{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T15:01:28Z","timestamp":1785164488761,"version":"3.55.0"},"reference-count":36,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T00:00:00Z","timestamp":1785110400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"INSA-VALOR: SANOFI-TBI","award":["28683"],"award-info":[{"award-number":["28683"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Understanding Clostridium tetani metabolism and physiology is essential for identifying factors controlling toxin synthesis. We compared a toxigenic C. tetani strain with a derivative strain lacking the toxin-encoding megaplasmid carrying tetR and tetX genes involved in toxin regulation and production. Both strains were cultured under identical strict anaerobic conditions in complex media. The toxigenic strain exhibited four successive fermentation phases, including rapid growth, toxin accumulation-associated slower growth, cell lysis, and toxin maturation. Cultivation yielded 0.45 g\/L of biomass, for a final toxin titer of 45 Lf\/mL. The megaplasmid-free strain demonstrated no toxin production or cell lysis, yielding a higher biomass concentration (0.75 g\/L) maintained for 150 h. Flow cytometry was applied for the first time to monitor C. tetani population dynamics throughout the culture. The toxigenic strain exhibited notable morphological heterogeneity and transient filamentous cells during rapid growth, followed by two smaller-cell subpopulations emerging during toxin production. The megaplasmid-free strain rapidly stabilized into a single homogeneous population. Membrane integrity remained largely preserved, while extracellular toxin became detectable, suggesting that extracellular toxin detection before cell lysis onset may involve an active export mechanism. Overall, these findings reveal the physiological transitions associated with megaplasmid presence and provide new insights into the cellular processes underlying tetanus toxin production.<\/jats:p>","DOI":"10.3390\/microorganisms14081640","type":"journal-article","created":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T14:08:14Z","timestamp":1785161294000},"page":"1640","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparative Analysis of Toxigenic and Megaplasmid-Free Clostridium tetani Strains Reveals Physiological Transitions Linked to Tetanus Toxin Production"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9889-5345","authenticated-orcid":false,"given":"Bastien","family":"Marie","sequence":"first","affiliation":[{"name":"Toulouse Biotechnology Institute, Universit\u00e9 de Toulouse, CNRS, INRAE, INSA, 31077 Toulouse Cedex 4, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5290-1316","authenticated-orcid":false,"given":"Nathalie","family":"Gorret","sequence":"additional","affiliation":[{"name":"Toulouse Biotechnology Institute, Universit\u00e9 de Toulouse, CNRS, INRAE, INSA, 31077 Toulouse Cedex 4, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3973-4725","authenticated-orcid":false,"given":"Rapha\u00ebl","family":"Esson","sequence":"additional","affiliation":[{"name":"Sanofi, 69280 Marcy L\u2019Etoile, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Romain","family":"Pizzato","sequence":"additional","affiliation":[{"name":"Sanofi, 69280 Marcy L\u2019Etoile, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominique","family":"Garnier","sequence":"additional","affiliation":[{"name":"Sanofi, 69280 Marcy L\u2019Etoile, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5175-9442","authenticated-orcid":false,"given":"St\u00e9phane E.","family":"Guillouet","sequence":"additional","affiliation":[{"name":"Toulouse Biotechnology Institute, Universit\u00e9 de Toulouse, CNRS, INRAE, INSA, 31077 Toulouse Cedex 4, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1409-4792","authenticated-orcid":false,"given":"Isabelle","family":"Meynial-Salles","sequence":"additional","affiliation":[{"name":"Toulouse Biotechnology Institute, Universit\u00e9 de Toulouse, CNRS, INRAE, INSA, 31077 Toulouse Cedex 4, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,27]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2020). Progress Towards Maternal and Neonatal Tetanus Elimination\u2014Worldwide, 2000\u20132018, World Health Organization. Weekly Epidemiological Record."},{"key":"ref_2","unstructured":"Bennett, J.E., Dolin, B., and Blaser, M.J. (2020). Tetanus (Clostridium tetani). Mandell, Douglas, and Bennett\u2019s Principles and Practice of Infectious Diseases, Elsevier."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1126\/science.6326263","article-title":"The Structural Gene for Tetanus Neurotoxin Is on a Plasmid","volume":"224","author":"Finn","year":"1984","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"623","DOI":"10.1093\/infdis\/142.4.623","article-title":"Plasmid-Associated Toxigenicity in Clostridium Tetani","volume":"142","author":"Laird","year":"1980","journal-title":"J. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Cohen, J.E., Wang, R., Shen, R.-F., Wu, W.W., and Keller, J.E. (2017). Comparative Pathogenomics of Clostridium Tetani. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0182909"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"763","DOI":"10.1128\/jb.43.6.763-772.1942","article-title":"Growth Requirements of Clostridium Tetani","volume":"43","author":"Mueller","year":"1941","journal-title":"J. Bacteriol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1128\/am.10.2.146-152.1962","article-title":"Tetanus Toxin Production in the Absence of Protein","volume":"10","author":"Latham","year":"1962","journal-title":"Appl. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"756","DOI":"10.1021\/bp049571b","article-title":"Effect of Medium Composition on the Production of Tetanus Toxin by Clostridium Tetani","volume":"21","author":"Fratelli","year":"2005","journal-title":"Biotechnol. Prog."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1016\/j.anaerobe.2016.07.006","article-title":"Tetanus Toxin Production Is Triggered by the Transition from Amino Acid Consumption to Peptides","volume":"41","author":"Steen","year":"2016","journal-title":"Anaerobe"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Govind, R., and Dupuy, B. (2012). Secretion of Clostridium Difficile Toxins A and B Requires the Holin-like Protein TcdE. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002727"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2600","DOI":"10.1128\/JB.00224-15","article-title":"Observations on the Role of TcdE Isoforms in Clostridium Difficile Toxin Secretion","volume":"197","author":"Govind","year":"2015","journal-title":"J. Bacteriol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e00580-20","DOI":"10.1128\/JB.00580-20","article-title":"Holin-Dependent Secretion of the Large Clostridial Toxin TpeL by Clostridium Perfringens","volume":"203","author":"Saadat","year":"2021","journal-title":"J. Bacteriol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Vidor, C.J., Hamiot, A., Wisniewski, J., Mathias, R.A., Dupuy, B., Awad, M., and Lyras, D. (2022). A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in Paeniclostridium Sordellii. Toxins, 14.","DOI":"10.3390\/toxins14020124"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1044","DOI":"10.1038\/s42003-024-06730-4","article-title":"A Clostridioides Difficile Endolysin Modulates Toxin Secretion without Cell Lysis","volume":"7","author":"Awad","year":"2024","journal-title":"Commun. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1016\/j.resmic.2015.01.002","article-title":"Genomics of Clostridium Tetani","volume":"166","author":"Brzuszkiewicz","year":"2015","journal-title":"Res. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"11220","DOI":"10.1038\/s41598-019-47551-4","article-title":"The Population Structure of Clostridium Tetani Deduced from Its Pan-Genome","volume":"9","author":"Plourde","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.biologicals.2020.02.001","article-title":"A Next-Generation Sequencing Based Method for Determining Genetic Stability in Clostridium Tetani Vaccine Strains","volume":"64","author":"Pennings","year":"2020","journal-title":"Biologicals"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"5604","DOI":"10.1128\/AEM.71.9.5604-5606.2005","article-title":"Molecular Characterization of Clostridium Tetani Strains by Pulsed-Field Gel Electrophoresis and Colony PCR","volume":"71","author":"Seguin","year":"2005","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"122883","DOI":"10.1016\/j.talanta.2021.122883","article-title":"Development and Validation of a Targeted LC-MS\/MS Quantitation Method to Monitor Cell Culture Expression of Tetanus Neurotoxin during Vaccine Production","volume":"236","author":"Francotte","year":"2022","journal-title":"Talanta"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Shapiro, H.M. (2003). Practical Flow Cytometry, John Wiley & Sons. [4th ed.].","DOI":"10.1002\/0471722731"},{"key":"ref_21","first-page":"1","article-title":"La floculation dans les m\u00e9langes de toxine et de s\u00e9rum antidipht\u00e9rique","volume":"37","author":"Ramon","year":"1923","journal-title":"Ann. Inst. Pasteur"},{"key":"ref_22","unstructured":"Roels, J.A. (1983). Energetics and Kinetics in Biotechnology, Elsevier Biomedical Press."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"San Millan, A., and MacLean, R.C. (2017). Fitness Costs of Plasmids: A Limit to Plasmid Transmission. Microbiol. Spectr., 5.","DOI":"10.1128\/microbiolspec.MTBP-0016-2017"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.biologicals.2008.05.002","article-title":"Calibration of Replacement International Standards of Diphtheria and Tetanus Toxoids for Use in Flocculation Test","volume":"36","author":"Rigsby","year":"2008","journal-title":"Biologicals"},{"key":"ref_25","unstructured":"World Health Organization (2013). Manual for Quality Control of Diphtheria, Tetanus, Pertussis and Combined Vaccines, Department of Immunization, Vaccines and Biologicals."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1128\/am.16.1.69-72.1968","article-title":"Continuous Production of Clostridium Tetani Toxin","volume":"16","author":"Zacharias","year":"1968","journal-title":"Appl. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"107781","DOI":"10.1016\/j.biotechadv.2021.107781","article-title":"Insights into Clostridium Tetani: From Genome to Bioreactors","volume":"54","author":"Garrigues","year":"2021","journal-title":"Biotechnol. Adv."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1316","DOI":"10.1073\/pnas.0335853100","article-title":"The Genome Sequence of Clostridium Tetani, the Causative Agent of Tetanus Disease","volume":"100","author":"Fricke","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.anaerobe.2003.08.001","article-title":"Insights in Metabolism and Toxin Production from the Complete Genome Sequence of Clostridium Tetani","volume":"10","author":"Gottschalk","year":"2004","journal-title":"Anaerobe"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1128\/jb.77.2.156-163.1959","article-title":"Glucose Dissimilation by Clostridium Tetani","volume":"77","author":"Martinez","year":"1959","journal-title":"J. Bacteriol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e01808-15","DOI":"10.1128\/mBio.01808-15","article-title":"A Quantitative System-Scale Characterization of the Metabolism of Clostridium Acetobutylicum","volume":"6","author":"Yoo","year":"2015","journal-title":"mBio"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Gregg, B.M., Gupta, S., Tepp, W.H., and Pellett, S. (2024). Expression of Recombinant Clostridial Neurotoxin by C. Tetani. Microorganisms, 12.","DOI":"10.3390\/microorganisms12122611"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1128\/jb.44.2.179-183.1942","article-title":"The Utilization of Amino Acids and Related Compounds by Clostridium Tetani","volume":"44","author":"Clifton","year":"1942","journal-title":"J. Bacteriol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1111\/j.2164-0947.1953.tb00369.x","article-title":"Participation of Peptides in Tetanus Toxin Production","volume":"16","author":"Mueller","year":"1953","journal-title":"Trans. N. Y. Acad. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1128\/jb.79.1.95-102.1960","article-title":"Formation and Action of a Peptidase Which Hydrolyzes Histidine Peptides Required in Tetanus Toxin Synthesis","volume":"79","author":"Miller","year":"1960","journal-title":"J. Bacteriol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"678","DOI":"10.1046\/j.1365-2672.1997.00299.x","article-title":"Specific Peptides of Casein Pancreatic Digestion Enhance the Production of Tetanus Toxin","volume":"83","author":"Prado","year":"1997","journal-title":"J. Appl. Microbiol."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1640\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,27]],"date-time":"2026-07-27T14:28:42Z","timestamp":1785162522000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/8\/1640"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,27]]},"references-count":36,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["microorganisms14081640"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14081640","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,27]]}}}