Botulism is a neuroparalytic disease caused by the botulinum toxin, which is produced by Clostridium botulinum. C. botulinum is physiologically divided into groups I–IV, and botulinum neurotoxins (BoNT) are classified into 7 types, BoNT/A–G. Human botulism is caused primarily by toxin types A, B, and E, and cases of human infection with C. botulinum group III, which produces toxin types C and D, are rare. Only 5 foodborne botulism outbreaks caused by C. botulinum group III (4 outbreaks caused by type C and 1 outbreak caused by type D) have been reported to date (1), and in Japan, only 1 infant botulism case caused by type C has been reported (2). C. botulinum group III is primarily known as an animal infection, and many of its toxin types have been reported as mosaic types (primarily in birds with toxin type CD and cattle with toxin type DC).

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Figure. Schematic diagrams of each functional domain between BoNT (C, CD, DC, D, and LC759602) in study of novel type C botulism strain in household outbreak, Japan. Gray shaded areas indicate...

In 2021, foodborne botulism occurred in Kumamoto, Japan. A meal eaten in a domestic residence was the assumed cause, and 4 patients were affected. Botulinum toxin and C. botulinum were detected in 3 of the 4 specimens. A commercially prepared chicken dish was suspected to be the cause, but because no food was remaining, we were unable to conduct tests on it. We neutralized the toxin present in the specimens with type C botulinum antitoxin serum, and the isolated strain was found to carry the bont/C gene using PCR targeting the bont genes (3). Next-generation sequencing data revealed full-length coding regions of the bont gene of the isolated strains (GenBank accession no. LC759602). The next-generation sequencing method was as follows: after treating C. botulinum with 20 mg/mL lysozyme in 20 mM Tris-HCl, 2 mM EDTA, and 1% Triton X-100 (pH 8.0), we extracted DNA using a QIAamp DNA Mini Kit (QIAGEN, https://www.qiagen.com). We prepared genome-sequencing libraries using the QIAseq FX DNA Library Kit (QIAGEN) and sequenced the samples on the Illumina iSeq 100 (https://www.illumina.com). We analyzed sequencing data using CLC Genomics Workbench 22.0.2 (QIAGEN). The obtained contig was assembled from reads of 59× coverage and 29 kbp in size. In comparison with other known bont genes, the bont gene of the strain sequenced in this study had the highest amino acid sequence similarity with the bont/C gene (90%) but was partially different from the reference bont/C gene (Table). Detailed analysis revealed that the bont gene (LC759602) had the bont/C gene or the bont/CD gene in the protease domain (LC) and the translocation domain (H_N), as well as the bont/DC gene in the receptor-binding domain (H_C) (Table; Figure). The bont gene (LC759602) has not been previously reported, and we propose its designation as a new subtype of C. botulinum toxin.

The H_C domain is involved in neurotoxin binding to specific receptors in peripheral nerve terminals. The bont gene (LC759602) possesses the bont/DC gene in the H_C domain, suggesting that human susceptibility to this gene might differ from that of the reference BoNT/C toxin. Unlike other BoNTs, BoNT/C interacts only with gangliosides, and no protein receptor for this toxin has been identified (4). However, BoNT/DC has been reported to interact with gangliosides and protein receptors (synaptotagmin I and II) (5).

The bont gene (LC759602) was determined to be BoNT/C using PCR, which can easily distinguish between types C, D, CD, and DC of C. botulinum group III (6). It should be noted that, because not all type C strains were subjected to sequencing, the presence of the bont gene (LC759602) as type C, as determined by typing PCR, might already exist in other samples. Further investigation is needed to determine the proportion of C. botulinum carrying the bont gene reported in this study. The risk for human infection with this new toxin type should also be investigated in future research. However, given that human infections with a similar toxin type, C. botulinum group III, have rarely occurred, this new toxin type might pose little threat to human health.

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