phylogenomic profiling of clostridium botulinum group ii isolates

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Phylogenomic profiling of Clostridium botulinum Group II isolates using whole 1

genome sequence data 2

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Running title: C. botulinum typing using WGS data 4

Journal: AEM 5

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KA Weedmark1, P Mabon1, KL Hayden1, D Lambert2,3, G Van Domselaar1, JW Austin2, 7

CR Corbett1# 8

1. National Microbiology Laboratory, Public Health Agency of Canada, 1015 Arlington 9

St., Winnipeg, Manitoba R3E 3R2, Canada 10

2. Bureau of Microbial Hazards, Health Products and Food Branch, Health Canada, 251 11

Sir Frederick Banting Driveway, Tunney's Pasture, Ottawa, Ontario K1A 0K9, Canada 12

3. Present address: Canadian Food Inspection Agency, 960 Carling Ave, Ottawa, Ontario 13

K1A 0C6, Canada 14

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Journal section: Evolutionary and genomic microbiology 16

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#To whom correspondence should be addressed: [email protected] 18

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AEM Accepted Manuscript Posted Online 26 June 2015Appl. Environ. Microbiol. doi:10.1128/AEM.01155-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 24

Clostridium botulinum Group II isolates (n=163) from different geographic regions, 25

outbreaks, and neurotoxin types and subtypes were characterized in silico using whole 26

genome sequence data. Two clusters representing a variety of botulinum neurotoxin 27

(BoNT) types/subtypes were identified by multilocus sequence typing (MLST) and core 28

Single Nucleotide Polymorphism (SNP) analysis. While one cluster included 29

BoNT/B4/F6/E9 and nontoxigenic members, the other comprised a wide variety of 30

different BoNT/E subtype isolates and a nontoxigenic strain. In silico MLST and core 31

SNP methods were consistent in terms of clade-level isolate classification, however core 32

SNP analysis showed higher resolution capability. Furthermore, core SNP analysis 33

correctly distinguished isolates by outbreak/location. This study illustrates the utility of 34

next-generation sequence-based typing approaches for isolate characterization and source 35

attribution, and identifies discrete SNP loci and MLST alleles for isolate comparison. 36

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INTRODUCTION 47

Clostridium botulinum is a group of spore-forming bacteria that produce botulinum 48

neurotoxins (BoNTs) – potent neurotoxins that cause botulism in humans and animals 49

(1). There are six phylogenetically distinct classes of clostridia that produce seven BoNT 50

serotypes (A-G). Group I (proteolytic) C. botulinum produce monovalent, and 51

occasionally bivalent, BoNTs of serotypes A/B/F, while Group II (non-proteolytic) C. 52

botulinum produce monovalent B, E, or F toxins. BoNT types C and D are produced by 53

Group III C. botulinum and type is produced by Group IV C. argentinense. Botulinogenic 54

C. butyricum (BoNT/E) and C. baratii (BoNT/F) have also been described (2, 3). 55

Human botulism in northern Canada and Alaska is frequently associated with the 56

consumption of high-risk traditional native foods, especially aged marine mammal 57

products, and a prevalence of C. botulinum Group II spores in the environment (4-10). 58

BoNT type E is the most frequent serotype associated with foodborne botulism in Canada 59

and accounts for 86% of all laboratory-confirmed foodborne botulism outbreaks 60

occurring between 1985–2005 (n=205) (6). In addition, C. botulinum Group II BoNT/E 61

strains are of particular concern for waterfowl health. Reports from the US Geological 62

Survey estimate that BoNT/E botulism outbreaks have killed up to 100,000 birds in and 63

around the Great Lakes since 2000 64

(http://cida.usgs.gov/glri/#/Browse/fahw/539773f8e4b0f7580bc0b420). 65

While the mouse bioassay remains the gold standard for laboratory confirmation 66

of BoNT detection, this method offers limited ability for toxin or strain characterization 67

beyond serotype. Several nucleic acid-based typing methods, including pulsed-field gel 68

electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), amplified 69


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fragment length polymorphism (AFLP), variable number tandem repeat (VNTR), 70

multiple locus sequence typing (MLST), DNA microarrays, and sequence analysis of the 71

bont gene and the flagellin variable region (flaVR) have all been used for genetic 72

characterization of C. botulinum Group II strains (11-24). 73

In the present study, whole genome sequence (WGS) data from 152 C. botulinum 74

Group II isolates were analyzed with 11 publicly available genomes (163 total isolates 75

characterized). The newly sequenced isolates were primarily derived from food and 76

clinical samples from outbreaks in northern Canada and from environmental sources in 77

the Nunavik region of northern Quebec (14, 24) and include a large number of BoNT/E 78

strains; BoNT/B4, BoNT/F6 and nontoxigenic isolates were also represented. Isolates 79

were characterized in silico by MLST and core SNP analyses. 80

Core SNP phylogeny analysis resolved isolates by outbreak and/or location of 81

origin. These results demonstrate the utility of in silico C. botulinum characterization 82

using next-generation sequence (NGS) data and provide discrete high quality SNP loci, 83

MLST alleles, and read data for 152 C. botulinum Group II isolates. 84

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METHODS 86

Culture conditions, DNA isolation, and genome sequencing. C. botulinum Group II 87

strains were cultured at 30 °C for 48-72 h under anaerobic conditions (AnaeroGen (Oxoid 88

Inc., Basingstoke, United Kingdom); or under an atmosphere of 10% H2, 10% CO2, and 89

80% N2) using MT-EYE (1.5% McClung-Toabe agar (Difco, Tucker, GA, USA), 5% egg 90

yolk extract, and 5% yeast extract (Difco)) plates. Single colonies were inoculated into 10 91

mL of TPGY (5% w/vol tryptone (Difco), 0.5% w/vol peptone (Difco), 0.4% w/vol 92


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glucose (Difco), 2% w/vol yeast extract (Difco), and 0.1% sodium thioglycolate (Sigma, 93

St. Louis, MO, USA)) medium for 24 h. For matched subcultures, a single colony was 94

serially cultured three times onto MT-EYE plates prior to TPGY inoculation. Genomic 95

DNA from C. botulinum isolates listed in Table 1 was extracted using the Qiagen 96

DNEasy Blood and Tissue kit (Qiagen, Mississauga, Canada). Libraries were prepared 97

using Nextera or TruSeq kits and sequenced using paired-end sequencing-by-synthesis 98

(2×250 cycles) on GAIIx or MiSeq instruments according to manufacturer protocols 99

(Illumina Inc., San Diego, USA). Average read coverage for all isolates exceeded 50-fold 100

based on the Alaska E43 reference genome size (3.66Mb). Virtual reads for publicly 101

available genomes were generated with Wombac v1.2 (length = 100; coverage = 50; 102

quality = 40) (www.vicbioinformatics.com/software.wombac.shtml). 103

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Identification of core SNPs. Reads were reference-mapped using SMALT v0.7.4 (word 105

length = 13, step = 1 (25), SNPs were identified in FreeBayes v0.9.6 (26) (map/base 106

quality ≥ 35; alternate fraction ≥ 0.75) and cross-referenced with SAMtools v0.1.18 (27) 107

(mpileup ≥ 20). SNPs identified using both methods were selected further analysis. 108

Complex SNP events are not distinguished in FreeBayes and were treated as individual 109

events. The subset of SNP loci present among all data sets was identified (core SNPs), 110

and a Perl script generated a meta-alignment for phylogeny analysis 111

(https://github.com/apetkau/core-phylogenomics; commit f132bf6). For in silico MLST, 112

sequence reads were mapped to a database of known MLST alleles (12, 23) for C. 113

botulinum Group II using SRST2 v2.1 (read mismatch = 10) (29). Consensus allele 114

sequences were concatenated and aligned with ClustalW v1.82 (clustalw-mpi, default 115


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parameters) (30). Alignments were visually inspected for accuracy and converted to 116

PHYLIP format (http://sequenceconversion.bugaco.com/converter/biology/sequences/). 117

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Phylogeny analyses. Maximum-likelihood phylogenetic trees were built using PhyML 119

v3.1 (31) using a GTR+G substitution model and a tree topology search for best 120

NNIs/SPRs and initial BioNJ tree. Branch support values for were estimated using the 121

approximate likelihood ratio test (32). Images were rendered in FigTree (v1.4.1) 122

(www.tree.bio.ed.ac.uk/software/figtree). For core SNP analyses, branch scales were 123

converted by multiplying the number of substitutions per position by the number of core 124

SNPs identified for the population. 125

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Nucleotide accession numbers. All sequence reads and MLST alleles included in this 127

study have been deposited in the NCBI Short Read Archive (SRP059342) and GenBank 128

(atpD, KT034476−KT034633; trpB, KT034634−KT034791; rpoB, KT034792− 129

KT035949; guaA, KT034950−KT035107; pta, KT035108−KT035265; ilvD, 130

KT035266−KT035423; lepA, KT035424−KT035581; gyrB, KT035582−KT035739; 131

recA, KT035740−KT035897; oppB, KT035898−KT036055; 23s rRNA rumA, 132

KT036056−KT036213; pyc, KT036214−KT036371)(http://www.ncbi.nlm.nih.gov/). 133

Public genomes included in analyses: Alaska E43, NC_010723; Eklund 17B, 134

NC_010674; pCLL, NC_010680; 202F, CP006903; pCBI, CP006904; E1 Beluga, 135

ACSC00000000; CDC66177, ALYJ00000000; CB11/1-1, AORM00000000; KAPB-3, 136

JQOK01000001; DB2, JQOJ01000001; NCTC 8226, CP010520; NCTC 8550, 137

CP010521; NCTC 11219, JXMR01000000 (23, 33-36). 138


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RESULTS AND DISCUSSION 140

In silico MLST profiling. To determine whether whole genome sequence data could be 141

used for en masse MLST profiling of 163 C. botulinum Group II isolates, and also to 142

compare profiles using a previously published typing scheme, consensus alleles were 143

determined in silico using SRST2 (v2.1) (29) for 15 MLST loci described by MacDonald 144

et al. (12). Concatenated alleles were aligned using ClustalW and maximum likelihood 145

phylogenetic trees were estimated using PhyML. 146

For this population, allelic heterogeneity produced ambiguous allele calls for 16S 147

(11 copies) and tuf (2 copies). tuf heterogeneity was observed for several isolates, where 148

the two alleles identified differed by a single SNP (data not shown). In addition, mutL 149

indels and elements showing high levels of identity resulted in consensus call 150

uncertainties (data not shown). Because of the ambiguous allele calls, these three loci 151

were excluded from the in silico MLST typing strategy. Despite this omission, the 152

MLST-12 phylogeny analysis resolved 25 distinct profiles for 41 previously 153

characterized isolates (Figure S1), which is consistent with previous reports using the 154

complete MLST-15 scheme (12), indicating that MLST resolution is comparable 155

irrespective of 16S, tuf, and mutL locus inclusion. 156

The MLST-12 scheme distinguished 29 taxonomic groups for the 163 isolates 157

analyzed (Figure 1). MLST profiles were consistent whether WGS read data or NCBI 158

genomic sequences were used, as observed for three strains: Eklund 17B, DB2, and 159

KAPB-3 (15-17, 35, 48)) (Figure S1). MLST profiles using WGS read data from 160

technical replicates (211 VH Dolman, SO329E2, SOKR-44E1) were also in agreement 161


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(data not shown). 162

Consistent with other reports (12, 23), a large cluster (20 taxa; n=149) that 163

included the Alaska E43 strain (NC_010723) represented a wide variety of BoNT/E toxin 164

subtypes found in C. botulinum Group II bacteria (E1-E3; E6-E8; E10-E11) (Figures 1 165

and S1). Interestingly, a nontoxic isolate from Saskatchewan, BFLY-1, also typed to this 166

large cluster. This isolate was derived from a waterfowl sample collected in 1980 as part 167

of an avian botulism investigation (outbreak 7) but tested negative for BoNT in 2012 168

(Table 1). A second population (9 taxa; n=14) clustered closer to the BoNT/B4 strain 169

Eklund 17B and included CDC66177, a BoNT/E9 isolate, as reported elsewhere (23). 170

Other BoNT/B4 isolates (Eklund 2B, DB2, KAPB-3, and KAPB-8), two BoNT/F6 171

isolates (202F, 610F) and several nontoxigenic isolates also grouped with the Eklund 17B 172

cluster. Two of the nontoxic isolates (BFLY-2, BFLY-6) in this cluster were derived 173

from the aforementioned waterfowl sample source (1980, outbreak 7) that was never 174

confirmed as toxigenic. However, three isolates (BE0211E1, BE0211E2, BE0211E3) 175

from a food sample (beluga) collected during a 2002 foodborne botulism outbreak in 176

Kuujjuaq (outbreak 30) originally tested positive for BoNT/E using the mouse bioassay 177

(Table 1 and data not shown). It is possible that these isolates have lost their neurotoxin 178

genes as reported for other group II C. botulinum strains (22, 48). 179

MLST profiling showed that the majority of taxa comprised isolates representing 180

a single BoNT subtype. Four taxa, however, included members carrying different toxin 181

subtypes (E1/E3; E3/E11; E3/E10) (Figure 1). 182

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Core SNP phylogeny profiling. Recombination and other forms of horizontal transfer 184


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can confound phylogeny analysis (37, 38). Although recent reports claim that bacterial 185

phylogenetic reconstruction from whole genome sequence data is relatively robust to 186

recombination (39), the effect of horizontal transfer events is potentially great for 187

phylogenetic estimations based on a relatively small portion of the genome. To assess 188

how genome-wide polymorphism profiling compares to the MLST analysis, isolates were 189

examined using core single nucleotide polymorphism (SNP) phylogeny, a method that 190

has been successfully adapted for many clinically and environmentally relevant bacterial 191

species undergoing horizontal gene transfer and recombination including clostridia (40-192

44). 193

Read mapping to the Alaska E43 reference genome (NC_010723) identified 194

69,321 core SNP loci for the C. botulinum Group II population (n=163), and, consistent 195

with MLST (Figure 1), maximum likelihood phylogeny analysis showed species-level 196

clustering into Eklund 17B (n=14) and Alaska E43 (n=149) subgroups (Figure 2). This 197

data confirms and extends reports by several groups which showed that genetic and/or 198

genomic diversity of C. botulinum group II strains formed two distinct subpopulations 199

(11, 22, 23). 200

Members of the Eklund 17B and Alaska E43 clusters differed by ≥ 65,598 SNPs 201

and could be distinguished by as little as one SNP 202

(https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv). 203

This clustering was also observed when other high quality finished genomes (Eklund 204

17B, 202F) were used as references (data not shown). 205

While the tree topologies differed between MLST and core SNP methods, profile 206

classifications between the two methods were remarkably consistent, with core SNP 207


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demonstrating an increased resolution over MLST. Core SNP phylogeny distinguished 31 208

clade profiles differing by > 60 core SNPs (Figure 2) and resolved subgroups for three 209

MLST taxa (Table S1). Two subgroups were identified for isolates in clade 7 by core 210

SNP phylogeny, and core SNP parsed BoNT/E3 and BoNT/E10 isolates from a common 211

MLST taxon (Figure 1) into clades 11 and 12, respectively (Figure 2, Table S1). In 212

addition, core SNP analysis distinguished SO309E2, a BoNT/E10 isolate from Ungava 213

Bay, from MU8903E, a BoNT/E3 isolate from the Northwest Territories (Figure 2), 214

which typed to the same profile by MLST (Figure 1). At this level of resolution, 215

however, core SNP phylogeny did not provide sufficient resolution to distinguish 216

BoNT/E10 from BoNT/E3 isolates in clade 13 (Figure 2). 217

As observed by MLST (Figure 1), food and clinical isolates collected during a 218

common outbreak (outbreaks 10, 14, 16-18, 20, 21, 26, 29, 32, 33, 35 as listed in Table 1) 219

typically typed within the same core SNP profile (Figure 2, Table S1). Likewise, 220

multiple strains recovered from a single environmental sample source were often typed to 221

a common clade by MLST and core SNP (FWSK02-01E2/3; FWSK02-06E1/2; TRK02-222

06E2/3; SO329E1/2; SWKR38E1/2; SO303E1/3/4/5; SO304E1/2; SO305E1/2; SOKR-223

22E1/3; TRK02-08E1/3) (Figures 1 and 2; Table S1). This observation would be 224

expected of isolates with a high level of relatedness in the population. 225

Exceptions were noted for two BoNT/E3 isolates derived from a food sample 226

collected during an outbreak in Tasiujaq (2004, outbreak 31), which generated different 227

clades (IG0410E3LC, clade 13; IG0410E2LC, clade 16), suggesting that these isolates 228

are divergent. In addition, a BoNT/E10 gastric liquid isolate, GA9706EMA, from 229

outbreak 19 (Tasiujaq, 1997), typed to clade 17, while an isolate from the suspected 230


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source food sample (MI9706E) grouped to clade 8 and encoded BoNT/E3. 231

Distinct clade classifications were also observed for multiple isolates derived 232

from 18 environmental samples collected from the Kuujjuaq or Tasiujaq regions of 233

Ungava Bay (TRK02-08E; TRK02-04E; TRK02-02E; SWKR0402E; SOKR-50E; 234

SOKR-49E; SOKR-46E; SOKR-44E; SOKR-34E; SOKR-25E; SOKR-24E; SOKR-23E; 235

RSKR-68E) (Figures 1- 3). For example, three isolates derived from a single coastal rock 236

sample from the Kuujjuaq region of Ungava Bay typed to clades 11 (RSKR-68E1), 17 237

(RSKR-68E2), and 7 (RSKR-68E3) (Figures 2 and 3). These results are consistent with 238

PFGE data (14), which also indicates isolate diversity within a single environmental 239

sample in these regions of northern Canada. Together, these results indicate MLST and 240

core SNP analysis can provide effective species-level characterization for broad-range 241

isolate inclusion/exclusion during an epidemiological investigation. 242

C. botulinum Group II in silico MLST and species-wide core SNP phylogeny 243

profiling are in agreement with flagellin (flaVR, flaB) typing methods (15, 16), but show 244

enhanced resolution capability. For instance, flagellin profiling groups several members 245

of the Eklund 17B cluster as flaVR9 and flaB- (Eklund 17B, 610F, KAPB-3, KAPB-8), 246

while members of the E43 cluster are flaB+ and flaVR8 (SO329E1 and SOKR38E2), 247

which type to two clades (7, 12), or flaB+ and flaVR10, (ex: Gordon, SW280E, 248

SO309E2, FE0005EJT; S9510E) which type to five clades (10/14/16/18/22) by MLST 249

and/or core SNP (15, 16). 250

High resolution core SNP analysis. Several core SNP clades were comprised solely of 251

isolates from the Ungava Bay region. This is not surprising given that the majority of 252

isolates studied originate from Ungava Bay specimens. Isolates from this area typed to a 253


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large proportion the profiles identified by core SNP and MLST (Figures 1 and 2). 254

However many profiles included specimens from multiple outbreaks (clades 7, 13, 15, 255

16, 21, 22) and regions (clades 16, 17, 21, 22). For instance, clade 16 included isolates 256

from twelve outbreaks (10, 12, 20, 25-27, 31, 33, 37) across eight communities in Canada 257

(regions: Ungava Bay, Baffin Island, Northwest Territories, British Columbia) and one 258

location in France (Paris) (Figure 3). 259

To determine whether closely related isolates could be resolved by outbreak or 260

region of origin, core SNP analysis was performed on isolates from clade 16 (Figure 2), 261

which includes the Alaska E43 reference genome. This analysis generated 20 unique 262

profiles for 27 isolates based on 263 core SNP loci (Figure 4). Two E1 isolates, NCTC 263

8550 and NCTC 8226, originating from France and British Columbia, respectively (50, 264

51), typed distal to the E3 isolates in this population (Figure 4). Isolates from the 265

Northwest Territories typed to a distinct cluster, and isolates from the communities of 266

Inuvik (outbreaks 25, 1999 and 26, 2000) and Aklavik (outbreak 27, 2001) differed by 267

one core SNP 268

(https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv, 269

NC_010723 position 1021249)(Figure 3). Three additional non-core SNP loci were 270

identified that discriminate between outbreaks 25, 26, and 27 (NC_010723 positions: 271

2263189, 2294119, 2295007) 272

(https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv). 273

Core SNP phylogeny also clustered isolates from the same outbreaks together as 274

observed for outbreaks 10 (Kangiqsualujjuaq, 1995; 0 SNPs), 20 (Kangiqsualujjuaq, 275

1997; ≤ 2 SNPs), and 33 (Baffin Island, 2000; 0 SNPs) (Figures 3, 4 and 276


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https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv). 277

Several of these isolates (outbreaks 10, 12, 20, 25, 26) were profiled previously by PFGE 278

methods (17). Compared to PFGE, the results in Figure 4 demonstrate that core SNP 279

analysis can provide enhanced typing discrimination among highly related isolates. 280

Despite the propensity for C. botulinum bacteria to undergo horizontal gene 281

transfer (11, 12, 23, 45-47), the data presented here indicate that core SNP analysis can 282

resolve even highly related isolates by outbreak and/or location and provides a useful tool 283

for epidemiological investigations. In addition, the deposition of sequence reads for 152 284

C. botulinum Group II isolates, as well as a catalog of in silico MLST alleles and SNP 285

locus calls, provides a significant resource to the scientific community. 286

287

ACKNOWLEDGEMENTS 288

This work was supported by Canadian Safety and Security Program project 07-219RD 289

from Defence Research and Development Canada and does not reflect the opinion of the 290

Government of Canada. 291

292

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butyricum type E strains. BMC Biol. 7:66. doi: 10.1186/1741-7007-7-66. 459

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2164-12-185. 466

48. Carter AT, Austin JW, Weedmark KA, Corbett C, Peck MW. 2014. Three 467

classes of plasmid (47-63 kb) carry the type B neurotoxin gene cluster of Group II 468

Clostridium botulinum. Genome Biol. Evol. . doi: evu164 [pii]. 469

49. Ball AP, Hopkinson RB, Farrell ID, Hutchison JG, Paul R, Watson RD, Page 470

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the Birmingham outbreak. Q. J. Med. 48:473-491. 473

50. Dolman CE, Kerr DE. 1947. Botulism in Canada, with report of a type E outbreak at 474

Nanaimo, B. C. Can. J. Public Health. 38:48-57. 475


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51. Prevot AR, Huet M. 1951. Existence in France of human botulism due to fish and to 476

Clostridium botulinum E. Bull. Acad. Natl. Med. 135:432-435. 477


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Figure 1. In silico MLST phylogeny for C. botulinum Group II isolates. Concatenated 478

consensus sequences for 12 MLST loci were aligned with ClustalW and analysed with 479

PhyML to estimate a maximum likelihood phylogeny for C. botulinum Group II isolates 480

(refer to Methods). Inset, MLST phylogeny for all isolates (n=163) showing two clusters 481

separated by a genetic distance of 0.034; outset, zoomed view of Alaska E43 cluster 482

isolates (n=149). Outbreak number, region of origin, and BoNT serotypes/variants for 483

isolates in the Alaska E43 cluster are indicated: BoNT/B4, BoNT/F6, BoNT/E1, 484

BoNT/E3, BoNT/E9, BoNT/E11, Nontoxigenic (NT) (details in Table 1). Eleven 485

publicly available genomes were included in analysis (*). Taxa that include multiple 486

BoNT serotypes/subtypes are indicated (yellow). Clades corresponding to core SNP 487

phylogeny (Figure 3) are indicated. Scale bars, number of nucleotide substitutions per 488

site. 489

490

Figure 2. Core SNP phylogenomic analysis of C. botulinum Group II isolates. 491

Maximum likelihood analysis of 69,321 core SNP loci identified among C. botulinum 492

Group II isolates by reference mapping to to Alaska E43 (NC_010723). Inset, core SNP 493

phylogeny for all isolates (n=163) showing two clusters separated by > 65,598 SNPs; 494

outset, zoomed view of Alaska E43 cluster isolates (n=149) showing clade-level 495

classifications (indicated, right). Outbreak number, region of origin, and BoNT 496

serotypes/variants for isolates in the Alaska E43 cluster are indicated: BoNT/B4, 497

BoNT/F6, BoNT/E1, BoNT/E3, BoNT/E9, BoNT/E11, Nontoxigenic (NT) 498

(details in Table 1). Eleven publicly available genomes were included in analysis (*). 499


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Clades that include multiple BoNT serotypes/subtypes are indicated (yellow) and clade 500

designations are indicated (right). 501

502

Figure 3. Geographic origins of C. botulinum Group II isolates studied. 503

Communities and regions where C. botulinum isolates originate (Table 1) plotted on a 504

world map (base map, © OpenStreetMap contributors 505

[http://www.openstreetmap.org/copyright]); image generated using CartoDB 506

[ ]). Inset: zoomed view of Canada’s Nunavik region. www.cartodb.com507

508

Figure 4. Core SNP analysis of Clade 16 isolates. Maximum likelihood phylogeny 509

based on 263 core SNPs for isolates typing to clade 16 (Figure 2) showing high-510

resolution topology for clinical and food isolates from communities in France (Paris) and 511

Canada (Nanaimo, BC (outbreak 37), Inuvik, NWT (outbreaks 25-26), Aklavik, NWT 512

(outbreak 27) Tasiujaq, QC (outbreaks 12, 30), Kangisualujjuaq, QC (outbreaks 10, 20), 513

Baffin Island, NU (outbreak 33) as well as Environmental isolates (*) from Kuujjuaq and 514

Tasiujaq. Alaska E43, reference genome (NC_010723). Distance bar, number of SNPs. 515

Branch lengths ≥ 20 SNPs are indicated. 516

517

Figure S1. In silico MLST-12 phylogeny for C. botulinum Group II isolates. 518

Concatenated consensus sequences for 12 MLST loci were aligned with ClustalW and 519

analysed with PhyML to estimate a maximum likelihood phylogeny for C. botulinum 520

Group II isolates (refer to Methods). The BoNT serotypes/variants for isolates are 521

indicated: BoNT/B4, BoNT/F6, BoNT/E1, BoNT/E2, BoNT/E3, BoNT/E6, 522


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BoNT/E7, BoNT/E8, BoNT/E9, BoNT/E10, BoNT/E11, Nontoxigenic (NT) 523

(details in Table 1). Eleven publicly available genomes are indicated (*). Scale bar, 524

number of nucleotide substitutions per site. Isolates described by MacDonald et al. (1) 525

are included to illustrate the same number of taxa classifications (25) for these isolates 526

using the MLST-15 and MLST-12 (loci excluded: 16S, tuf, mutL). MLST profiles were 527

identical for technical replicates (211 VH Dolman, SO329E2, SOKR-44E1) as well as for 528

strains with read data and NCBI reference genomes (Eklund 17B, KAPB-3, and DB2). 529

530

Supplemental Figure S1 References: 531

1. Macdonald TE, Helma CH, Shou Y, Valdez YE, Ticknor LO, Foley BT, Davis 532

SW, Hannett GE, Kelly-Cirino CD, Barash JR, Arnon SS, Lindstrom M, Korkeala 533

H, Smith LA, Smith TJ, Hill KK. 2011. Analysis of Clostridium botulinum serotype E 534

strains by using multilocus sequence typing, amplified fragment length polymorphism, 535

variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing. 536

Appl. Environ. Microbiol. 77:8625-8634. doi: 10.1128/AEM.05155-11. 537


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Table 1. Clostridium botulinum Group II Isolates Studied.

Isolate OBa BoNTb Year Sample Type Origin Region Location Source 202F* F6 1965 Environmental Marine sediment PAC Pacific Coast, USA CP006903, CP006904(34)

211 VH Dolman 2 E3 1949 Food Pickled herring PAC Vancouver, BC NML(24) 610F 3 F6 1966 Food Salmon PAC Oregon, USA BRS(16-17, 48) Alaska E43* E3 ND Food Salmon Eggs PAC Alaska, USA NC_010723(12) BE0211E1 30 NTc 2002 Food Beluga UB Kuujjuaq, QC BRS

BE0211E2 30 NTc 2002 Food Beluga UB Kuujjuaq, QC BRS

BE0211E3 30 NTc 2002 Food Beluga UB Kuujjuaq, QC BRS

BE9708E1 17 E3 1997 Food Beluga WHB Arviat, NU BRS(24) BFLY-1 7 NTd 1980 Avian Duck carcasse SK Little Quill Lake, SK NML

BFLY-2 7 NTd 1980 Avian Duck carcasse SK Little Quill Lake, SK NML

BFLY-6 7 NTd 1980 Avian Duck carcasse SK Little Quill Lake, SK NML

CA9708E1 17 E3 1997 Food Caribou WHB Arviat, NU BRS(24)

CB11/1-1* E1 1999 Food Whitefish roe EUR Finland AORM00000000(33)

CDC66177* E9 1993 Environmental Soil SAM Dolavon, Argentina ALYJ00000000(23)

DB2* B4 1968 Environmental Sediment PAC USA JQOJ01000001(35, 48)

E-RUSS 1 E1 ~1936 Food Sturgeon intestine EUR Sea of Azov, Ukraine BRS(24)

E1 Beluga* E1 1951 Food Fermented Whale Flippers PAC USA ACSC00000000(12)

E1 Dolman 6 E1 <1980 Clinical ND PAC ND NML(23)

Eklund 17B* B4 1965 Environmental Marine sediment PAC Washington, USA BRS(15-17); NC_010674, NC_010680Eklund 2B B4 1965 Environmental Marine sediment PAC USA BRS(15-17)

F9508EPB 12 E3 1995 Clinical Feces UB Tasiujaq, QC BRS(24)

FE0005EJT 26 E3 2000 Clinical Feces NWT Inuvik, NWT BRS(24) FE0201E1BC 29 E3 2002 Clinical Feces UB Tasiujaq, QC BRS(24) FE0202E1TC 29 E3 2002 Clinical Feces UB Tasiujaq, QC BRS(24) FE0801E1IT 33 E3 2008 Clinical Feces BI Kimmirut, NU BRS(24) FE1010E1JL 35 E3 2010 Clinical Feces UB Kuujjuaq, QC BRS(24) FE9507EEA 10 E3 1995 Clinical Feces UB Kangiqsualujjuaq, QC BRS(24) FE9604ENT 14 E3 1996 Clinical Feces UB Quaqtaq, QC BRS(24)

FE9708E1JI 17 E3 1997 Clinical Feces WHB Arviat, NU BRS(24) FE9708E1PI 17 E3 1997 Clinical Feces WHB Arviat, NU BRS(24)

FE9709EBB 20 E3 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS(24)

FE9709EBB2 21 E10 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS(24)

FE9709ELB 20 E3 1997 Clinical Feces UB Kangiqsualujjuaq, QC BRS(24) FE9908EDL 24 E3 1999 Clinical Feces UB Aupaluk, QC BRS(24)

FE9909ERG 25 E3 1999 Clinical Feces NWT Inuvik, NWT BRS(24)

FWKR02E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24)

FWKR11E1 E10 2004 Environmental Freshwater UB Kuujjuaq, QC BRS(24)

FWSK02-01E2 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24) FWSK02-01E3 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24) FWSK02-02E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24) FWSK02-04E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24) FWSK02-05E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24) FWSK02-05E2 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24) FWSK02-06E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24) FWSK02-06E2 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24)

FWSK02-07E1 E3 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24)

FWSK02-07E3 E10 2004 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24)

FWSKR40E1 E10 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24)

FWSKR4802E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24)

GA0108EJC 28 E3 2001 Clinical Gastric liquid UB Tasiujaq, QC BRS(24)

GA0202E1TS 29 E3 2002 Clinical Gastric liquid UB Tasiujaq, QC BRS(24) GA0702E1 32 E3 2007 Clinical Gastric liquid UB Kuujjuaq, QC BRS(24) GA0702E1CS 32 E3 2007 Clinical Gastric liquid UB Kuujjuaq, QC BRS(24) GA0808EPA 34 E3 2008 Clinical Gastric liquid UB Kuujjuaq, QC BRS(24) GA0811E1IT 33 E3 2008 Clinical Gastric liquid BI Baffin Island, NU BRS(24) GA1101E1BB 36 E10 2011 Clinical Gastric liquid UB Kuujjuaq, QC BRS(24) GA9604EAK 14 E3 1996 Clinical Gastric liquid UB Quaqtaq, QC BRS(24) GA9604ESM 14 E3 1996 Clinical Gastric liquid UB Quaqtaq, QC BRS(24) GA9608EPB 16 E3 1996 Clinical Gastric liquid UB Tasiujaq, QC BRS(24) GA9706EMA 19 E10 1997 Clinical Gastric liquid UB Tasiujaq, QC BRS(24) GA9709EHS 20 E3 1997 Clinical Gastric liquid UB Kangiqsualujjuaq, QC BRS(24) GA9709EJA 20 E3 1997 Clinical Gastric liquid UB Kangiqsualujjuaq, QC BRS(24) GA9709ENS 20 E3 1997 Clinical Gastric liquid UB Kangiqsualujjuaq, QC BRS(24) GA9811E2MS 22 E3 1998 Clinical Gastric liquid UB Kuujjuaq, QC BRS(24)

Gordon 5 E3 1975 Clinical Clinical specimen UB Kuujjuaq, QC BRS(24)

IG0201E2BC 29 E3 2002 Food Walrus igunaq UB Tasiujaq, QC BRS(24)

IG0202E1 29 E3 2002 Food Walrus igunaq UB Tasiujaq, QC BRS(24)

IG0410E2LC 31 E3 2004 Food Igunaq UB Tasiujaq, QC BRS(24)

IG0410E3LC 31 E3 2004 Food Igunaq UB Tasiujaq, QC BRS(24)

IN01SE63E1 E3 2001 Marine Mammal Seal Intestine UB Kuujjuaq, QC BRS(24)

INWB2202E1 E3 2002 Marine Mammal Seal Intestine UB Kangiqsujuaq, QC BRS(24)

KAPB-3* 8 B4 1981 Food Salted whitefish PAC California, USA JQOK01000001(35, 48)

KAPB-8 8 B4 1981 Food Salted whitefish PAC California, USA BRS (48)

ME0702E1CS 32 E3 2007 Food Seal meat in oil UB Kuujjuaq, QC BRS(24)

ME1010E1JL 35 E3 2010 Food Meat UB Kuujjuaq, QC BRS(24)

MI19709E 21 E10 1997 Food Seal igunaq UB Kangiqsualujjuaq, QC BRS(24)



MI9507E 10 E3 1995 Food Seal misiraq UB Kangiqsualujjuaq, QC BRS(24)

MI9608ESM 15 E3 1996 Food Seal meat UB Tasiujaq, QC BRS(24)

MI9706E 19 E3 1997 Food Igunaq UB Tasiujaq, QC BRS(24)

MSKR5102E2 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS(24)

MU0005EJT 26 E3 2000 Food Muktuk NWT Inuvik, NWT BRS(24)

MU0103EMS 27 E3 2001 Food Muktuk oil NWT Aklavik, NT BRS(24)


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*Publicly available genome; ND: No data; aOB, outbreak; bBoNT serotype/subtype; cIsolate originally tested positive for BoNT/E in 2002; dIsolated from original (1980) specimen in 2012 and tested negative for BoNT using the mouse bioassay; eSample collected in 1980 tested negative for BoNT in 2012; NT, Nontoxigenic; BRS, Botulism Reference Service, Health Canada; NML, National Microbiology Laboratory, Public Health Agency of Canada; Regions: EUR, Europe; PAC, Pacific; SK, Saskatchewan; WHB, West Hudson's Bay; EHB, East Hudson's Bay; SAM, South America; BI, Baffin Island; UB, Ungava Bay; BC, British Columbia; QC, Quebec; NU, Nunavut.

MU8903E 9 E3 1989 Food Muktuk NWT Paulatuk, NWT BRS(24)

MU9708EJG-F235 18 E3 1997 Food Muktuk NWT Aklavik, NWT BRS(24)

MU9708EJG-F236 18 E3 1997 Food Muktuk NWT Aklavik, NWT BRS(24)

NCTC 11219* 4 E3 1974 Food Canned Salmon PAC Alaska, USA JXMR01000000(36, 49)

NCTC 8266* 37 E1 1944 Food Canned Salmon PAC Nanaimo, BC CP010520(36, 50)

NCTC 8550* E1 1952 Food Fish EUR France CP010521(36, 51)

PBKR-41E1 E10 2002 Environmental Peat Bog UB Kuujjuaq, QC BRS(24)

RSKR-68E1 E3 2004 Environmental Coastal rock UB Kuujjuaq, QC BRS(24)



S9510E 13 E3 1995 Food Seal meat UB Kuujjuaq, QC BRS(24)

SE9908E E3 1999 Marine Mammal Seal Intestine UB Kuujjuaq, QC BRS(24)

SO303E1 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS(24)




SO304E1 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS(24)




SO307E1 E10 2003 Environmental Shoreline soil EHB Puvirnituq, QC BRS(24)

SO309E2 E10 2003 Environmental Shoreline soil EHB Puvirnituq, QC BRS(24)

SO321E1 E3 2001 Environmental Shoreline soil UB Kangirsuk, QC BRS(24)

SO325E E3 2001 Environmental Shoreline soil UB Tasiujaq, QC BRS(24)

SO326E1 E3 2001 Environmental Shoreline soil UB Tasiujaq, QC BRS(24)

SO329E1 E11 2001 Environmental Shoreline soil UB Kuujjuaq, QC BRS(24)

SO329E2 E11 2001 Environmental Shoreline soil UB Kuujjuaq, QC BRS(24)

SOKR-19E1 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS(24)

SOKR-20E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS(24)



SOKR-23E1 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS(24)




SOKR-25E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24)



SOKR-33E1 E10 2002 Environmental Peat bog UB Kuujjuaq, QC BRS(24)


SOKR-34E5 E10 2002 Environmental Sediment UB Kuujjuaq, QC BRS(24)

SOKR-3602E1 E3 2002 Environmental Shoreline soil UB Tasiujaq, QC BRS(24)


SOKR-42E1 E10 2002 Environmental Shoreline soil UB Tasiujaq, QC BRS(24)











SP417E-Alc E10 2001 Environmental Coastal rock EHB Puvirnituq, QC BRS(24)

SP417E-NT E10 2001 Environmental Coastal rock EHB Puvirnituq, QC BRS(24)

SP457-458E E3 2002 Environmental Coastal rock UB Kuujjuaq, QC BRS(24)

SW279E E3 2001 Environmental Seawater UB Kuujjuaq, QC BRS(24)

SW280E E11 2001 Environmental Seawater UB Kuujjuaq, QC BRS(24)

SWKR0402E1 E3 2004 Environmental Seawater UB Kuujjuaq, QC BRS(24) SWKR0402E2 E3 2004 Environmental Seawater UB Kuujjuaq, QC BRS(24) SWKR07E1 E3 2004 Environmental Seawater UB Kuujjuaq, QC BRS(24) SWKR24E1 E11 2004 Environmental Seawater UB Kuujjuaq, QC BRS(24)

SWKR38E1 E10 2004 Environmental Seawater UB Kuujjuaq, QC BRS(24) SWKR38E2 E10 2004 Environmental Seawater UB Kuujjuaq, QC BRS(24) TRK02-02E1 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) TRK02-02E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) TRK02-04E1 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) TRK02-04E3 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) TRK02-06E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) TRK02-06E3 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) TRK02-07E1 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS(24) TRK02-08E1 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) TRK02-08E3 E10 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS(24) V9804E 23 E3 1998 Food Seal meat UB Kuujjuaq, QC BRS(24) VI9508E 11 E3 1995 Food Seal igunaq UB Kuujjuaq, QC BRS(24) VI9608EPB 16 E3 1996 Food Seal meat UB Tasiujaq, QC BRS(24) VO0202E1TC 29 E3 2002 Food Gastric liquid UB Tasiujaq, QC BRS(24)


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