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. 2015 Aug 7;81(17):5938–5948. doi: 10.1128/AEM.01155-15

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

K A Weedmark a, P Mabon a, K L Hayden a, D Lambert b,*, G Van Domselaar a, J W Austin b, C R Corbett a,✉
Editor: M W Griffiths
PMCID: PMC4551264  PMID: 26116673

Abstract

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.

INTRODUCTION

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 laboratory-confirmed foodborne botulism outbreaks occurring between 1985 and 2005 (n = 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 used for genetic characterization of C. botulinum group II strains (11–24).

In the present study, whole-genome sequence (WGS) data from 152 C. botulinum group II isolates were analyzed with 11 publicly available genomes (163 total isolates characterized). 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.

Core SNP phylogeny analysis resolved isolates by outbreak and/or location of origin. These results demonstrate the utility of in silico C. botulinum characterization using next-generation sequence (NGS) data and provide discrete high-quality SNP loci, MLST alleles, and read data for 152 C. botulinum group II isolates.

MATERIALS AND METHODS

Culture conditions, DNA isolation, and genome sequencing.

C. botulinum group II strains were cultured at 30°C for 48 to 72 h under anaerobic conditions (AnaeroGen [Oxoid Inc., Basingstoke, United Kingdom] or under an atmosphere of 10% H2, 10% CO2, and 80% N2) using MT-EYE (1.5% McClung-Toabe agar [Difco, Tucker, GA], 5% egg yolk extract, and 5% yeast extract [Difco]) plates. Single colonies were inoculated into 10 ml of TPGY (5% [wt/vol] tryptone [Difco], 0.5% [wt/vol] peptone [Difco], 0.4% [wt/vol] glucose [Difco], 2% [wt/vol] yeast extract [Difco], and 0.1% sodium thioglycolate [Sigma, St. Louis, MO]) medium for 24 h. For matched subcultures, a single colony was serially cultured three times onto MT-EYE plates prior to TPGY inoculation. Genomic DNA from C. botulinum isolates listed in Table 1 was extracted using the Qiagen DNeasy blood and tissue kit (Qiagen, Mississauga, Canada). Libraries were prepared using Nextera or TruSeq kits and sequenced using paired-end sequencing by synthesis (2 × 250 cycles) on GAIIx or MiSeq instruments according to manufacturer protocols (Illumina Inc., San Diego, CA). Average read coverage for all isolates exceeded 50-fold based on the Alaska E43 reference genome size (3.66 Mb). Virtual reads for publicly available genomes were generated with Wombac v1.2 (length = 100; coverage = 50; quality = 40) (http://www.vicbioinformatics.com/software.wombac.shtml).

TABLE 1.

Clostridium botulinum group II isolates studieda

Isolate OB BoNT serotype or subtype Yr Sample type Origin Region Location Accession number(s), reference(s), and/or source
202Fb F6 1965 Environmental Marine sediment PAC Pacific Coast, USA CP006903, CP006904 (33)
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, 36)
Alaska E43b 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)
CB11/1-1b E1 1999 Food Whitefish roe EUR Finland AORM00000000 (32)
CDC66177b E9 1993 Environmental Soil SAM Dolavon, Argentina ALYJ00000000 (23)
DB2b B4 1968 Environmental Sediment PAC USA JQOJ01000000 (34, 36)
E-RUSS 1 E1 ∼1936 Food Sturgeon intestine EUR Sea of Azov, Ukraine BRS (24)
E1 Belugab E1 1951 Food Fermented whale flippers PAC USA ACSC00000000 (12)
E1 Dolman 6 E1 <1980 Clinical ND PAC ND NML (23)
Eklund 17Bb B4 1965 Environmental Marine sediment PAC Washington, USA BRS (15–17); NC_010674, NC_010680
Eklund 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-3b 8 B4 1981 Food Salted whitefish PAC California, USA JQOK01000000 (34, 36)
KAPB-8 8 B4 1981 Food Salted whitefish PAC California, USA BRS (36)
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)
MI59709E 21 E10 1997 Food Seal igunaq UB Kangiqsualujjuaq, QC BRS (24)
MI69709E 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)
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 11219b 4 E3 1974 Food Canned salmon PAC Alaska, USA JXMR01000000 (35, 50)
NCTC 8266b 37 E1 1944 Food Canned salmon PAC Nanaimo, BC CP010520 (35, 45)
NCTC 8550b E1 1952 Food Fish EUR France CP010521 (35, 46)
PBKR-41E1 E10 2002 Environmental Peat bog UB Kuujjuaq, QC BRS (24)
RSKR-68E1 E3 2004 Environmental Coastal rock UB Kuujjuaq, QC BRS (24)
RSKR-68E2 E10 2004 Environmental Coastal rock UB Kuujjuaq, QC BRS (24)
RSKR-68E3 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)
SO303E3 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS (24)
SO303E4 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS (24)
SO303E5 E10 2001 Environmental Shoreline soil EHB Umiujaq, QC BRS (24)
SO304E1 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS (24)
SO304E2 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS (24)
SO305E1 E10 2003 Environmental Shoreline soil EHB Inukjuak, QC BRS (24)
SO305E2 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-22E1 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (24)
SOKR-22E3 E3 2002 Environmental Freshwater sediment UB Kuujjuaq, QC BRS (24)
SOKR-23E1 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (24)
SOKR-23E3 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (24)
SOKR-24E2 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (24)
SOKR-24E3 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (24)
SOKR-25E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (24)
SOKR-25E3 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (24)
SOKR-27E1 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (24)
SOKR-33E1 E10 2002 Environmental Peat bog UB Kuujjuaq, QC BRS (24)
SOKR-34E2 E10 2002 Environmental Shoreline soil 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-38E2 E3 2002 Environmental Marine sediment UB Kuujjuaq, QC BRS (24)
SOKR-42E1 E10 2002 Environmental Shoreline soil UB Tasiujaq, QC BRS (24)
SOKR-43E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (24)
SOKR-44E1 E11 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (24)
SOKR-44E2 E11 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (24)
SOKR-44E3 E3 2002 Environmental Shoreline soil UB Kuujjuaq, QC BRS (24)
SOKR-46E1 E11 2004 Environmental Marine sediment UB Kuujjuaq, QC BRS (24)
SOKR-46E3 E3 2004 Environmental Marine sediment UB Kuujjuaq, QC BRS (24)
SOKR-49E1 E10 2002 Environmental Sediment UB Kuujjuaq, QC BRS (24)
SOKR-49E2 E10 2002 Environmental Sediment UB Kuujjuaq, QC BRS (24)
SOKR-50E1 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, QC BRS (24)
SOKR-50E2 E3 2002 Environmental Terrestrial soil UB Kuujjuaq, 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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a

Abbreviations: OB, outbreak; ND, no data; NT, nontoxigenic; BRS, Botulism Reference Service, Health Canada; NML, National Microbiology Laboratory, Public Health Agency of Canada; 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.

b

Publicly available genome.

c

Isolate originally tested positive for BoNT/E in 2002.

d

Isolated from original (1980) specimen in 2012 and tested negative for BoNT using the mouse bioassay.

e

Sample collected in 1980 tested negative for BoNT in 2012.

Identification of core SNPs.

Reads were reference mapped using SMALT v0.7.4 (word length = 13, step = 1 (25), SNPs were identified in FreeBayes v0.9.6 (26) (map/base quality ≥ 35; alternate fraction ≥ 0.75) and cross-referenced with SAMtools v0.1.18 (27) (mpileup ≥ 20). SNPs identified using both methods were selected for further analysis. Complex SNP events are not distinguished in FreeBayes and were treated as individual events. The subset of SNP loci present among all data sets was identified (core SNPs), and a Perl script generated a meta-alignment for phylogeny analysis (https://github.com/apetkau/core-phylogenomics; commit f132bf6). For in silico MLST, sequence reads were mapped to a database of known MLST alleles (12, 23) for C. botulinum group II using SRST2 v2.1 (read mismatch = 10) (28). Consensus allele sequences were concatenated and aligned with ClustalW v1.82 (clustalw-mpi, default parameters) (29). Alignments were visually inspected for accuracy and converted to PHYLIP format (http://sequenceconversion.bugaco.com/converter/biology/sequences/).

Phylogeny analyses.

Maximum likelihood phylogenetic trees were built using PhyML v3.1 (30) using a GTR+G substitution model and a tree topology search for best nearest-neighbor interactions/subtree prunings and regraftings (NNIs/SPRs) and initial BioNJ tree. Branch support values for were estimated using the approximate likelihood ratio test (31). Images were rendered in FigTree (v1.4.1) (http://tree.bio.ed.ac.uk/software/figtree). For core SNP analyses, branch scales were converted by multiplying the number of substitutions per position by the number of core SNPs identified for the population.

Nucleotide sequence accession numbers.

All sequence reads and MLST alleles included in this study have been deposited in the NCBI Sequence Read Archive (SRP059342) and GenBank (atpD, KT034476 to KT034633; trpB, KT034634 to KT034791; rpoB, KT034792 to KT034949; guaA, KT034950 to KT035107; pta, KT035108 to KT035265; ilvD, KT035266 to KT035423; lepA, KT035424 to KT035581; gyrB, KT035582 to KT035739; recA, KT035740 to KT035897; oppB, KT035898 to KT036055; 23S rRNA rumA, KT036056 to KT036213; pyc, KT036214 to KT036371) (http://www.ncbi.nlm.nih.gov/) databases. Public genomes included in analyses were Alaska E43, NC_010723; Eklund 17B, NC_010674; pCLL, NC_010680; 202F, CP006903; pCBI, CP006904; E1 Beluga, ACSC00000000; CDC66177, ALYJ00000000; CB11/1-1, AORM00000000; KAPB-3, JQOK01000000; DB2, JQOJ01000000; NCTC 8266, CP010520; NCTC 8550, CP010521; and NCTC 11219, JXMR01000000 (23, 32–35).

RESULTS AND DISCUSSION

In silico MLST profiling.

To determine whether whole-genome sequence data could be used for en masse MLST profiling of 163 C. botulinum group II isolates, and also to compare profiles using a previously published typing scheme, consensus alleles were determined in silico using SRST2 (v2.1) (28) for 15 MLST loci described by Macdonald et al. (12). Concatenated alleles were aligned using ClustalW, and maximum likelihood phylogenetic trees were estimated using PhyML.

For this population, allelic heterogeneity produced ambiguous allele calls for 16S (11 copies) and tuf (2 copies). tuf heterogeneity was observed for several isolates, where the two alleles identified differed by a single SNP (data not shown). In addition, mutL indels and elements showing high levels of identity resulted in consensus call uncertainties (data not shown). Because of the ambiguous allele calls, these three loci were excluded from the in silico MLST typing strategy. Despite this omission, the MLST-12 phylogeny analysis resolved 25 distinct profiles for 41 previously characterized isolates (see Fig. S1 in the supplemental material), which is consistent with previous studies using the complete MLST-15 scheme (12), indicating that MLST resolution is comparable irrespective of 16S, tuf, and mutL locus inclusion.

The MLST-12 scheme distinguished 29 taxonomic groups for the 163 isolates analyzed (Fig. 1). MLST profiles were consistent whether WGS read data or NCBI genomic sequences were used, as observed for three strains: Eklund 17B, DB2, and KAPB-3 (15–17, 34, 36) (see Fig. S1 in the supplemental material). MLST profiles using WGS read data from technical replicates (211 VH Dolman, SO329E2, and SOKR-44E1) were also in agreement (data not shown).

FIG 1.

FIG 1

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In silico MLST phylogeny for C. botulinum group II isolates. Concatenated consensus sequences for 12 MLST loci were aligned with ClustalW and analyzed with PhyML to estimate a maximum likelihood phylogeny for C. botulinum group II isolates (refer to Materials and Methods). Inset, MLST phylogeny for all isolates (n = 163) showing two clusters separated by a genetic distance of 0.034; outset, zoomed view of Alaska E43 cluster isolates (n = 149). Outbreak number, region of origin, and BoNT serotypes or variants for isolates in the Alaska E43 cluster are indicated as follows: orange, BoNT/B4; turquoise, BoNT/F6; purple, BoNT/E1; red, BoNT/E3; pink, BoNT/E9; royal blue, BoNT/E10; green, BoNT/E11; and black, nontoxigenic (NT) (details in Table 1). Eleven publicly available genomes were included in the analysis (*). Taxa that include multiple BoNT serotypes or subtypes are indicated (yellow). Clades corresponding to core SNP phylogeny (Fig. 3) are indicated. Scale bars, number of nucleotide substitutions per site.

Consistent with other reports (12, 23), a large cluster (20 taxa; n = 149) that included the Alaska E43 strain (NC_010723) represented a wide variety of BoNT/E toxin subtypes found in C. botulinum group II bacteria (E1 to E3, E6 to E8, and E10 and E11) (Fig. 1; see also Fig. S1). Interestingly, a nontoxic isolate from Saskatchewan, BFLY-1, also typed to this large cluster. This isolate was derived from a waterfowl sample collected in 1980 as part of an avian botulism investigation (outbreak 7) but tested negative for BoNT in 2012 (Table 1). A second population (9 taxa; n = 14) clustered closer to the BoNT/B4 strain Eklund 17B and included CDC66177, a BoNT/E9 isolate, as reported elsewhere (23). Other BoNT/B4 isolates (Eklund 2B, DB2, KAPB-3, and KAPB-8), two BoNT/F6 isolates (202F and 610F), and several nontoxigenic isolates also grouped with the Eklund 17B cluster. Two of the nontoxic isolates (BFLY-2 and BFLY-6) in this cluster were derived from the aforementioned waterfowl sample source (1980, outbreak 7) that was never confirmed as toxigenic. However, three isolates (BE0211E1, BE0211E2, and BE0211E3) from a food sample (beluga) collected during a 2002 foodborne botulism outbreak in Kuujjuaq (outbreak 30) originally tested positive for BoNT/E using the mouse bioassay (Table 1 and data not shown). It is possible that these isolates have lost their neurotoxin genes, as reported for other group II C. botulinum strains (22, 36).

MLST profiling showed that the majority of taxa comprised isolates representing a single BoNT subtype. Four taxa, however, included members carrying different toxin subtypes (E1/E3, E3/E11, and E3/E10) (Fig. 1).

Core SNP phylogeny profiling.

Recombination and other forms of horizontal transfer can confound phylogeny analysis (37, 38). Although recent reports claim that bacterial phylogenetic reconstruction from whole-genome sequence data is relatively robust 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 (40–44).

Read mapping to the Alaska E43 reference genome (NC_010723) identified 69,321 core SNP loci for the C. botulinum group II population (n = 163), and consistent with MLST (Fig. 1), maximum likelihood phylogeny analysis showed species-level clustering into Eklund 17B (n = 14) and Alaska E43 (n = 149) subgroups (Fig. 2). These data confirm and extend reports by several groups which showed that genetic and/or genomic diversity of C. botulinum group II strains formed two distinct subpopulations (11, 22, 23).

FIG 2.

FIG 2

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Core SNP phylogenomic analysis of C. botulinum group II isolates. Shown is a maximum likelihood analysis of 69,321 core SNP loci identified among C. botulinum group II isolates by reference mapping to Alaska E43 (NC_010723). Inset, core SNP phylogeny for all isolates (n = 163) showing two clusters separated by >65,598 SNPs; outset, zoomed view of Alaska E43 cluster isolates (n = 149) showing clade-level classifications (indicated on the right). Outbreak number, region of origin, and BoNT serotypes or variants for isolates in the Alaska E43 cluster are indicated as for Fig. 1 (details in Table 1). Eleven publicly available genomes were included in the analysis (*). Clades that include multiple BoNT serotypes or subtypes are indicated (yellow), and clade designations are indicated (right).

Members of the Eklund 17B and Alaska E43 clusters differed by ≥65,598 SNPs and could be distinguished by as little as one SNP (https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv). This clustering was also observed when other high-quality finished genomes (Eklund 17B, 202F) were used as references (data not shown).

While the tree topologies differed between MLST and core SNP methods, profile classifications between the two methods were remarkably consistent, with core SNP demonstrating an increased resolution over MLST. Core SNP phylogeny distinguished 31 clade profiles differing by >60 core SNPs (Fig. 2) and resolved subgroups for three MLST taxa (see Table S1 in the supplemental material). Two subgroups were identified for isolates in clade 7 by core SNP phylogeny, and core SNP parsed BoNT/E3 and BoNT/E10 isolates from a common MLST taxon (Fig. 1) into clades 11 and 12, respectively (Fig. 2; see also Table S1). In addition, core SNP analysis distinguished SO309E2, a BoNT/E10 isolate from Ungava Bay, from MU8903E, a BoNT/E3 isolate from the Northwest Territories (Fig. 2), which typed to the same profile by MLST (Fig. 1). At this level of resolution, however, core SNP phylogeny did not provide sufficient resolution to distinguish BoNT/E10 from BoNT/E3 isolates in clade 13 (Fig. 2).

As observed by MLST (Fig. 1), food and clinical isolates collected during a common outbreak (outbreaks 10, 14, 16 to 18, 20, 21, 26, 29, 32, 33, and 35 as listed in Table 1) typically typed within the same core SNP profile (Fig. 2; see also Table S1). Likewise, multiple strains recovered from a single environmental sample source were often typed to a common clade by MLST and core SNP (FWSK02-01E2/3, FWSK02-06E1/2, TRK02-06E2/3, SO329E1/2, SWKR38E1/2, SO303E1/3/4/5, SO304E1/2, SO305E1/2, SOKR-22E1/3, and TRK02-08E1/3) (Fig. 1 and 2; see also Table S1). This observation would be expected of isolates with a high level of relatedness in the population.

Exceptions were noted for two BoNT/E3 isolates derived from a food sample collected during an outbreak in Tasiujaq (2004, outbreak 31), which generated different clades (IG0410E3LC, clade 13, and IG0410E2LC, clade 16), suggesting that these isolates are divergent. In addition, a BoNT/E10 gastric liquid isolate, GA9706EMA, from outbreak 19 (Tasiujaq, 1997) typed to clade 17, while an isolate from the suspected source food sample (MI9706E) grouped to clade 8 and encoded BoNT/E3.

Distinct clade classifications were also observed for multiple isolates derived from 18 environmental samples collected from the Kuujjuaq or Tasiujaq region of Ungava Bay (TRK02-08E, TRK02-04E, TRK02-02E, SWKR0402E, SOKR-50E, SOKR-49E, SOKR-46E, SOKR-44E, SOKR-34E, SOKR-25E, SOKR-24E, SOKR-23E, and RSKR-68E) (Fig. 1 to 3). For example, three isolates derived from a single coastal rock sample from the Kuujjuaq region of Ungava Bay typed to clades 11 (RSKR-68E1), 17 (RSKR-68E2), and 7 (RSKR-68E3) (Fig. 2 and 3). These results are consistent with PFGE data (14), which also indicates isolate diversity within a single environmental sample in these regions of northern Canada. Together, these results indicate that MLST and core SNP analysis can provide effective species-level characterization for broad-range isolate inclusion or exclusion during an epidemiological investigation.

FIG 3.

FIG 3

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Geographic origins of C. botulinum group II isolates studied. Communities and regions where C. botulinum isolates originate (Table 1) were plotted on a world map (base map, © OpenStreetMap contributors [http://www.openstreetmap.org/]; image generated using CartoDB [https://kweedmark.cartodb.com/viz/48f29664-d89c-11e4-9548-0e018d66dc29/embed_map]). Inset, zoomed view of Canada's Nunavik region.

C. botulinum group II in silico MLST and species-wide core SNP phylogeny profiling are in agreement with flagellin (flaVR, flaB) typing methods (15, 16) but show enhanced resolution capability. For instance, flagellin profiling groups several members of the Eklund 17B cluster as flaVR9 and negative for flaB (Eklund 17B, 610F, KAPB-3, KAPB-8), while members of the E43 cluster are flaVR8 and positive for flaB (SO329E1 and SOKR38E2), which type to two clades (7 and 12), or flaVR10 and positive for flaB (e.g., Gordon, SW280E, SO309E2, FE0005EJT, and S9510E), which type to five clades (10, 14, 16, 18, and 22) by MLST and/or core SNP (15, 16).

High-resolution core SNP analysis.

Several core SNP clades were comprised solely of isolates from the Ungava Bay region. This is not surprising given that the majority of isolates studied originate from Ungava Bay specimens. Isolates from this area typed to a large proportion the profiles identified by core SNP and MLST (Fig. 1 and 2). However, many profiles included specimens from multiple outbreaks (clades 7, 13, 15, 16, 21, and 22) and regions (clades 16, 17, 21, and 22). For instance, clade 16 included isolates from 12 outbreaks (10, 12, 20, 25–27, 30, 32, 37) across eight communities in Canada (regions: Ungava Bay, Baffin Island, Northwest Territories, and British Columbia) and one location in France (Paris) (Fig. 3).

To determine whether closely related isolates could be resolved by outbreak or region of origin, core SNP analysis was performed on isolates from clade 16 (Fig. 2), which includes the Alaska E43 reference genome. This analysis generated 20 unique profiles for 27 isolates based on 263 core SNP loci (Fig. 4). Two E1 isolates, NCTC 8550 and NCTC 8226, originating from France and British Columbia, respectively (45, 46), typed distal to the E3 isolates in this population (Fig. 4). Isolates from the Northwest Territories typed to a distinct cluster, and isolates from the communities of Inuvik (outbreaks 25 [1999] and 26 [2000]) and Aklavik (outbreak 27 [2001]) differed by one core SNP (https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv; accession number NC_010723 position 1021249) (Fig. 3). Three additional non-core SNP loci were identified that discriminate between outbreaks 25, 26, and 27 (accession number NC_010723 positions 2263189, 2294119, and 2295007) (https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv). Core SNP phylogeny also clustered isolates from the same outbreaks together, as observed for outbreaks 10 (Kangiqsualujjuaq, 1995; 0 SNPs), 20 (Kangiqsualujjuaq, 1997; ≤2 SNPs), and 33 (Baffin Island, 2000; 0 SNPs) (Fig. 3 and 4) (https://www.corefacility.ca/supplementary_data/AEM01155_SupplementalDataset1_vS.tsv). Several of these isolates (outbreaks 10, 12, 20, 25, and 26) were profiled previously by PFGE methods (17). Compared to PFGE, the results in Fig. 4 demonstrate that core SNP analysis can provide enhanced typing discrimination among highly related isolates.

FIG 4.

FIG 4

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Core SNP analysis of clade 16 isolates. Shown is a maximum likelihood phylogeny based on 263 core SNPs for isolates typing to clade 16 (Fig. 2) showing high-resolution topology for clinical and food isolates from communities in France (Paris) and Canada (Nanaimo, BC [outbreak 37], Inuvik, NWT [outbreaks 25 and 26], Aklavik, NWT [outbreak 27], Tasiujaq, QC [outbreaks 12 and 30], Kangisualujjuaq, QC [outbreaks 10 and 20], and Baffin Island, NU [outbreak 33)] as well as environmental isolates (*) from Kuujjuaq and Tasiujaq. Alaska E43, reference genome (NC_010723). Distance bar, number of SNPs. Branch lengths of ≥20 SNPs are indicated.

Despite the propensity for C. botulinum bacteria to undergo horizontal gene transfer (11, 12, 23, 47–49), the data presented here indicate that core SNP analysis can resolve even highly related isolates by outbreak and/or location and provide a useful tool for epidemiological investigations. In addition, the deposition of sequence reads for 152 C. botulinum group II isolates, as well as a catalog of in silico MLST alleles and SNP locus calls, provides a significant resource to the scientific community.

Supplementary Material

Supplemental material
supp_81_17_5938__index.html (1.1KB, html)

ACKNOWLEDGMENTS

This work was supported by Canadian Safety and Security Program project 07-219RD from Defense Research and Development Canada.

This work does not reflect the opinion of the Government of Canada.

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01155-15.

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