Pan-Genomic Analysis of Clostridium botulinum Group II (Non-Proteolytic C. botulinum) Associated with Foodborne Botulism and Isolated from the Environment

Abstract

The neurotoxin formed by Clostridium botulinum Group II is a major cause of foodborne botulism, a deadly intoxication. This study aims to understand the genetic diversity and spread of C. botulinum Group II strains and their neurotoxin genes. A comparative genomic study has been conducted with 208 highly diverse C. botulinum Group II strains (180 newly sequenced strains isolated from 16 countries over 80 years, 28 sequences from Genbank). Strains possessed a single type B, E, or F neurotoxin gene or were closely related strains with no neurotoxin gene. Botulinum neurotoxin subtype variants (including novel variants) with a unique amino acid sequence were identified. Core genome single-nucleotide polymorphism (SNP) analysis identified two major lineages-one with type E strains, and the second dominated by subtype B4 strains with subtype F6 strains. This study revealed novel details of population structure/diversity and established relationships between whole-genome lineage, botulinum neurotoxin subtype variant, association with foodborne botulism, epidemiology, and geographical source. Additionally, the genome sequences represent a valuable resource for the research community (e.g., understanding evolution of C. botulinum and its neurotoxin genes, dissecting key aspects of C. botulinum Group II biology). This may contribute to improved risk assessments and the prevention of foodborne botulism.

Keywords: Clostridium botulinum; botulism; foodborne; neurotoxin; non-proteolytic.

Figure 1

Geographical locations and heat map of newly sequenced isolates used in this study. A total of 113 newly sequenced isolates were attributed to a geographical location. Further details about individual isolates are given in Table S1.

Figure 2

Phylogeny of subtypes of botulinum neurotoxin type B. Further details about individual sequences and isolates are given in Table S2, where they appear in the same order as in this figure. The sources of the isolates are given in Table S1. The toxin protein sequences were aligned with the Muscle module of MEGA7 [59] algorithm and the phylogenetic tree was generated using the Neighbour-Joining method. Three subtype B4 neurotoxin variants are present in C. botulinum Group II. Scale bar represents the number of amino acid substitutions per site. A total of 71 amino acid sequences were analysed. White blocks represent absence of information regarding source. Black blocks represent reference neurotoxins [16].

Figure 3

Phylogeny of subtypes of botulinum neurotoxin type E. Further details about individual sequences and isolates are given in Table S2, where they appear in the same order as in this figure. The sources of the isolates are given in Table S1. The toxin protein sequences were aligned with the Muscle module of MEGA7 [59] algorithm and the phylogenetic tree was generated using the Neighbour-Joining method. The new variants of subtype E1 are highlighted in a red box. Scale bar represents the number of amino acid substitutions per site. A total of 112 amino acid sequences were analysed. White blocks represent absence of information regarding source. Black blocks represent reference neurotoxins [16].

Figure 4

Phylogeny of subtypes of botulinum neurotoxin type F. Further details about individual sequences and isolates are given in Table S2, where they appear in the same order as in this figure. The sources of the isolates are specified in Table S1. The toxin protein sequences were aligned with the Muscle module of MEGA7 [59] algorithm and the phylogenetic tree was generated using the Neighbour-Joining method. Scale bar represents the number of amino acid substitutions per site. A total of 23 amino acid sequences were analysed. White blocks represent absence of information regarding source. Black blocks represent reference neurotoxins [16].

Figure 5

Phylogeny of C. botulinum Group II genomes. Two major lineages (type E neurotoxin gene lineage and type B/E/F neurotoxin gene lineage) were identified. The phylogenetic tree was created by comparison of core single-nucleotide polymorphisms identified using the ParSNP program [63]. Treegraph v2 [64], MEGA7 [59] and Figtree were used to annotate and visualize the phylogenetic tree. Accessory genes were identified using BLAST, using reference sequences for the ha and orfX neurotoxin cluster configurations. The distance bar (0.1) represents the number of nucleotide substitutions per site for a given branch, based on the number of SNPs found in the core genome. Further details about individual sequences and strains are given in Tables S1 and S3, where they appear in the same order as in this figure.

Figure 6

Pan-genome comparison of the two major lineages of C. botulinum Group II. The two major lineages (type E neurotoxin gene lineage and type B/E/F neurotoxin gene lineage) are shown in Figure 5. (A) Distribution of features per genome, with many features only present in a few genomes. The core features are shown at the right-hand side, and the type E toxin gene lineage and type B/E/F toxin gene lineage-specific genes are found in the centre. (B) Graphical representation of feature distribution linked to the SNP-based phylogenetic tree also used in Figure 5. The features marked by the blue square represent the core genome, the pink square represents the genes specific for the type E toxin gene lineage, and the red square represents the genes specific for the type B/E/F toxin gene lineage. (C) The lineage-specific genes are distributed throughout the genome, as shown using representative genomes for the type E toxin gene lineage (Alaska E43) and type B/E/F toxin gene lineage (Eklund 17B-NRP). Dark blue diamonds represent type E toxin gene lineage features, and red diamonds represent type B/E/F toxin lineage features.