Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Abstract

c Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA ؉ OrfX ؊ ) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA ؊ OrfX ؉ ) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA ؉ OrfX ؊ cluster (69A and 32A) and one strain with the HA ؊ OrfX ؉ cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data

“…Although recent reports claim that bacterial phylogenetic reconstruction from whole-genome sequence data is relatively ro-bust to recombination (39), the effect of horizontal transfer events is potentially great for phylogenetic estimations based on a relatively small portion of the genome. To assess how genome-wide polymorphism profiling compares to the MLST analysis, isolates were examined using core SNP phylogeny, a method that has been successfully adapted for many clinically and environmentally relevant bacterial species undergoing horizontal gene transfer and recombination, including clostridia (40)(41)(42)(43)(44).…”

Clostridial neurotoxins

“…Since a 100-fold greater concentration of arsenic occurs in certain areas of Finland versus other European sites, acquisition of arsenic resistance possibly represent an adaptive response to grow in specific environments [20]. Moreover, whole genome phylogenic single nucleotide polymorphism (SNP) analysis revealed five distinct lineages among C. botulinum strains of group I, according to type/subtypes and/or botulinum locus organization with ha or orfX operon (as discussed later in this chapter) [21]. Although group II C. botulinum strains are genetically closely related as monitored by DNA microarrays, two subsets are individualized: one encompassing type B and F strains, and another one containing the type E strains.…”

Genomic Epidemiology of Clostridium botulinum Isolates from Temporally Related Cases of Infant Botulism in New South Wales, Australia

“…SNPs were excluded if they were in regions with a minimum fold coverage of Ͻ10, within 10 bp of another SNP or Ͻ15 bp from the end of a contig. The analysis also included isolates belonging to each of the five lineages of C. botulinum group I strains, recently described by Gonzalez-Escalona et al (3). BoNT-complex sequences were extracted from de novo-assembled contigs and compared to publically available BoNT cluster sequences using BLASTn.…”

“…enomic epidemiology has provided novel insights into the genetic characteristics and phylogenetic diversity of botulinum neurotoxin (BoNT)-producing Clostridium species (1)(2)(3)(4)(5)(6)(7)(8).…”