Two Novel Toxin Variants Revealed by Whole-Genome Sequencing of 175 Clostridium botulinum Type E Strains

Abstract

We sequenced 175 Clostridium botulinum type E strains isolated from food, clinical, and environmental sources from northern Canada and analyzed their botulinum neurotoxin (bont) coding sequences (CDSs). In addition to bont/E1 and bont/E3 variant types, neurotoxin sequence analysis identified two novel BoNT type E variants termed E10 and E11. Strains producing type E10 were found along the eastern coastlines of Hudson Bay and the shores of Ungava Bay, while strains producing type E11 were only found in the Koksoak River region of Nunavik. Strains producing BoNT/E3 were widespread throughout northern Canada, with the exception of the coast of eastern Hudson Bay. B otulism, a rare and severe disease characterized by a descending flaccid paralysis, is caused by botulinum neurotoxin (BoNT), the most potent toxin known. BoNT is produced by phylogenetically distinct anaerobic spore-forming bacteria grouped under the taxonomic designation of Clostridium botulinum. Rare botulinogenic strains of related clostridia, such as C. baratii, C. butyricum, and C. argentinense, have also been observed (1, 2). Seven serologically distinct BoNTs (A to G) can be distinguished based on neutralization of toxicity with specific antisera. Recently, a strain of C. botulinum producing botulinum neurotoxin type B (BoNT/B) and another BoNT that is not neutralized by antitoxins to BoNTs A to G has been isolated from a case of infant botulism (3). It has been proposed that this novel neurotoxin is an eighth serotype, BoNT/H (3,4). The botulinum neurotoxins are comprised of three structural domains (translocation [H N ], receptor binding [H C ], and catalytic [LC]). These toxins target different SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins in the neuromuscular junction to block neurotransmitter release (5,6).Heterogeneity within the BoNTs has led to a classification of BoNTs into subtypes. Within a toxin serotype, differences in amino acid sequences can range from 0.9% to 25% (2, 7). Hill and Smith (8) point out that the newer subtypes have been defined based on their DNA sequence and propose the use of the term "subtype/genetic variant" to avoid confusion with the historical use of "subtype," utilized to designate immunological or enzymatic differences among neurotoxins. Two approaches have been used to define new BoNT variants. The first uses a cutoff value of 2.5% difference in amino acid composition (9-11), whereas the second relies on a phylogenetic approach in which variants correspond to clades formed by the clustering of bont sequences (1, 7, 12) Based on these methods, bont/A1 to bont/A5, bont/B1 to bont/ B7, bont/E1 to bont/E9, and bont/F1 to bont/F7 gene variants have been described (1,2,8,10,(12)(13)(14)(15)(16)(17).In Canada, C. botulinum type E is the predominant BoNT serotype associated with food-borne botulism, accounting for 86.2% of all laboratory-confirmed food-borne botulism outbreaks reported between 1985 and 2005 (18). Although variant types E1, E3, and E7 have been identified in t...

Impact of Clostridium botulinum genomic diversity on food safety

“…Group II type E strains are frequently present in arctic and subarctic regions, but interestingly have now also been found in the southern hemisphere [57]. In a detailed study of Group II type E in northern Canada, strains forming subtype E3 neurotoxin were widespread in northern Canada with the exception of eastern Hudson Bay, subtype E10 strains were present along the shores of Ungava Bay and eastern Hudson Bay, and subtype E11 strains were restricted to the Koksoak River region of Nunavik [69 • ]. …”

“…Information is now becoming available to begin to inform these decisions. For example, a thousand-fold difference has been reported in the quantity of neurotoxin formed by type E strains [69 • ], and differences have been identified in carbohydrate utilisation patterns between the three Group II lineages, but not in growth response at chill temperature or at high NaCl concentrations [55]. Additionally, Wachnicka et al .…”

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

“…The newly sequenced isolates were primarily derived from food and clinical samples from outbreaks in northern Canada and from environmental sources in the Nunavik region of northern Quebec (14,24) and included a large number of BoNT/E strains; BoNT/ B4, BoNT/F6, and nontoxigenic isolates were also represented. Isolates were characterized in silico by MLST and core single nucleotide polymorphism (SNP) analyses.…”

“…Several nucleic acid-based typing methods, including pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), variable number tandem repeat (VNTR), multiple-locus sequence typing (MLST), DNA microarrays, and sequence analysis of the bont gene and the flagellin gene variable region (flaVR), have all been used for genetic characterization of C. botulinum group II strains (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

An Atypical Outbreak of Food-Borne Botulism Due to Clostridium botulinum Types B and E from Ham

“…C. botulinum E strain was isolated from a home-cured ham, and it was speculated that the contamination could have occurred from the seasoning, possibly sea salt, since other products (sausage and bacon) from the same pig were not contaminated (30). Since strain 84-10 shows a different subtype and MLST profile from strains isolated from fish and marine animals (8,13,31), it could represent a particular C. botulinum variant adapted to a specific environment or a specific host, such as pigs.…”