Evaluating disinfectant efficacy on mixed biofilms comprising Shiga toxigenic Escherichia coli, lactic acid bacteria, and spoilage microorganisms.

This study aimed to investigate the impact of temperature and the presence of other microorganisms on the susceptibility of STEC to biocides. Mature biofilms were formed at both 10°C and 25°C. An inoculum of planktonic bacteria comprising 106 CFU/mL of spoilage bacteria and 103 CFU/mL of a single E. coli strain (O157, O111, O103, and O12) was used to form mixed biofilms. The following bacterial combinations were tested: T1: Carnobacterium piscicola + Lactobacillus bulgaricus + STEC, T2: Comamonas koreensis + Raoultella terrigena + STEC, and T3: Pseudomonas aeruginosa + C. koreensis + STEC. Tested biocides included quaternary ammonium compounds (Quats), sodium hypochlorite (Shypo), sodium hydroxide (SHyd), hydrogen peroxide (HyP), and BioDestroy®-organic peroxyacetic acid (PAA). Biocides were applied to 6-day-old biofilms. Minimum Bactericidal Concentrations (MBC) and Biofilm Eradication Concentrations (BEC) were determined. Planktonic cells and single-species biofilms exhibited greater susceptibility to sanitizers (p < 0.0001). Lactobacillus and Carnobacterium were more susceptible than the rest of the tested bacteria (p < 0.0001). Single species biofilms formed by E. coli O111, O121, O157, and O45 showed resistance (100%) to Shypo sanitizer (200 ppm) at 25°C. From the most effective to the least effective, sanitizer performance on single-species biofilms was PAA > Quats > HyP > SHyd > Shypo. In multi-species biofilms, spoilage bacteria within T1, T2, and T3 biofilms showed elevated resistance to SHyd (30%), followed by quats (23.25%), HyP (15.41%), SHypo (9.70%), and BioDestroy® (3.42%; p < 0.0001). Within T1, T2, and T3, the combined STEC strains exhibited superior survival to Quats (23.91%), followed by HyP (19.57%), SHypo (18.12%), SHyd (16.67%), and BioDestroy® (4.35%; p < 0.0001). O157:H7-R508 strains were less tolerant to Quats and Shypo when combined with T2 and T3 (p < 0.0001). O157:H7 and O103:H2 strains in mixed biofilms T1, T2, and T3 exhibited higher biocide resistance than the weak biofilm former, O145:H2 (p < 0.0001). The study shows that STEC within multi-species biofilms' are more tolerant to disinfectants.

Use of a taxon-specific reference database for accurate metagenomics-based pathogen detection of Listeria monocytogenes in turkey deli meat and spinach.

BACKGROUND: The reliability of culture-independent pathogen detection in foods using metagenomics is contingent on the quality and composition of the reference database. The inclusion of microbial sequences from a diverse representation of taxonomies in universal reference databases is recommended to maximize classification precision for pathogen detection. However, these sizable databases have high memory requirements that may be out of reach for some users. In this study, we aimed to assess the performance of a foodborne pathogen (FBP)-specific reference database (taxon-specific) relative to a universal reference database (taxon-agnostic). We tested our FBP-specific reference database's performance for detecting Listeria monocytogenes in two complex food matrices-ready-to-eat (RTE) turkey deli meat and prepackaged spinach-using three popular read-based DNA-to-DNA metagenomic classifiers: Centrifuge, Kraken 2 and KrakenUniq. RESULTS: In silico host sequence removal led to substantially fewer false positive (FP) classifications and higher classification precision in RTE turkey deli meat datasets using the FBP-specific reference database. No considerable improvement in classification precision was observed following host filtering for prepackaged spinach datasets and was likely a consequence of a higher microbe-to-host sequence ratio. All datasets classified with Centrifuge using the FBP-specific reference database had the lowest classification precision compared to Kraken 2 or KrakenUniq. When a confidence-scoring threshold was applied, a nearly equivalent precision to the universal reference database was achieved for Kraken 2 and KrakenUniq. Recall was high for both reference databases across all datasets and classifiers. Substantially fewer computational resources were required for metagenomics-based detection of L. monocytogenes using the FBP-specific reference database, especially when combined with Kraken 2. CONCLUSIONS: A universal (taxon-agnostic) reference database is not essential for accurate and reliable metagenomics-based pathogen detection of L. monocytogenes in complex food matrices. Equivalent classification performance can be achieved using a taxon-specific reference database when the appropriate quality control measures, classification software, and analysis parameters are applied. This approach is less computationally demanding and more attainable for the broader scientific and food safety communities.

Impact of the COVID-19 Pandemic on the Reported Incidence of Select Bacterial Enteric Diseases in Canada, 2020.

The aim of this study was to describe the impact of the COVID-19 pandemic on reported cases and clusters of select enteric diseases in Canada, for the period of March 2020 to December 2020. Weekly counts of laboratory confirmed cases of Salmonella, Shigella, Shiga toxin-producing Escherichia coli (STEC), and Listeria monocytogenes were obtained from laboratory surveillance data. These data were supplemented with epidemiological information on the suspected source of illness, collected for cases identified within whole genome sequencing clusters. Incidence rate ratios were calculated for each pathogen. All data were compared with a prepandemic reference period. Decreases in the number of reported cases in 2020 compared with the previous 5-year period were noted for Salmonella, Shigella, Escherichia coli O157, and non-O157 STEC. Reported number of cases for L. monocytogenes in 2020 remained similar to those of the previous 5-year period. There was a considerable decline (59.9%) in the number of cases associated with international travel compared with a 10% decline in the number of domestic cases. Comparison of reported incidence rates of clustered versus sporadic cases for each pathogen showed little variation. This study represents the first formal assessment of the impact of COVID-19 on reported enteric diseases in Canada. Reported case counts across several pathogens saw notable declines in 2020 compared with prepandemic levels, with restrictions on international travel playing a key role. Additional research is needed to understand how limitations on social gatherings, lock downs, and other public health measures have impacted enteric diseases.

Estimated Reduction in the Burden of Nontyphoidal Salmonella Illness in Canada Circa 2019.

Nontyphoidal Salmonella (NTS) is a leading cause of acute gastrointestinal illness in Canada, and reported cases have been on the rise since the early 2000s. To address this trend, agri-food industry partners and government have worked to identify and implement interventions, guided by the enhanced information provided by whole-genome sequencing, to reduce the incidence of NTS. A substantial reduction in the number of NTS cases reported occurred in 2019. Due to underreporting and underdiagnosis factors, the observed decrease in the number of reported cases represents a fraction of the true number of illnesses averted in the community. The objective of this study was to: (1) use burden of illness estimation methodologies to estimate the true number of NTS illnesses, hospitalizations, and deaths prevented, and (2) estimate the economic savings associated with the prevention of these cases. Compared with the previous 5 years, there were an estimated 25,821 fewer illnesses, 213 fewer hospitalizations, and 2 fewer deaths attributable to NTS in 2019. This corresponds to an estimated reduction of 26.9 million Canadian dollars in the economic burden of NTS. Although causality cannot be proven by this study, the findings are suggestive that the strategically implemented suite of public health actions, including genomic-based surveillance, policy changes, and interventions by the government and industry, were successful in reducing the economic and health burden of NTS infections in Canada.

Formation and Transfer of Multi-Species Biofilms Containing E. coli O103:H2 on Food Contact Surfaces to Beef.

Interactions of Shiga toxin-producing E. coli (STEC; O103:H2) with lactic acid bacteria (LAB) or spoilage bacteria (SP) multispecies biofilms on polyurethane (TPU) and stainless-steel (SS) were assessed at 10 and 25°C under wet and dry conditions after 6, 30, and 60 days of storage. One LAB T1: Carnobacterium piscicola + Lactobacillus bulgaricus, and two SP T2: Comamonas koreensis + Raoultella terrigena; T3: Pseudomonas aeruginosa + C. koreensis were assessed for their ability to form multispecies biofilms with O103:H2. O103:H2 single-species biofilms served as a control positive (T4). Coupons were stored dry (20-50% relative humidity; RH) or moist (60-90% RH) for up to 60 days, at which point O103:H2 transfer to beef and survival was evaluated. At 25°C, T3 decreased beef contamination with O103:H2 by 2.54 log10 CFU/g (P < 0.001). Overall, at 25°C contamination of beef with O103:H2 decreased (P < 0.001) from 3.17 log10 CFU/g on Day 6 to 0.62 log10 CFU/g on Day 60. With 60 days dry biofilms on TPU, an antagonistic interaction was observed among O103:H2 and multispecies biofilm T1 and T3. E. coli O103:H2 was not recovered from T1 and T3 after 60 days but it was recovered (33%) from T2 and T4 dry biofilms. At 10°C, contamination of beef with O103:H2 decreased (P < 0.001) from 1.38 log10 CFU/g after 6 days to 0.47 log10 CFU/g after 60 days. At 10°C, recovery of O103:H2 from 60 days dry biofilms could only be detected after enrichment and was always higher for T2 than T4 biofilms. Regardless of temperature, the transfer of O103:H2 to beef from the biofilm on TPU was greater (P < 0.001) than SS. Moist biofilms also resulted in greater (P < 0.001) cell transfer to beef than dry biofilms at 10 and 25°C. Development of SP or LAB multispecies biofilms with O103:H2 can either increase or diminish the likelihood of beef contamination. Environmental conditions such as humidity, contact surface type, as well as biofilm aging all can influence the risk of beef being contaminated by STEC within multi-species biofilms attached to food contact surfaces.

PulseNet International Survey on the Implementation of Whole Genome Sequencing in Low and Middle-Income Countries for Foodborne Disease Surveillance.

PulseNet International (PNI) is a global network of 88 countries who work together through their regional and national public health laboratories to track foodborne disease around the world. The vision of PNI is to implement globally standardized surveillance using whole genome sequencing (WGS) for real-time identification and subtyping of foodborne pathogens to strengthen preparedness and response and lower the burden of disease. Several countries in North America and Europe have experienced significant benefits in disease mitigation after implementing WGS. To broaden the routine use of WGS around the world, challenges and barriers must be overcome. We conducted this study to determine the challenges and barriers countries are encountering in their attempts to implement WGS and to identify how PNI can provide support to improve and become a better integrated system overall. A survey was designed with a set of qualitative questions to capture the status, challenges, barriers, and successes of countries in the implementation of WGS and was administered to laboratories in Africa, Asia-Pacific, Latin America and the Caribbean, and Middle East. One-third of respondents do not use WGS, and only 8% reported using WGS for routine, real-time surveillance. The main barriers for implementation of WGS were lack of funding, gaps in expertise, and training, especially for data analysis and interpretation. Features of an ideal system to facilitate implementation and global surveillance were identified as an all-in-one software that is free, accessible, standardized and validated. This survey highlights the minimal use of WGS for foodborne disease surveillance outside the United States, Canada, and Europe to date. Although funding remains a major barrier to WGS-based surveillance, critical gaps in expertise and availability of tools must be overcome. Opportunities to seek sustainable funding, provide training, and identify solutions for a globally standardized surveillance platform will accelerate implementation of WGS worldwide.

Correlation between Phenotypic and In Silico Detection of Antimicrobial Resistance in Salmonella enterica in Canada Using Staramr.

Whole genome sequencing (WGS) of Salmonella supports both molecular typing and detection of antimicrobial resistance (AMR). Here, we evaluated the correlation between phenotypic antimicrobial susceptibility testing (AST) and in silico prediction of AMR from WGS in Salmonella enterica (n = 1321) isolated from human infections in Canada. Phenotypic AMR results from broth microdilution testing were used as the gold standard. To facilitate high-throughput prediction of AMR from genome assemblies, we created a tool called Staramr, which incorporates the ResFinder and PointFinder databases and a custom gene-drug key for antibiogram prediction. Overall, there was 99% concordance between phenotypic and genotypic detection of categorical resistance for 14 antimicrobials in 1321 isolates (18,305 of 18,494 results in agreement). We observed an average sensitivity of 91.2% (range 80.5-100%), a specificity of 99.7% (98.6-100%), a positive predictive value of 95.4% (68.2-100%), and a negative predictive value of 99.1% (95.6-100%). The positive predictive value of gentamicin was 68%, due to seven isolates that carried aac(3)-IVa, which conferred MICs just below the breakpoint of resistance. Genetic mechanisms of resistance in these 1321 isolates included 64 unique acquired alleles and mutations in three chromosomal genes. In general, in silico prediction of AMR in Salmonella was reliable compared to the gold standard of broth microdilution. WGS can provide higher-resolution data on the epidemiology of resistance mechanisms and the emergence of new resistance alleles.

A multi-provincial Salmonella Typhimurium outbreak in Canada associated with exposure to pet hedgehogs, 2017-2020.

Background: In October 2020, an investigation began in Canada on an outbreak of Salmonella Typhimurium infections of the same strain as a concomitant outbreak in the United States (US) that was linked to pet hedgehogs. The objective of this article is to identify the source of the outbreak, determine if there was a link between the Canadian and US outbreaks and identify risk factors for infection to inform public health interventions. Methods: Cases were identified through whole genome sequencing of S. Typhimurium isolates. Information was collected on case exposures, including animal contact. Hedgehog and environmental specimens were tested for S. Typhimurium and a trace back investigation was conducted. Results: There were 31 cases in six provinces, with illness onset dates from June 1, 2017, to October 15, 2020. Median case age was 20 years and 52% were female. Isolates grouped together between 0-46 whole genome multi locus sequence typing allele differences. Of 23 cases with available exposure information, 19 (83%) reported contact with hedgehogs in the seven days prior to symptoms; 15/18 (83%) reported direct contact and 3/18 (17%) reported indirect contact. Trace back investigation did not identify a common source of hedgehogs but uncovered an industry with a complex distribution network. The outbreak strain was detected in samples collected from a hedgehog in one case's home and from a hedgehog in a Québec zoo. Conclusion: Direct and indirect contact with hedgehogs was identified as the source of this S. Typhimurium outbreak. Public health communications aimed to increase awareness about the risks of zoonoses from hedgehogs and shared key hygienic practices to reduce disease transmission.

Population structure, case clusters, and genetic lesions associated with Canadian Salmonella 4,[5],12:i:- isolates.

Monophasic Salmonella 4,[5]:12:i:- are a major public health problem because they are one of the top five Salmonella serotypes isolated from clinical cases globally and because they can carry resistance to multiple antibiotics. A total of 811 Salmonella 4,[5]:12:i:- and S. Typhimurium whole genome sequences (WGS) were generated. The various genetic lesions causing the Salmonella 4,[5]:12:i:- genotype were identified and assessed with regards to their distribution in the population of 811 Salmonella 4,[5]:12:i:- and S. Typhimurium isolates, their geographical and temporal distribution, and their association with non-human sources. Several clades were identified in the population structure, and the largest two were associated almost exclusively with a short prophage insertion and insertion of a mobile element carrying loci encoding antibiotic and mercury resistance. IS26-mediated deletions and fljB point mutants appeared to spread clonally. 'Inconsistent' Salmonella 4,[5]:12:i:- isolates associated with specific, single amino acid changes in fljA and hin were found in a single clade composed of water, shellfish, and avian isolates. Inclusion of isolates from different case clusters identified previously by PFGE validated some of the clusters and invalidated others. Some wgMLST clusters of clinical isolates composed of very closely related isolates contained an isolate(s) with a different genetic lesion, suggesting continuing mobility of the implicated element responsible. Such cases may need to be left out of epidemiological investigations until sufficient numbers of isolates are included that statistical significance of association with sources is not impaired. Non-human sources were frequently found in or near clinical case clusters. Prospective surveillance and WGS of non-human sources and retrospective analysis by WGS of isolates from existing culture collections provides data critical for epidemiological investigations of food- and waterborne outbreaks.

Economic evaluation of whole genome sequencing for pathogen identification and surveillance - results of case studies in Europe and the Americas 2016 to 2019.

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

Distribution of heavy metal resistance elements in Canadian Salmonella 4,[5],12:i:- populations and association with the monophasic genotypes and phenotype.

Salmonella 4,[5],12:i:- are monophasic S. Typhimurium variants incapable of producing the second-phase flagellar antigen. They have emerged since the mid-1990s to become one of the most prevalent Salmonella serotypes causing human disease world-wide. Multiple genetic events associated with different genetic elements can result in the monophasic phenotype. Several jurisdictions have reported the emergence of a Salmonella 4,[5],12:i:- clone with SGI-4 and a genetic element (MREL) encoding a mercury resistance operon and antibiotic resistance loci that disrupts the second phase antigen region near the iroB locus in the Salmonella genome. We have sequenced 810 human and animal Canadian Salmonella 4,[5],12:i:- isolates and determined that isolates with SGI-4 and the mercury resistance element (MREL; also known as RR1&RR2) constitute several global clades containing various proportions of Canadian, US, and European isolates. Detailed analysis of the data provides a clearer picture of how these heavy metal elements interact with bacteria within the Salmonella population to produce the monophasic phenotype. Insertion of the MREL near iroB is associated with several deletions and rearrangements of the adjacent flaAB hin region, which may be useful for defining human case clusters that could represent outbreaks. Plasmids carrying genes encoding silver, copper, mercury, and antimicrobial resistance appear to be derived from IS26 mediated acquisition of these genes from genomes carrying SGI-4 and the MREL. Animal isolates with the mercury and As/Cu/Ag resistance elements are strongly associated with porcine sources in Canada as has been shown previously for other jurisdictions. The data acquired in these investigations, as well as from the extensive literature on the subject, may aid source attribution in outbreaks of the organism and interventions to decrease the prevalence of this clone and reduce its impact on human disease.

Source Tracking Based on Core Genome SNV and CRISPR Typing of Salmonella enterica Serovar Heidelberg Isolates Involved in Foodborne Outbreaks in Québec, 2012.

Whole-genome sequencing (WGS) is the method of choice for bacterial subtyping and it is rapidly replacing the more traditional methods such as pulsed-field gel electrophoresis (PFGE). Here we used the high-resolution core genome single nucleotide variant (cgSNV) typing method to characterize clinical and food from Salmonella enterica serovar Heidelberg isolates in the context of source attribution. Additionally, clustered regularly interspaced short palindromic repeats (CRISPR) analysis was included to further support this method. Our results revealed that cgSNV was highly discriminatory and separated the outbreak isolates into distinct clusters (0-4 SNVs). CRISPR analysis was also able to distinguish outbreak strains from epidemiologically unrelated isolates. Specifically, our data clearly demonstrated the strength of these two methods to determine the probable source(s) of a 2012 epidemiologically characterized outbreak of S. Heidelberg. Using molecular cut-off of 0-10 SNVs, the cgSNV analysis of 246 clinical and food isolates of S. Heidelberg collected in Québec, in the same year of the outbreak event, revealed that retail and abattoir chicken isolates likely represent an important source of human infection to S. Heidelberg. Interestingly, the isolates genetically related by cgSNV also harbored the same CRISPR as outbreak isolates and clusters. This indicates that CRISPR profiles can be useful as a complementary approach to determine source attribution in foodborne outbreaks. Use of the genomic analysis also allowed to identify a large number of cases that were missed by PFGE, indicating that most outbreaks are probably underestimated. Although epidemiological information must still support WGS-based results, cgSNV method is a highly discriminatory method for the resolution of outbreak events and the attribution of these events to their respective sources. CRISPR typing can serve as a complimentary tool to this analysis during source tracking.

Emergence of a Novel Salmonella enterica Serotype Reading Clonal Group Is Linked to Its Expansion in Commercial Turkey Production, Resulting in Unanticipated Human Illness in North America.

Two separate human outbreaks of Salmonella enterica serotype Reading occurred between 2017 and 2019 in the United States and Canada, and both outbreaks were linked to the consumption of raw turkey products. In this study, a comprehensive genomic investigation was conducted to reconstruct the evolutionary history of S. Reading from turkeys and to determine the genomic context of outbreaks involving this infrequently isolated Salmonella serotype. A total of 988 isolates of U.S. origin were examined using whole-genome-based approaches, including current and historical isolates from humans, meat, and live food animals. Broadly, isolates clustered into three major clades, with one apparently highly adapted turkey clade. Within the turkey clade, isolates clustered into three subclades, including an "emergent" clade that contained only isolates dated 2016 or later, with many of the isolates from these outbreaks. Genomic differences were identified between emergent and other turkey subclades, suggesting that the apparent success of currently circulating subclades is, in part, attributable to plasmid acquisitions conferring antimicrobial resistance, gain of phage-like sequences with cargo virulence factors, and mutations in systems that may be involved in beta-glucuronidase activity and resistance towards colicins. U.S. and Canadian outbreak isolates were found interspersed throughout the emergent subclade and the other circulating subclade. The emergence of a novel S Reading turkey subclade, coinciding temporally with expansion in commercial turkey production and with U.S. and Canadian human outbreaks, indicates that emergent strains with higher potential for niche success were likely vertically transferred and rapidly disseminated from a common source.IMPORTANCE Increasingly, outbreak investigations involving foodborne pathogens are difficult due to the interconnectedness of food animal production and distribution, and homogeneous nature of industry integration, necessitating high-resolution genomic investigations to determine their basis. Fortunately, surveillance and whole-genome sequencing, combined with the public availability of these data, enable comprehensive queries to determine underlying causes of such outbreaks. Utilizing this pipeline, it was determined that a novel clone of Salmonella Reading has emerged that coincided with increased abundance in raw turkey products and two outbreaks of human illness in North America. The rapid dissemination of this highly adapted and conserved clone indicates that it was likely obtained from a common source and rapidly disseminated across turkey production. Key genomic changes may have contributed to its apparent continued success in commercial turkeys and ability to cause illness in humans.

Whole Genome Sequencing Differentiates Presumptive Extended Spectrum Beta-Lactamase Producing Escherichia coli along Segments of the One Health Continuum.

Antimicrobial resistance (AMR) has important implications for the continued use of antibiotics to control infectious diseases in both beef cattle and humans. AMR along the One Health continuum of the beef production system is largely unknown. Here, whole genomes of presumptive extended-spectrum ß-lactamase E. coli (ESBL-EC) from cattle feces (n = 40), feedlot catch basins (n = 42), surrounding streams (n = 21), a beef processing plant (n = 4), municipal sewage (n = 30), and clinical patients (n = 25) are described. ESBL-EC were isolated from ceftriaxone selective plates and subcultured on ampicillin selective plates. Agreement of genotype-phenotype prediction of AMR ranged from 93.2% for ampicillin to 100% for neomycin, trimethoprim/sulfamethoxazole, and enrofloxacin resistance. Overall, ß-lactam (100%; blaEC, blaTEM-1, blaSHV, blaOXA, blaCTX-M-), tetracycline (90.1%; tet(A), tet(B)) and folate synthesis (sul2) antimicrobial resistance genes (ARGs) were most prevalent. The ARGs tet(C), tet(M), tet(32), blaCTX-M-1, blaCTX-M-14, blaOXA-1, dfrA18, dfrA19, catB3, and catB4 were exclusive to human sources, while blaTEM-150, blaSHV-11-12, dfrA12, cmlA1, and cmlA5 were exclusive to beef cattle sources. Frequently encountered virulence factors across all sources included adhesion and type II and III secretion systems, while IncFIB(AP001918) and IncFII plasmids were also common. Specificity and prevalence of ARGs between cattle-sourced and human-sourced presumptive ESBL-EC likely reflect differences in antimicrobial use in cattle and humans. Comparative genomics revealed phylogenetically distinct clusters for isolates from human vs. cattle sources, implying that human infections caused by ESBL-EC in this region might not originate from beef production sources.

Shared genome analyses of notable listeriosis outbreaks, highlighting the critical importance of epidemiological evidence, input datasets and interpretation criteria.

The persuasiveness of genomic evidence has pressured scientific agencies to supplement or replace well-established methodologies to inform public health and food safety decision-making. This study of 52 epidemiologically defined Listeria monocytogenes isolates, collected between 1981 and 2011, including nine outbreaks, was undertaken (1) to characterize their phylogenetic relationship at finished genome-level resolution, (2) to elucidate the underlying genetic diversity within an endemic subtype, CC8, and (3) to re-evaluate the genetic relationship and epidemiology of a CC8-delimited outbreak in Canada in 2008. Genomes representing Canadian Listeria outbreaks between 1981 and 2010 were closed and manually annotated. Single nucleotide variants (SNVs) and horizontally acquired traits were used to generate phylogenomic models. Phylogenomic relationships were congruent with classical subtyping and epidemiology, except for CC8 outbreaks, wherein the distribution of SNV and prophages revealed multiple co-evolving lineages. Chronophyletic reconstruction of CC8 evolution indicates that prophage-related genetic changes among CC8 strains manifest as PFGE subtype reversions, obscuring the relationship between CC8 isolates, and complicating the public health interpretation of subtyping data, even at maximum genome resolution. The size of the shared genome interrogated did not change the genetic relationship measured between highly related isolates near the tips of the phylogenetic tree, illustrating the robustness of these approaches for routine public health applications where the focus is recent ancestry. The possibility exists for temporally and epidemiologically distinct events to appear related even at maximum genome resolution, highlighting the continued importance of epidemiological evidence.

Evaluation of whole-genome sequencing for outbreak detection of Verotoxigenic Escherichia coli O157:H7 from the Canadian perspective.

BACKGROUND: Rapid and accurate identification of Verotoxigenic Escherichia coli (VTEC) O157:H7 is dependent on well-established, standardized and highly discriminatory typing methods. Currently, conventional subtyping tests for foodborne bacterial pathogen surveillance are rapidly being replaced with whole-genome sequencing (WGS) in public health laboratories. The capacity of WGS to revolutionize global foodborne disease surveillance has positioned this tool to become the new gold standard; however, to ensure evidence standards for public health decision making can still be achieved, the performance of WGS must be thoroughly validated against current gold standard methods prior to implementation. Here we aim to verify the performance of WGS in comparison to pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) for eight retrospective outbreaks of VTEC O157:H7 from the Canadian perspective. Since real-time implementation and routine use of WGS in public health laboratories is highly reliant on standardized data analysis tools, we also provide a comparative analysis of two popular methodologies for WGS analyses; an in-house developed single nucleotide variant phylogenomics (SNVPhyl) pipeline and the BioNumerics whole genome multilocus sequence typing (wgMLST) tool. To provide a useful and consistent starting point for examining laboratory-based surveillance data for VTEC O157:H7 in Canada, we also aim to describe the number of genetic differences observed among outbreak-associated isolates. RESULTS: WGS provided enhanced resolution over traditional subtyping methods, and accurately distinguished outbreak-related isolates from non-outbreak related isolates with high epidemiological concordance. WGS also illuminated potential linkages between sporadic cases of illness and contaminated food, and isolates spanning multiple years. The topologies generated by SNVPhyl and wgMLST were highly congruent with strong statistical support. Few genetic differences were observed among outbreak-related isolates (≤5 SNVs/ < 10 wgMLST alleles) unless the outbreak was suspected to be multi-strain. CONCLUSIONS: This study validates the superiority of WGS and indicates the BioNumerics wgMLST schema is suitable for surveillance and cluster detection of VTEC O157:H7. These findings will provide a useful and consistent starting point for examining WGS data for prospective laboratory-based surveillance of VTEC O157:H7, but however, the data will continue to be interpreted according to context and in combination with epidemiological and food safety evidence to inform public-health decision making in Canada.

Comparison of advanced whole genome sequence-based methods to distinguish strains of Salmonella enterica serovar Heidelberg involved in foodborne outbreaks in Québec.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145 S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks.

Impact of the choice of reference genome on the ability of the core genome SNV methodology to distinguish strains of Salmonella enterica serovar Heidelberg.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. The core genome single nucleotide variant pipeline (cgSNV) is one of several whole genome based sequence typing methods used for the laboratory investigation of foodborne pathogens. SNV detection using this method requires a reference genome. The purpose of this study was to investigate the impact of the choice of the reference genome on the cgSNV-informed phylogenetic clustering and inferred isolate relationships. We found that using a draft or closed genome of S. Heidelberg as reference did not impact the ability of the cgSNV methodology to differentiate among 145 S. Heidelberg isolates involved in foodborne outbreaks. We also found that using a distantly related genome such as S. Dublin as choice of reference led to a loss in resolution since some sporadic isolates were found to cluster together with outbreak isolates. In addition, the genetic distances between outbreak isolates as well as between outbreak and sporadic isolates were overall reduced when S. Dublin was used as the reference genome as opposed to S. Heidelberg.

Comprehensive assessment of the quality of Salmonella whole genome sequence data available in public sequence databases using the Salmonella in silico Typing Resource (SISTR).

Public health and food safety institutions around the world are adopting whole genome sequencing (WGS) to replace conventional methods for characterizing Salmonella for use in surveillance and outbreak response. Falling costs and increased throughput of WGS have resulted in an explosion of data, but questions remain as to the reliability and robustness of the data. Due to the critical importance of serovar information to public health, it is essential to have reliable serovar assignments available for all of the Salmonella records. The current study used a systematic assessment and curation of all Salmonella in the sequence read archive (SRA) to assess the state of the data and their utility. A total of 67 758 genomes were assembled de novo and quality-assessed for their assembly metrics as well as species and serovar assignments. A total of 42 400 genomes passed all of the quality criteria but 30.16 % of genomes were deposited without serotype information. These data were used to compare the concordance of reported and predicted serovars for two in silico prediction tools, multi-locus sequence typing (MLST) and the Salmonella in silico Typing Resource (SISTR), which produced predictions that were fully concordant with 87.51 and 91.91 % of the tested isolates, respectively. Concordance of in silico predictions increased when serovar variants were grouped together, 89.25 % for MLST and 94.98 % for SISTR. This study represents the first large-scale validation of serovar information in public genomes and provides a large validated set of genomes, which can be used to benchmark new bioinformatics tools.