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...

This study examined the phylogenetic structure of serotype a Haemophilus influenzae (Hia) isolates recovered from patients in Canada. Hia isolates from 490 separate patients and an American Type Culture Collection (ATCC) strain were analyzed by multilocus sequence typing (MLST), with 18 different sequence types (STs) identified. Most (85.7%) Hia patient isolates were typed as ST-23 and another 12.7% belonged to 14 different STs with 6, 5, or 4 MLST gene loci related to ST-23 (ST-23 complex). Core genome single-nucleotide variation phylogeny (SNVPhyl) on whole genome sequence (WGS) data of 121 Hia patient isolates representing all identified STs and the ATCC strain revealed 2 phylogenetic populations, with all the ST-23 complex isolates within 1 population. The other phylogenetic population contained only the ATCC strain and 3 patient isolates. Concatenated hitABC sequences retrieved from WGS data and analyzed by MEGA (Molecular Evolutionary Genetic Analysis) alignment confirmed the phylogeny obtained by SNVPhyl. The sodC gene was found only in isolates in the minor phylogenetic population. The 2 phylogenetic populations of the Canadian Hia isolates are similar to the 2 clonal divisions described for serotype b H. influenzae. Combining MLST, core SNVPhyl, and hitABC gene sequence alignment showed that most (99.4%) Canadian Hia patient isolates belonged to 1 major phylogenetic population.

The finding of an increase in genetic diversity in the strains in this study and an increase in macrolide resistance compared with previous Canadian reports highlighted the need for continued surveillance including strain characterization.

Background: Syphilis is a sexually transmitted disease that can have atypical clinical presentations. Conventional laboratory tests to confirm the diagnosis are not rapid enough to affect clinical decision on treatment and contact tracing. Rapid point-of-care tests (POCT) can be useful for control of infectious diseases; however, no POCT for syphilis detection is currently available in Canada. The aim of this study is to evaluate two POCTs (RevealTM Rapid TP (Treponema pallidum) Antibody test and DPP® Syphilis Screen and Confirm test) for detection of infectious syphilis. Methods: One hundred serum samples with known syphilis serological status, based on treponemal and non-treponemal test results, were analysed in the laboratory with two POCTs by two independent operators in a blind fashion. Results were analysed to evaluate their ability to detect infectious syphilis. Results: The Reveal Rapid TP Antibody POCT showed an overall sensitivity of 95.0% and a specificity of 83.3%, while the DPP Syphilis Screen and Confirm POCT showed a sensitivity of 87.5% and a specificity of 98.3%. Both POCTs gave a sensitivity of 100% on active syphilis samples with Venereal Disease Research Laboratory (VDRL) titres of greater than 1:4, but their sensitivities decreased for samples with low VDRL titres. Both POCTs gave weakly or very weakly reactive results on 11.3%–25.0% of the treponemal antibody positive samples. Conclusion: This laboratory evaluation has shown promising results for both POCTs to detect infectious syphilis. Further evaluations in the field would be required to confirm this preliminary finding.