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Carter A.T.,UK Institute of Food Research | Austin J.W.,Bureau of Microbial Hazards | Weedmark K.A.,Public Health Agency of Canada | Peck M.W.,UK Institute of Food Research
Genome biology and evolution | Year: 2016

Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxin-encoding genes were located on plasmids. Seven closely related (134-144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


Taboada E.N.,Public Health Agency of Canada | Clark C.G.,Public Health Agency of Canada | Sproston E.L.,Bureau of Microbial Hazards | Carrillo C.D.,Canadian Food Inspection Agency
Journal of Microbiological Methods | Year: 2013

Campylobacter remains one of the most common bacterial causes of gastroenteritis worldwide. Tracking sources of this organism is challenging due to the large numbers of human cases, and the prevalence of this organism throughout the environment due to growth in a wide range of animal species. Many molecular subtyping methods have been developed to characterize Campylobacter species, but only a few are commonly used in molecular epidemiology studies. This review examines the applicability of these methods, as well as the role that emerging whole genome sequencing technologies will play in tracking sources of Campylobacter spp. infection. © 2013.


Mattison K.,Bureau of Microbial Hazards | Sebunya T.K.,University of Botswana | Shukla A.,Bureau of Microbial Hazards | Noliwe L.N.,University of Botswana | Bidawid S.,Bureau of Microbial Hazards
Journal of Medical Virology | Year: 2010

Stool specimenswere collected from100 children in Botswana. RT-PCR analysis detected noroviruses (NoVs) in 24% of samples tested. Genogroup I and genogroup II strains were identified. There was no association between NoV detection and age or gender. This study is the first indication that NoVs circulate widely in Botswana. © 2009 Wiley-Liss, Inc.


Dixon B.,Bureau of Microbial Hazards | Parrington L.,Bureau of Microbial Hazards | Cook A.,Public Health Agency of Canada | Pollari F.,Public Health Agency of Canada | Farber J.,Bureau of Microbial Hazards
Journal of Food Protection | Year: 2013

Numerous foodborne outbreaks of diarrheal illness associated with the consumption of produce contaminated with protozoan parasites have been reported in North America in recent years. The present study reports on the presence of Cyclospora, Cryptosporidium, and Giardia in precut salads and leafy greens purchased at retail in Ontario, Canada. A total of 544 retail samples were collected between April 2009 and March 2010 and included a variety of salad blends and individual leafy greens. Most of these products were grown in the United States, with some from Canada and Mexico. Parasites were eluted and concentrated before detection by PCR and immunofluorescence microscopy. DNA sequences were aligned with reference sequences in GenBank. Cyclospora spp. were identified by PCR-restriction fragment length polymorphism in nine (1.7%) samples and by DNA sequence analysis. Cryptosporidium spp. were identified in 32 (5.9%) samples; 29 were sequenced and aligned with the zoonotic species Cryptosporidium parvum. Giardia duodenalis was identified in 10 (1.8%) samples, and of the 9 samples successfully sequenced, 7 aligned with G. duodenalis assemblage B and 2 with assemblage A, both of which are also zoonotic. The presence of Cryptosporidium oocysts and Giardia cysts was confirmed in some of the PCR-positive samples using microscopy, while Cyclospora-like oocysts were observed in most of the Cyclospora PCR-positive samples. The relatively high prevalence of these parasites in packaged salads and leafy greens establishes a baseline for further studies and suggests a need for additional research with respect to the possible sources of contamination of these foods, the determination of parasite viability and virulence, and means to reduce foodborne transmission to humans. Copyright ©, International Association for Food Protection.


Kozak G.K.,Bureau of Microbial Hazards | Macdonald D.,Public Health Agency of Canada | Landry L.,Public Health Agency of Canada | Farber J.M.,Bureau of Microbial Hazards
Journal of Food Protection | Year: 2013

Foodborne disease outbreaks associated with fresh fruits and vegetables have been increasing in occurrence worldwide. Canada has one of the highest per capita consumption rates of fresh fruits and vegetables in the world. In this article, we review the foodborne disease outbreaks linked to produce consumption in Canada from 2001 through 2009. The 27 produce-related outbreaks included an estimated 1,549 cases of illness. Bacterial infection outbreaks represented 66% of the total. Among these, Salmonella was the most frequent agent (50% of outbreaks) followed by Escherichia coli (33%) and Shigella (17%). Cyclospora cayetanensis was the only parasite detected and was associated with seven outbreaks. Among the foodborne viruses, only hepatitis A was implicated in two outbreaks. The food vehicles most commonly implicated in outbreaks were leafy greens and herbs (26% of outbreaks), followed by seed sprouts (11%). Contamination sources and issues related to the future control of fresh produce-related foodborne disease outbreaks also are discussed. Copyright ©, International Association for Food Protection.


Ronholm J.,Bureau of Microbial Hazards | Nasheri N.,Bureau of Microbial Hazards | Petronella N.,Bureau of Food Surveillance and Science Integration | Pagotto F.,Bureau of Microbial Hazards | Pagotto F.,Listeriosis Reference Center
Clinical Microbiology Reviews | Year: 2016

The epidemiological investigation of a foodborne outbreak, including identification of related cases, source attribution, and development of intervention strategies, relies heavily on the ability to subtype the etiological agent at a high enough resolution to differentiate related from nonrelated cases. Historically, several different molecular subtyping methods have been used for this purpose; however, emerging techniques, such as single nucleotide polymorphism (SNP)-based techniques, that use whole-genome sequencing (WGS) offer a resolution that was previously not possible. WithWGS,unlike traditional subtyping methods that lack complete information, data can be used to elucidate phylogenetic relationships and disease-causing lineages can be tracked and monitored over time. The subtyping resolution and evolutionary context provided by WGS data allow investigators to connect related illnesses that would be missed by traditional techniques. The added advantage of data generated by WGS is that these data can also be used for secondary analyses, such as virulence gene detection, antibiotic resistance gene profiling, synteny comparisons, mobile genetic element identification, and geographic attribution. In addition, several software packages are now available to generate in silico results for traditional molecular subtyping methods from the whole-genome sequence, allowing for efficient comparison with historical databases. Metagenomic approaches using next-generation sequencing have also been successful in the detection of nonculturable foodborne pathogens. This review addresses state-of-the-art techniques in microbial WGS and analysis and then discusses how this technology can be used to help support food safety investigations. Retrospective outbreak investigations using WGS are presented to provide organism-specific examples of the benefits, and challenges, associated withWGSin comparison to traditional molecular subtyping techniques. © Crown copyright 2016.


Tracking of sources of sporadic cases of campylobacteriosis remains challenging, as commonly used molecular typing methods have limited ability to unambiguously link genetically related strains. Genomics has become increasingly prominent in the public health response to enteric pathogens as methods enable characterization of pathogens at an unprecedented level of resolution. However, the cost of sequencing and expertise required for bioinformatic analyses remains prohibitive, and these comprehensive analyses are limited to a few priority strains. Although several molecular typing methods are currently widely used for epidemiological analysis of campylobacters, it is not clear how accurately these methods reflect true strain relationships. To address this, we have developed a framework and associated computational tools to rapidly analyze draft genome sequence data for the assessment of molecular typing methods against a "gold standard" based on the phylogenetic analysis of highly conserved core (HCC) genes with high sequence quality. We analyzed 104 publicly available whole genome sequences (WGS) of C. jejuni and C. coli. In addition to in silico determination of multi-locus sequence typing (MLST), flaA, and porA type, as well as comparative genomic fingerprinting (CGF) type, we inferred a "reference" phylogeny based on 389 HCC genes. Molecular typing data were compared to the reference phylogeny for concordance using the adjusted Wallace coefficient (AWC) with confidence intervals. Although MLST targets the sequence variability in core genes and CGF targets insertions/deletions of accessory genes, both methods are based on multi-locus analysis and provided better estimates of true phylogeny than methods based on single loci (porA, flaA). A more comprehensive WGS dataset including additional genetically related strains, both epidemiologically linked and unlinked, will be necessary to more comprehensively assess the performance of subtyping methods for outbreak investigations and surveillance activities. Analyses of the strengths and weaknesses of widely used typing methodologies in inferring true strain relationships will provide guidance in the interpretation of this data for epidemiological purposes.


MacLean L.L.,NRC Institute for Biological Sciences | Vinogradov E.,NRC Institute for Biological Sciences | Pagotto F.,Bureau of Microbial Hazards | Perry M.B.,NRC Institute for Biological Sciences
Canadian Journal of Microbiology | Year: 2012

Cronobacter dublinensis (formerly Enterobacter sakazakii) HPB 3169 is a pathogenic Gram-negative bacterium that produces a smooth-type lipopolysaccharide in which the antigenic O-polysaccharide component was determined to be a repeating pentasaccharide unit composed of L-rhamnose; 2-acetamido-2-deoxy-D-glucose; 3,6-dideoxy-3-(R)-3-hydroxybutyramido-Dglucose; and 3-deoxy-manno-oct-2-ulosonic acid in the respective molar ratio 2:1:1:1. Chemical and 2D NMR analyses of the O-polysaccharide and a pentasaccharide derived by the mild acid hydrolysis of the ketosyl linkage of the Kdo (3-deoxy-D-manno-2-octulosonic acid) residue in the O-polysaccharide established that the O-antigen is a high molecular mass unbranched polymer of a repeating pentasaccharide unit and has the structure where Bu is a (R)-3-hydroxybutanoyl substituent. The O-antigen is structurally similar to that of the recently reported Cronobacter sakazakii strain G706 (designated as serotype O5), except that in strain G706 the D-Qui3N is in its N-acetyl form, in contrast to its presence as a 3-deoxy-3-(R)-3-hydroxybutyramido derivative in the C. sakazakii HPB 3169 strain O-antigen.


Dixon B.R.,Bureau of Microbial Hazards
Current Opinion in Food Science | Year: 2016

Foodborne transmission of protozoan parasites is an emerging issue in developed countries around the world. Specifically, the parasites Cryptosporidium, Giardia and Cyclospora have been linked to numerous foodborne outbreaks of diarrheal illness. Many of these outbreaks have been associated with the consumption of fresh produce imported from developing regions, where water quality, hygiene and sanitation may be sub-optimal, and where numerous surveillance studies have demonstrated the presence of these parasites on fruits and vegetables. However, illness outbreaks have also been associated with the direct contamination of fresh produce at the food handler/consumer level. The implementation of control measures at pre-harvest and post-harvest, as well as at the food handler/consumer level, will be crucial in minimizing the foodborne transmission of these parasites. © 2016.


Gill A.,Bureau of Microbial Hazards
Journal of Microbiological Methods | Year: 2014

The growth characteristics of 96 shiga toxin-producing Escherichia coli (STEC) strains representing 36 different O-types (including priority O types O26, O45, O103, O111, O121, O145 and O157) on commercial and in-house agar media were studied. The ability of the strains to grow on agar media with varying selective supplement formulations was evaluated using MacConkey Agar (MAC); Rainbow® Agar O157 (RBA); Rainbow® Agar O157 with manufacturer-recommended selective supplements (RBA-NT); Rainbow® Agar O157 with USDA-recommended selective supplements (RBA-USDA); CHROMagar STEC™ (CH STEC); Tryptone Bile agar containing cefixime and tellurite (TBA-CT); Tryptone Bile agar containing cefixime, tellurite, eosin and methylene blue (TBA-EM); and VTEC agar. All of the strains were able to grow on MAC, RBA and VTEC agar, whereas a number of strains (including some non-O157 priority O types) were unable to grow on the highly selective media CH STEC, RBA-NT, RBA-USDA, TBA-EM and TBA-CT. Only RBA-NT and CH STEC exhibited significant inhibition of background flora from ground beef enrichment. Significant inhibition of background flora from beef trim enrichment was observed with RBA-NT, RBA-USDA, CH STEC, TBA-EM and VTEC agar. With exception of E. coli O157, several different colony morphologies were observed on the differential plating media among strains of the same O type, indicating that this colony morphology is not a reliable means of identifying target STEC. These results suggest that an approach to maximize the recovery of target STEC from beef enrichment cultures is dual plating on lesser (RBA, MAC, VTEC agar) and more highly (RBA-NT, CH STEC) selective agars. © 2013.

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