Dual-Toxin-Producing Clostridium botulinum Strain Isolated from a Foodborne Botulism Case in Korea: Genomic and Functional Insights

Abstract

Clostridium botulinum produces one of the most potent biological toxins and causes botulism, a rare but potentially fatal neuroparalytic disease. In 2014, a foodborne botulism case was reported in Korea, and a strain (CB-2014001) was isolated. Initial characterization identified it as a BoNT/B-producing strain based on mouse bioassay and conventional PCR. However, subsequent genomic analysis revealed the presence of dual BoNT gene clusters, bont/B and bont/F, corresponding to subtypes B5 and F2, respectively. Therefore, we aimed to analyze the genetic characteristics and toxin expression profiles of the isolated strain. The strain showed high sequence identity with Bf-type strains such as CDC 3281 and An436. Functional assays confirmed simultaneous expression of both BoNT/B and /F toxins at 35 °C, and temperature-dependent assays revealed predominant expression of BoNT/F at 30 °C and BoNT/B at 37 °C, indicating that toxin expression is influenced by environmental temperature. These findings highlight the potential for differential pathogenicity based on culture conditions and underscore the importance of developing diagnostic tools capable of detecting multiple bont genes. To our knowledge, this is the first report of a dual-toxin-producing C. botulinum strain associated with foodborne botulism in Korea, providing important insights into botulism diagnosis, treatment strategies, and public health preparedness.

Keywords: Clostridium botulinum; bont/b; bont/f; botulinum toxins; dual-toxin-producing strain; foodborne botulism.

Figure 1

bont gene cluster arrangements in strain CB-2014001 and other known representative strains, including (A) Subtype B5; (B) subtype F2. Arrows indicate the gene location and orientation; gene names are shown above the arrows; the numbers between genes below the arrows indicate intergenic spacing. The percentage values below each gene represent Average Nucleotide Identity (ANI) compared to the corresponding gene in strain CB-2014001. Dotted lines connect homologous genes across strains to highlight sequence conservation patterns. GenBank accession numbers are CB-2014001 (B: OP019001, F: OP019002), CDC 3281 (B: Y13630, F: Y13631), Bf (ABDP00000000), An436 (LFON00000000), CDC69057 (QVAH00000000), and PA9508B (CP102918).

Figure 2

Phylogenetic tree of the bont/B (A) and the bont/F (B) nucleotide sequences. Maximum likelihood (ML) tree inferred under the GTR+GAMMA model and rooted via midpoint-rooting. The branches are scaled in terms of the expected number of substitutions per site. The numbers above the branches are support values when > 60% from the ML (left) and maximum parsimony (MP) (right) bootstrapping. Detailed information for bont genes is provided in Supplemental Table S1.