{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T04:33:58Z","timestamp":1791520438456,"version":"4.3.4"},"reference-count":52,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2023,5,10]],"date-time":"2023-05-10T00:00:00Z","timestamp":1683676800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Taiwan Fisheries Agency of Fund","award":["111-Adjust-Fishery-01(06)"],"award-info":[{"award-number":["111-Adjust-Fishery-01(06)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Bacillus cereus is ubiquitous in the environment and a well-known causative agent of foodborne disease. Surprisingly, more and more emerging strains of atypical B. cereus have been identified and related to severe disease in humans and mammals such as chimpanzees, apes, and bovine. Recently, the atypical B. cereus isolates, which mainly derive from North America and Africa, have drawn great attention due to the potential risk of zoonosis. The cluster of B. cereus carries several anthrax-like virulent genes that are implicated in lethal disease. However, in non-mammals, the distribution of atypical B. cereus is still unknown. In this study, we conducted a retrospective screening of the 32 isolates of Bacillus spp. from diseased Chinese soft-shelled turtles from 2016 to 2020. To recognize the causative agent, we used various methods, such as sequencing analysis using PCR-amplification of the 16S rRNA gene, multiplex PCR for discriminating, and colony morphology by following previous studies. Furthermore, the digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated, respectively, below the 70 and 96% cutoff to define species boundaries. According to the summarized results, the pathogen is taxonomically classified as Bacillus tropicus str. JMT (previous atypical Bacillus cereus). Subsequently, analyses such as targeting the unique genes using PCR and visual observation of the bacteria under various staining techniques were implemented in our study. Our findings show that all (32\/32, 100%) isolates in this retrospective screening share similar phenotypical properties and carry the protective antigen (PA), edema factor (EF), hyaluronic acid (HA), and exopolysaccharide (Bps) genes on their plasmids. In this study, the results indicate that the geographic distribution and host range of B. tropicus were previously underestimated.<\/jats:p>","DOI":"10.3390\/pathogens12050693","type":"journal-article","created":{"date-parts":[[2023,5,11]],"date-time":"2023-05-11T03:22:19Z","timestamp":1683775339000},"page":"693","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Retrospective Screening of Anthrax-like Disease Induced by Bacillus tropicus str. JMT from Chinese Soft-Shell Turtles in Taiwan"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1426-2576","authenticated-orcid":false,"given":"Jia-Ming","family":"Tsai","sequence":"first","affiliation":[{"name":"Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan"},{"name":"Fish Doctor Veterinary Clinic, Pingtung 94042, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hsin-Wei","family":"Kuo","sequence":"additional","affiliation":[{"name":"General Research Service Center, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Winton","family":"Cheng","sequence":"additional","affiliation":[{"name":"Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,5,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.aquaculture.2013.05.006","article-title":"First Case of Soft Shell Disease in Chinese Soft-shelled Turtle (Trionyx sinens) Associated with Aeromonas sobria\u2013A. veronii complex","volume":"406","author":"Chen","year":"2013","journal-title":"Aquaculture"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/j.aquaculture.2006.07.005","article-title":"Pathological and PCR Detection of Mycobacteriosis in Pond-cultured Chinese Soft Shell Turtles, Trionyx sinensis","volume":"261","author":"Hsieh","year":"2006","journal-title":"Aquaculture"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"474","DOI":"10.1136\/vr.152.15.474","article-title":"Mycobacterium kansasii Infection in Chinese Soft Shell Turtle (Pelodiscus sinensis)","volume":"152","author":"Jensen","year":"2003","journal-title":"Vet. Rec."},{"key":"ref_4","first-page":"115","article-title":"Isolation and Identification of Bacillus cereus from Trionyx sinensis","volume":"20","author":"Tan","year":"2011","journal-title":"Guangdong Agric. Sci."},{"key":"ref_5","unstructured":"Yeh, S.-H. (2013). Pathological and Molecular Studies on Bacillus spp. of Soft-Shell Turtle (Trionyx sinensis). [Master Thesis, National Pingtung University of Science and Technology]."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.aquaculture.2014.02.018","article-title":"First Reported Fatal Bacillus thuringiensis Infections in Chinese Soft-shelled Turtles (Trionyx sinensis)","volume":"428","author":"Chen","year":"2014","journal-title":"Aquaculture"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1515","DOI":"10.1111\/jfd.13473","article-title":"Genotype and Virulence Gene Analyses of Bacillus cereus Group Clinical Isolates from the Chinese Softshell Turtle (Pelodiscus sinensis) in Taiwan","volume":"44","author":"Cheng","year":"2021","journal-title":"J. Fish Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"14082","DOI":"10.1038\/srep14082","article-title":"Genomic Insights into the Taxonomic Status of the Bacillus cereus group","volume":"5","author":"Liu","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2499","DOI":"10.1099\/ijsem.0.001821","article-title":"Proposal of Nine Novel Species of the Bacillus cereus Group","volume":"67","author":"Liu","year":"2017","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2236","DOI":"10.1001\/jama.287.17.2236","article-title":"Anthrax as a Biological Weapon, 2002: Updated Recommendations for Management","volume":"287","author":"Inglesby","year":"2002","journal-title":"Jama"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"213","DOI":"10.4315\/0022-2747-35.4.213","article-title":"Bacillus cereus: Food Poisoning Organism","volume":"35","author":"Goepfert","year":"1972","journal-title":"A Review. J. Milk Food Technol."},{"key":"ref_12","first-page":"242","article-title":"Insecticidal Crystal Proteins of Bacillus thuringiensis","volume":"53","author":"Whiteley","year":"1989","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2500","DOI":"10.1007\/s13197-014-1344-4","article-title":"Bacillus cereus Food Poisoning: International and Indian Perspective","volume":"52","author":"Tewari","year":"2015","journal-title":"J. Food Sci. Technol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8449","DOI":"10.1073\/pnas.0402414101","article-title":"Identification of Anthrax Toxin Genes in a Bacillus cereus Associated with an Illness Resembling Inhalation Anthrax","volume":"101","author":"Hoffmaster","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1309","DOI":"10.1016\/S0002-9440(10)61218-7","article-title":"Bacillus anthracis Edema Toxin Causes Extensive Tissue Lesions and Rapid Lethality in Mice","volume":"167","author":"Firoved","year":"2005","journal-title":"Am. J. Pathol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2291","DOI":"10.1073\/pnas.90.6.2291","article-title":"Pathology of Inhalational Anthrax in 42 Cases from the Sverdlovsk Outbreak of 1979","volume":"90","author":"Abramova","year":"1993","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"414","DOI":"10.1086\/510429","article-title":"Fatal Pneumonia among Metalworkers Due to Inhalation Exposure to Bacillus cereus Containing Bacillus anthracis Toxin Genes","volume":"44","author":"Avashia","year":"2007","journal-title":"Clin. Infect. Dis."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3352","DOI":"10.1128\/JCM.00561-06","article-title":"Characterization of Bacillus cereus Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to Bacillus anthracis and Harbor B. anthracis Virulence Genes","volume":"44","author":"Hoffmaster","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Klee, S.R., Brzuszkiewicz, E.B., Nattermann, H., Bruggemann, H., Dupke, S., Wollherr, A., Franz, T., Pauli, G., Appel, B., and Liebl, W. (2010). The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0010986"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5333","DOI":"10.1128\/JB.00303-06","article-title":"Characterization of Bacillus anthracis-like Bacteria Isolated from Wild Great Apes from Cote d\u2019Ivoire and Cameroon","volume":"188","author":"Klee","year":"2006","journal-title":"J. Bacteriol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1038\/nature02722","article-title":"Anthrax Kills Wild Chimpanzees in a Tropical Rainforest","volume":"430","author":"Leendertz","year":"2004","journal-title":"Nature"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5818","DOI":"10.1128\/AEM.00074-11","article-title":"Bovine Bacillus anthracis in Cameroon","volume":"77","author":"Pilo","year":"2011","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1731","DOI":"10.3389\/fmicb.2020.01731","article-title":"You Can\u2019t B. cereus\u2013A Review of Bacillus cereus Strains That Cause Anthrax-like Disease","volume":"11","author":"Baldwin","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_24","first-page":"536","article-title":"Bacillus tropicus, a New Species Isolated from Man and Animals Described and Compared with Other Bacilli Resembling, Bacillus anthracis","volume":"2","author":"Heaslip","year":"1941","journal-title":"Med. J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1100","DOI":"10.1099\/ijsem.0.000760","article-title":"OrthoANI: An Improved Algorithm and Software for Calculating Average Nucleotide Identity","volume":"66","author":"Lee","year":"2016","journal-title":"Int. J. Syst. Evol. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Meier-Kolthoff, J.P., Auch, A.F., Klenk, H.-P., and G\u00f6ker, M. (2013). Genome Sequence-based Species Delimitation with Confidence Intervals and Improved Distance Functions. BMC Bioinform., 14.","DOI":"10.1186\/1471-2105-14-60"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/S0022-2011(02)00018-6","article-title":"Usefulness of Staining Parasporal Bodies When Screening for Bacillus thuringiensis","volume":"79","author":"Ammons","year":"2002","journal-title":"J. Invertebr. Pathol."},{"key":"ref_28","unstructured":"Goodfellow, E.S.A.M. (1991). Nucleic Acid Techniques in Bacterial Systematic, John Wiley and Sons."},{"key":"ref_29","first-page":"1177","article-title":"Simultaneous Detection and Identification of Bacillus cereus Group Bacteria Using Multiplex PCR","volume":"17","author":"Park","year":"2007","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"883","DOI":"10.3389\/fmicb.2019.00883","article-title":"Differentiation of Bacillus thuringiensis From Bacillus cereus Group Using a Unique Marker Based on Real-Time PCR","volume":"10","author":"Wei","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1111\/j.1574-6968.1996.tb08548.x","article-title":"Identification and Characterization of Bacillus anthracis by Multiplex PCR Analysis of Sequences on Plasmids pXO1 and pXO2 and Chromosomal DNA","volume":"145","author":"Ramisse","year":"1996","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1016\/j.jbiotec.2006.04.038","article-title":"Single-step Purification of Recombinant Anthrax Lethal Factor from Periplasm of Escherichia coli","volume":"126","author":"Chang","year":"2006","journal-title":"J. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1186\/s13059-016-1103-0","article-title":"The Oxford Nanopore MinION: Delivery of Nanopore Sequencing to the Genomics Community","volume":"17","author":"Jain","year":"2016","journal-title":"Genome Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1089\/cmb.2012.0021","article-title":"SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-cell Sequencing","volume":"19","author":"Bankevich","year":"2012","journal-title":"J. Comput. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1101\/gr.092759.109","article-title":"Circos: An Information Aesthetic for Comparative Genomics","volume":"19","author":"Krzywinski","year":"2009","journal-title":"Genome Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"D801","DOI":"10.1093\/nar\/gkab902","article-title":"TYGS and LPSN: A Database Tandem for Fast and Reliable Genome-based Classification and Nomenclature of Prokaryotes","volume":"50","author":"Carbasse","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1978","DOI":"10.3389\/fmicb.2019.01978","article-title":"Comparative Genomic and Phylogenomic Analyses Clarify Relationships within and between Bacillus cereus and Bacillus thuringiensis: Proposal for the Recognition of Two Bacillus thuringiensis Genomovars","volume":"10","author":"Baek","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_38","first-page":"570","article-title":"Isolation, Identification and Pathogenicity Analysis of Bacillus cereus from Chinese Soft-shelled Turtles, Pelodiscus sinensis","volume":"43","author":"Meng","year":"2019","journal-title":"Acta Hydrobiol. Sin."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"324","DOI":"10.1007\/s10965-020-02296-x","article-title":"Biodegradation Efficacy of Soil Inherent Novel sp. Bacillus tropicus (MK318648) onto Low Density Polyethylene Matrix","volume":"27","author":"Samanta","year":"2020","journal-title":"J. Polym. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1590\/1678-4324-2021200319","article-title":"Isolation and Characterization of Alkaline Pectinase Productive Bacillus tropicus from Fruit and Vegetable Waste Dump Soil","volume":"64","author":"Thakur","year":"2021","journal-title":"Braz. Arch. Biol. Technol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"113755","DOI":"10.1016\/j.jenvman.2021.113755","article-title":"Biosequestration of Lignin in Municipal Landfill Leachate by Tailored Cationic Lipoprotein Biosurfactant through Bacillus tropicus Valorized Tannery Solid Waste","volume":"300","author":"Uddin","year":"2021","journal-title":"J. Environ. Manag."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Brezillon, C., Haustant, M., Dupke, S., Corre, J.P., Lander, A., Franz, T., Monot, M., Couture-Tosi, E., Jouvion, G., and Leendertz, F.H. (2015). Capsules, Toxins and AtxA as Virulence Factors of Emerging Bacillus cereus Biovar anthracis. PLoS Negl. Trop. Dis., 9.","DOI":"10.1371\/journal.pntd.0003455"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3426","DOI":"10.1128\/JCM.00873-06","article-title":"Capsule Production in Bacillus cereus Strains Associated with Severe Pneumonia","volume":"44","author":"Sue","year":"2006","journal-title":"J. Clin. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1111\/j.1365-2958.2011.07582.x","article-title":"Two Capsular Polysaccharides Enable Bacillus cereus G9241 to Cause Anthrax-like Disease","volume":"80","author":"Oh","year":"2011","journal-title":"Mol. Microbiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"462","DOI":"10.3389\/fimmu.2020.00462","article-title":"Bacillus anthracis Poly-\u03b3-d-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation","volume":"11","author":"Sharma","year":"2020","journal-title":"Front. Immunol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.mimet.2013.01.009","article-title":"A Simple, Reliable M\u2019Fadyean Stain for Visualizing the Bacillus anthracis Capsule","volume":"92","author":"Owen","year":"2013","journal-title":"J. Microbiol. Methods"},{"key":"ref_47","first-page":"197234","article-title":"Identification of pBC218\/pBC210 Genes of Bacillus cereus G9241 in Five Florida Soils Using qPCR","volume":"2014","author":"Luna","year":"2014","journal-title":"Int. Sch. Res. Not."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Marston, C.K., Ibrahim, H., Lee, P., Churchwell, G., Gumke, M., Stanek, D., Gee, J.E., Boyer, A.E., Gallegos-Candela, M., and Barr, J.R. (2016). Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-like Eschar. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0156987"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e00065-18","DOI":"10.1128\/mSystems.00065-18","article-title":"Genomic Characterization and Copy Number Variation of Bacillus anthracis Plasmids pXO1 and pXO2 in a Historical Collection of 412 Strains","volume":"3","author":"Marston","year":"2018","journal-title":"MSystems"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1128\/AAC.00706-06","article-title":"Poly-\u03b3-glutamate Capsule-Degrading Enzyme Treatment Enhances Phagocytosis and Killing of Encapsulated Bacillus anthracis","volume":"51","author":"Scorpio","year":"2007","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"1459","DOI":"10.1099\/mic.0.035857-0","article-title":"Capsule Depolymerase Overexpression Reduces Bacillus anthracis Virulence","volume":"156","author":"Scorpio","year":"2010","journal-title":"Microbiology"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"3012","DOI":"10.1128\/IAI.00205-11","article-title":"Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves Like Attenuated Toxigenic Nonencapsulated B. anthracis Sterne in Rabbits and Mice","volume":"79","author":"Wilson","year":"2011","journal-title":"Infect. Immun."}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/12\/5\/693\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:32:07Z","timestamp":1760124727000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/12\/5\/693"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,10]]},"references-count":52,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2023,5]]}},"alternative-id":["pathogens12050693"],"URL":"https:\/\/doi.org\/10.3390\/pathogens12050693","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,5,10]]}}}