The recent widespread application of whole-genome sequencing (WGS) for microbial disease investigations has spurred the development of new bioinformatics tools, including a notable proliferation of phylogenomics pipelines designed for infectious disease surveillance and outbreak investigation. Transitioning the use of WGS data out of the research laboratory and into the front lines of surveillance and outbreak response requires user-friendly, reproducible and scalable pipelines that have been well validated. Single Nucleotide Variant Phylogenomics (SNVPhyl) is a bioinformatics pipeline for identifying high-quality single-nucleotide variants (SNVs) and constructing a whole-genome phylogeny from a collection of WGS reads and a reference genome. Individual pipeline components are integrated into the Galaxy bioinformatics framework, enabling data analysis in a user-friendly, reproducible and scalable environment. We show that SNVPhyl can detect SNVs with high sensitivity and specificity, and identify and remove regions of high SNV density (indicative of recombination). SNVPhyl is able to correctly distinguish outbreak from non-outbreak isolates across a range of variant-calling settings, sequencing-coverage thresholds or in the presence of contamination. SNVPhyl is available as a Galaxy workflow, Docker and virtual machine images, and a Unix-based command-line application. SNVPhyl is released under the Apache 2.0 license and available at http://snvphyl.readthedocs.io/ or at https://github.com/phac-nml/snvphyl-galaxy.

22Motivation: The recent widespread application of whole-genome sequencing (WGS) for microbial 23

Pulsed-field gel electrophoresis and DNA sequence analysis of 26 strains of Group II (nonproteolytic) Clostridium botulinum type B4 showed that 23 strains carried their neurotoxin gene cluster on a 47–63 kb plasmid (three strains lacked any hybridization signal for the neurotoxin gene, presumably having lost their plasmid). Unexpectedly, no neurotoxin genes were found on the chromosome. This apparent constraint on neurotoxin gene transfer to the chromosome stands in marked contrast to Group I C. botulinum, in which neurotoxin gene clusters are routinely found in both locations. The three main classes of type B4 plasmid identified in this study shared different regions of homology, but were unrelated to any Group I or Group III plasmid. An important evolutionary aspect firmly links plasmid class to geographical origin, with one class apparently dominant in marine environments, whereas a second class is dominant in European terrestrial environments. A third class of plasmid is a hybrid between the other two other classes, providing evidence for contact between these seemingly geographically separated populations. Mobility via conjugation has been previously demonstrated for the type B4 plasmid of strain Eklund 17B, and similar genes associated with conjugation are present in all type B4 plasmids now described. A plasmid toxin–antitoxin system pemI gene located close to the neurotoxin gene cluster and conserved in each type B4 plasmid class may be important in understanding the mechanism which regulates this unique and unexpected bias toward plasmid-borne neurotoxin genes in Group II C. botulinum type B4.

Clostridium botulinum group II isolates (n ‫؍‬ 163) from different geographic regions, outbreaks, and neurotoxin types and subtypes were characterized in silico using whole-genome sequence data. Two clusters representing a variety of botulinum neurotoxin (BoNT) types and subtypes were identified by multilocus sequence typing (MLST) and core single nucleotide polymorphism (SNP) analysis. While one cluster included BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core SNP methods were consistent in terms of clade-level isolate classification; however, core SNP analysis showed higher resolution capability. Furthermore, core SNP analysis correctly distinguished isolates by outbreak and location. This study illustrated the utility of next-generation sequence-based typing approaches for isolate characterization and source attribution and identified discrete SNP loci and MLST alleles for isolate comparison. Clostridium botulinum is a group of spore-forming bacteria that produce botulinum neurotoxins (BoNTs), potent neurotoxins that cause botulism in humans and animals (1). There are six phylogenetically distinct classes of clostridia that produce seven BoNT serotypes (A to G). Group I (proteolytic) C. botulinum organisms produce monovalent, and occasionally bivalent, BoNTs of serotypes A, B, and F, while group II (nonproteolytic) C. botulinum organisms produce monovalent B, E, or F toxins. BoNT types C and D are produced by group III C. botulinum, and type G is produced by group IV C. argentinense. Botulinogenic C. butyricum (BoNT/E) and C. baratii (BoNT/F) have also been described (2, 3).Human botulism in northern Canada and Alaska is frequently associated with the consumption of high-risk traditional native foods, especially aged marine mammal products, and a prevalence of C. botulinum group II spores in the environment (4-10). BoNT type E is the most frequent serotype associated with foodborne botulism in Canada and accounts for 86% of all laboratoryconfirmed foodborne botulism outbreaks occurring between 1985 and 2005 (n ϭ 205) (6). In addition, C. botulinum group II BoNT/E strains are of particular concern for waterfowl health. Reports from the U.S. Geological Survey estimate that BoNT/E botulism outbreaks have killed up to 100,000 birds in and around the Great Lakes since 2000 (http://cida.usgs.gov/glri/#/Browse /fahw/539773f8e4b0f7580bc0b420).While the mouse bioassay remains the "gold standard" for laboratory confirmation of BoNT detection, this method offers limited ability for toxin or strain characterization beyond serotype. 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 ...

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...