Foodborne botulism is a rare but highly lethal disease. The botulinum neurotoxin (BoNT) that causes botulism is produced by Clostridium botulinum (C. botulinum) and other clostridia. In this study, we characterized C. botulinum strains isolated during food poisoning events in Sichuan Province from 1990 to 2024 by analyzing whole-genome sequencing data. Statistical analyses of the geographical distribution food sources of strains suggested that the primary sources of contamination were associated with specific regions and food types. The isolates were further compared with reference strains using average nucleotide identity analysis to reveal their genetic diversity and evolutionary relationships. Among the eight strains, five strains belonged to Group I, and the other three strains belonged to Group II. All strains belonging to Group I (SC001, SC002, SC003, SC006, and SC009) were assigned to five totally different recognized ST types (ST-2 to ST-51). In addition, analysis of BoNTs subtypes demonstrated that the types of BoNTs causing botulism in Sichuan were mainly types A, B, and E. Among them, some rare subtypes of BoNT reported for the first time in China, such as BoNT/B4, BoNT/E12, and BoNT/A5(B3), and no specific subtypes were predominant in the botulism incidents. This study is critical for disease surveillance and early warning systems, while also providing a basis for food safety regulation, clinical diagnosis, and treatment in the future.
ArnonS, SchechterR, InglesbyT, et al.Botulinum toxin as a biological weapon: Medical and public health management. JAMA, 2001; 285(8):1059–1070; doi: 10.1001/jama.285.8.1059
2.
BellinzonaG, NardiT, CastelliM, et al.Comparative genomics of Cryptosporidium parvum reveals the emergence of an outbreak-associated population in Europe and its spread to the United States. Genome Res, 2024; 34(6):877–887; doi: 10.1101/gr.278830.123
3.
CarterAT, PearsonBM, CrossmanLC, et al.Complete genome sequence of the proteolytic Clostridium botulinum type A5 (B3′) strain H04402 065. J Bacteriol, 2011; 193(9):2351–2352; doi: 10.1128/jb.00072-11
4.
ChenY, KorkealaH, LindénJ, et al.Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature. Appl Environ Microbiol, 2008; 74(19):6132–6137; doi: 10.1128/aem.00469-08
5.
ChristineR-E, EmmanuelL, MichelRP. Human Botulism in France, 1875–2016. Toxins (Basel), 2020; 12(5); doi: 10.3390/toxins12050338
6.
ClaireJPS, TimothyAM, AlvinLC. FbpA—a bacterial transferrin with more to offer. Biochim Biophys Acta, 2011; 1820(3); doi: 10.1016/j.bbagen.2011.09.001
DoverN, BarashJR, ArnonSS. Identical novel A5(B3′) botulinum neurotoxin gene arrangements isolated from widely disparate geographical and patient sources suggest their independent origins. J Clin Microbiol, 2010a;48(5):1989; doi: 10.1128/jcm.00468-10
9.
DoverN, BarashJR, HillKK, et al.Novel structural elements within the nonproteolytic clostridium botulinum type F toxin gene cluster. Appl Environ Microbiol, 2010b;77(5):1904–1906; doi: 10.1128/aem.02422-10
10.
GaoQY, HuangYF, WuJG, et al.A review of botulism in China. Biomed Environ Sci, 1990; 3(3).
11.
GregoryKS, AcharyaKR. A comprehensive structural analysis of Clostridium botulinum Neurotoxin A cell-binding domain from different subtypes. Toxins (Basel), 2023; 15(2):92; doi: 10.3390/toxins15020092
12.
HasanK, Mehmet ResatC, HasanB, et al.Foodborne botulism in Turkey, 1983 to 2017. Infect Dis (Lond), 2019; 51(2):91–96.
13.
HeilmannA, LacyA, KoyfmanA, et al.High risk and low prevalence diseases: Botulism. Am J Emerg Med, 2024; 82:174–182; doi: 10.1016/j.ajem.2024.06.018
14.
HennequinC, CollignonA, KarjalainenT. Analysis of expression of GroEL (Hsp60) of Clostridium difficile in response to stress. Microb Pathog, 2001; 31(5):255–260; doi: 10.1006/mpat.2001.0468
15.
HillK, XieG, FoleyB, et al.Genetic diversity within the botulinum neurotoxin-producing bacteria and their neurotoxins. Toxicon, 2015; 107(Pt A):2–8; doi: 10.1016/j.toxicon.2015.09.011
16.
HosomiK, SakaguchiY, KohdaT, et al.Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan. Mol Genet Genomics, 2014; 289(6):1267–1274; doi: 10.1007/s00438-014-0887-4
17.
JasonB, AndrewTC, SandraCS, et al.Identification of a novel botulinum neurotoxin gene cluster in Enterococcus. FEBS Lett, 2018; 592(3); doi: 10.1002/1873-3468.12969
18.
KeithAJ, JamesEB, MartinCJM. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res, 2018; 3(0); doi: 10.12688/wellcomeopenres.14826.1
19.
LearoydTP. Underreporting or failed notification? global botulism reporting, 2000-2022. Health Secur, 2024; 22(3):203–209; doi: 10.1089/hs.2023.0081
20.
LiH, GuoY, TianT, et al.Epidemiological analysis of foodborne botulism outbreaks - China, 2004–2020. China CDC Wkly, 2022; 4(35):788–792; doi: 10.46234/ccdcw2022.114
21.
LonatiD, SchicchiA, CrevaniM, et al.Foodborne botulism: Clinical diagnosis and medical treatment. Toxins (Basel), 2020; 12(8):509; doi: 10.3390/toxins12080509
22.
LoreVD, NatasjaC, ChanaC, et al.Protein truncating variants of colA in Clostridium perfringens Type G Strains. Front Cell Infect Microbiol, 2021; 11(0); doi: 10.3389/fcimb.2021.645248
23.
LuZ, FengJ, LuoX, et al.Tracing and characterization of foodborne botulism caused by the new MLST type Clostridium botulinum A2 in Hebei province, China. Front Microbiol, 2025; 16:1567360; doi: 10.3389/fmicb.2025.1567360
24.
MazuetC, SautereauJ, LegeayC, et al.An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham. J Clin Microbiol, 2015; 53(2):722–726; doi: 10.1128/jcm.02942-14
25.
MichaelWP, TheresaJS, FabrizioA, et al.Historical perspectives and guidelines for Botulinum neurotoxin subtype nomenclature. Toxins (Basel), 2017; 9(1); doi: 10.3390/toxins9010038
26.
Muhammad TanveerM, NarjesM, LaurentG, et al.Physical treatments to control Clostridium botulinum hazards in food. Foods, 2023; 12(8); doi: 10.3390/foods12081580
27.
Prevention USCfDCa. National Botulism Surveillance Summary Published. 2017. Available from: https://stacks.cdc.gov/view/cdc/152164 [Last accessed: December, 15].
28.
PriceT, RealegenoS, MirasolR, et al.Validation, implementation, and clinical utility of whole genome sequence-based bacterial identification in the clinical microbiology laboratory. J Mol Diagn, 2021; 23(11):1468–1477; doi: 10.1016/j.jmoldx.2021.07.020
29.
PriceV, NgwiraL, LewisJ, et al.A systematic review of economic evaluations of whole-genome sequencing for the surveillance of bacterial pathogens. Microb Genom, 2023; 9(2):mgen000947; doi: 10.1099/mgen.0.000947
30.
RawsonA, DempsterA, HumphreysC, et al.Pathogenicity and virulence of Clostridium botulinum. Virulence, 2023; 14(1):2205251; doi: 10.1080/21505594.2023.2205251
RichardH, ChristineT, NatalieP, et al.A summary of surveillance, morbidity and microbiology of laboratory-confirmed cases of infant botulism in Canada, 1979–2019. Can Commun Dis Rep, 2021; 47(78); doi: 10.14745/ccdr.v47i78a05
33.
SakaguchiY, SuzukiT, YamamotoY, et al.Genomics of Clostridium botulinum group III strains. Res Microbiol, 2015; 166(4):318–325; doi: 10.1016/j.resmic.2014.07.016
34.
SebaihiaM, PeckMW, MintonNP, et al.Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. Genome Res, 2007; 17(7):1082–1092; doi: 10.1101/gr.6282807
35.
ShanquanL, TanZ, ZhihaoL, et al.Response mechanism of Vibrio parahaemolyticus at high pressure revealed by transcriptomic analysis. Appl Microbiol Biotechnol, 2022; 106(17); doi: 10.1007/s00253-022-12082-y
TaoL, NianzhiN, AngeloI, et al.Novel putative transposable element associated with the Subtype E5 Botulinum toxin gene cluster of neurotoxigenic Clostridium butyricum Type E strains from China. Int J Mol Sci, 2022; 23(2):906; doi: 10.3390/ijms23020906
38.
ThomasEM, CharlesHH, YulinS, et al.Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing. Appl Environ Microbiol, 2011; 77(24); doi: 10.1128/aem.05155-11
39.
TomaszG, AnnaG, AnnaJ, et al.Opportunistic features of Non-Clostridium botulinum strains containing bont gene cluster. Pathogens, 2024; 13(9); doi: 10.3390/pathogens13090780
40.
UmedaK, HiraiY, NakamuraH, et al.Comparative whole-genome sequence analysis of a BoNT/B5-producing Clostridium botulinum isolate from an infant botulism case of unknown source in Osaka, Japan. FEMS Microbiol Lett, 2022; 369(1):fnac069; doi: 10.1093/femsle/fnac069
41.
WangY-L, YangX, HaoL-N. Phenology of vegetation and its response to climate change inthe Western Sichuan Plateau. Journal of Changjiang River Scientific Research Institute, 2023; 40(5):77–84; doi: 10.11988/ckyyb.20220041
42.
WeedmarkKA, MabonP, HaydenKL, et al.Clostridium botulinum Group II Isolate phylogenomic profiling using whole-genome sequence data. Appl Environ Microbiol, 2015; 81(17):5938–5948; doi: 10.1128/aem.01155-15
43.
WeiyingY, DongyueJ, RuiruiL, et al.Food-borne botulism from homemade sauce leading to cardiac arrest: A family case series with literature review. Toxicon, 2023; 235(0):107326; doi: 10.1016/j.toxicon.2023.107326
44.
YinpingD, WeiW, TaoJ, et al.Whole genome sequencing of Clostridium butyricum that caused the first infant botulism in China. Disease Surveillance, 2022; 1:38–44; doi: 10.3784/jbjc.202108040431
45.
YuliH, QianH, RongW, et al.The XRE family transcriptional regulator SrtR in Streptococcus suis is involved in oxidant tolerance and virulence. Front Cell Infect Microbiol, 2018; 8(0):452; doi: 10.3389/fcimb.2018.00452
46.
YuminZ, SongL, ZihaoP, et al.XRE family transcriptional regulator XtrSs modulates Streptococcus suis fitness under hydrogen peroxide stress. Arch Microbiol, 2022; 204(5):244; doi: 10.1007/s00203-022-02854-5
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
