmission, and zoonotic potential of this lineage.\n                  <\/jats:p>","DOI":"10.1111\/1462-2920.70332","type":"journal-article","created":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T10:06:51Z","timestamp":1780049211000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["A Single Amino Acid Substitution in\n                    <scp>CspA<\/scp>\n                    Increases Germination Sensitivity and Broadens Bile Salt Germinant Specificity in\n                    <i>Clostridioides difficile<\/i>\n                    Spores"],"prefix":"10.1111","volume":"28","author":[{"given":"Isabel","family":"Roseiro","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier  Oeiras Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6449-8053","authenticated-orcid":false,"given":"Alexandra","family":"Nunes","sequence":"additional","affiliation":[{"name":"Genomics and Bioinformatics Unit, Department of Infectious Diseases National Institute of Health Doutor Ricardo Jorge  Lisboa Portugal"},{"name":"Veterinary and Animal Research Center (CECAV), Faculty of Veterinary Medicine, Lus\u00f3fona University, University Center of Lisbon  Lisboa Portugal"},{"name":"Research in Veterinary Medicine (I\u2010MVET), Faculty of Veterinary Medicine, Lus\u00f3fona University, University Center of Lisbon  Lisboa Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1119-0594","authenticated-orcid":false,"given":"Diogo","family":"Martins","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier  Oeiras Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1664-4471","authenticated-orcid":false,"given":"Frederico","family":"Alves","sequence":"additional","affiliation":[{"name":"National Reference Laboratory of Gastrointestinal Infections, Department of Infectious Diseases National Institute of Health Doutor Ricardo Jorge  Lisboa Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S\u00f8ren","family":"Persson","sequence":"additional","affiliation":[{"name":"Department of Bacteria, Parasites and Fungi Statens Serum Institut  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4292-4802","authenticated-orcid":false,"given":"Adriano O.","family":"Henriques","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier  Oeiras Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6360-2576","authenticated-orcid":false,"given":"M\u00f3nica","family":"Oleastro","sequence":"additional","affiliation":[{"name":"National Reference Laboratory of Gastrointestinal Infections, Department of Infectious Diseases National Institute of Health Doutor Ricardo Jorge  Lisboa Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3335-3219","authenticated-orcid":false,"given":"M\u00f3nica","family":"Serrano","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica Ant\u00f3nio Xavier  Oeiras Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,5,29]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-024-07487-w"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003165"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciae429"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.3389\/fpubh.2022.1070258"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2022.858310"},{"key":"e_1_2_10_7_1","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0187658","article-title":"Prevalence of Binary Toxin Positive \n                     Clostridium difficile\n                   in Diarrhoeal Humans in the Absence of Epidemic Ribotype 027","volume":"12","author":"AM M. 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