{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T08:05:40Z","timestamp":1771833940146,"version":"3.50.1"},"reference-count":21,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T00:00:00Z","timestamp":1771804800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100014440","name":"Ministerio de Ciencia, Innovaci\u00f3n y Universidades","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100014440","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Infect. Microbiol."],"abstract":"<jats:p>\n                    Botulism is a rare but severe neurological disease caused by botulinum neurotoxins (BoNTs). Standard diagnostic methods including the mouse bioassay (SMB) and\n                    <jats:italic>bont<\/jats:italic>\n                    gene type-specific PCR, are often limited by the high genetic diversity among\n                    <jats:italic>bont<\/jats:italic>\n                    subtypes, which can lead to false-negative results. The nontoxin-nonhemagglutinin (\n                    <jats:italic>ntnh)<\/jats:italic>\n                    gene is highly conserved and exclusively co-located with the\n                    <jats:italic>bont<\/jats:italic>\n                    gene complex. Thus, this study evaluates the use of\n                    <jats:italic>ntnh<\/jats:italic>\n                    gene as a complementary diagnostic tool for botulism and assesses its association with BoNT types. The\n                    <jats:italic>ntnh<\/jats:italic>\n                    gene was detected in a prospective BoNT-diagnostic group (n=88) and a BoNT-historical group (n=54). Toxin cluster proteins were identified in GenBank and RefSeq\n                    <jats:italic>Clostridium<\/jats:italic>\n                    proteomes using MMSeqs2.\n                    <jats:italic>ntnh<\/jats:italic>\n                    gene detection reinforced positive results from SMB or\n                    <jats:italic>bont<\/jats:italic>\n                    gene tests in 26 cases (35.62% of the total confirmed cases) of foodborne and infant botulism. In two foodborne cases from the BoNT-diagnostic group, the\n                    <jats:italic>ntnh<\/jats:italic>\n                    gene was detected despite negative results from both SMB and\n                    <jats:italic>bont<\/jats:italic>\n                    gene tests, highlighting its potential to identify missed cases. An\n                    <jats:italic>in silico<\/jats:italic>\n                    analysis of 3,250 RefSeq and 2,494 GenBank annotated\n                    <jats:italic>Clostridium<\/jats:italic>\n                    proteomes was conducted. respectively. So, NTNH showed a high co-presence pattern with BoNT. Moreover, NTNH sequences were far more conserved than BoNT sequences in inter-type comparisons (67.2 vs.39.7), which highlights its applicability as a disease biomarker. The\n                    <jats:italic>ntnh<\/jats:italic>\n                    gene analysis is a valuable complementary tool enhancing the diagnosis of botulism. The study highlights the need for clear guidelines to interpret positive\n                    <jats:italic>ntnh<\/jats:italic>\n                    results when direct toxin or\n                    <jats:italic>bont<\/jats:italic>\n                    gene confirmation are negative.\n                  <\/jats:p>","DOI":"10.3389\/fcimb.2026.1758429","type":"journal-article","created":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T07:20:07Z","timestamp":1771831207000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["The unsung hero: ntnh gene as complementary botulism marker"],"prefix":"10.3389","volume":"16","author":[{"given":"Sylvia","family":"Valdezate","sequence":"first","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"M\u00f3nica","family":"Valiente","sequence":"additional","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sergio","family":"D\u00edaz-Ram\u00f3n","sequence":"additional","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gema","family":"Carrasco","sequence":"additional","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mar\u00eda J.","family":"Medina-Pascual","sequence":"additional","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Javier","family":"Pardos","sequence":"additional","affiliation":[{"name":"Veterinary Unit, Animal Department, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mar\u00eda Isabel","family":"Hurtado","sequence":"additional","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noelia","family":"Garrido","sequence":"additional","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pilar","family":"Villal\u00f3n","sequence":"additional","affiliation":[{"name":"Reference and Research Laboratory for Taxonomy, National Centre of Microbiology, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Antonio J.","family":"Mart\u00edn-Galiano","sequence":"additional","affiliation":[{"name":"Proteomics Unit, Core Scientific and Technical Units, Instituto de Salud Carlos III","place":["Majadahonda, Madrid, Spain"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2026,2,23]]},"reference":[{"key":"B1","article-title":"Botulism in the United States, 1899\u20131996","volume-title":"handbook for epidemiologists, clinicians, and laboratory workers","year":"1998"},{"key":"B2","doi-asserted-by":"publisher","first-page":"e0013920","DOI":"10.1128\/JCM.00139-20","article-title":"Current developments in diagnostic assays for laboratory confirmation and investigation of botulism","volume":"60","author":"Centurioni","year":"2022","journal-title":"J. Clin. Microbiol."},{"key":"B3","doi-asserted-by":"publisher","first-page":"1105","DOI":"10.1099\/00207713-46-4-1105","article-title":"Organization and phylogenetic interrelationships of genes encoding components of the botulinum toxin complex in proteolytic Clostridium botulinum types A, B, and F: evidence of chimeric sequences in the gene encoding the nontoxic nonhemagglutinin component","volume":"46","author":"East","year":"1996","journal-title":"Int. J. Syst. Bacteriol"},{"key":"B4","doi-asserted-by":"publisher","first-page":"465","DOI":"10.1111\/j.1365-2672.2009.04215.x","article-title":"Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum","volume":"107","author":"Fach","year":"2009","journal-title":"J. Appl. Microbiol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"672","DOI":"10.1111\/febs.17004","article-title":"NTNH protein: more than a bodyguard for botulinum neurotoxins","volume":"291","author":"Gao","year":"2024","journal-title":"FEBS J."},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1099\/mgen.0.001169","article-title":"Complete genomes of Clostridium botulinum type B(F) isolates associated with a 1995 foodborne botulism outbreak from commercial p\u00e2t\u00e9 reveals a recombination event disrupting the ntnh gene","volume":"10","author":"Harris","year":"2024","journal-title":"Microb. 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Microbiol."}],"container-title":["Frontiers in Cellular and Infection Microbiology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2026.1758429\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,2,23]],"date-time":"2026-02-23T07:20:08Z","timestamp":1771831208000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcimb.2026.1758429\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,23]]},"references-count":21,"alternative-id":["10.3389\/fcimb.2026.1758429"],"URL":"https:\/\/doi.org\/10.3389\/fcimb.2026.1758429","relation":{},"ISSN":["2235-2988"],"issn-type":[{"value":"2235-2988","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,23]]},"article-number":"1758429"}}