Neptune: a bioinformatics tool for rapid discovery of genomic variation in bacterial populations.

The ready availability of vast amounts of genomic sequence data has created the need to rethink comparative genomics algorithms using 'big data' approaches. Neptune is an efficient system for rapidly locating differentially abundant genomic content in bacterial populations using an exact k-mer matching strategy, while accommodating k-mer mismatches. Neptune's loci discovery process identifies sequences that are sufficiently common to a group of target sequences and sufficiently absent from non-targets using probabilistic models. Neptune uses parallel computing to efficiently identify and extract these loci from draft genome assemblies without requiring multiple sequence alignments or other computationally expensive comparative sequence analyses. Tests on simulated and real datasets showed that Neptune rapidly identifies regions that are both sensitive and specific. We demonstrate that this system can identify trait-specific loci from different bacterial lineages. Neptune is broadly applicable for comparative bacterial analyses, yet will particularly benefit pathogenomic applications, owing to efficient and sensitive discovery of differentially abundant genomic loci. The software is available for download at: http://github.com/phac-nml/neptune.

Microfluidic filtration and extraction of pathogens from food samples by hydrodynamic focusing and inertial lateral migration.

Detecting pathogenic bacteria in food or other biological samples with lab-on-a-chip (LOC) devices requires several sample preparation steps prior to analysis which commonly involves cleaning complex sample matrices of large debris. This often underestimated step is important to prevent these larger particles from clogging devices and to preserve initial concentrations when LOC techniques are used to concentrate or isolate smaller target microorganisms for downstream analysis. In this context, we developed a novel microfluidic system for membrane-free cleaning of biological samples from debris particles by combining hydrodynamic focusing and inertial lateral migration effects. The microfluidic device is fabricated using thermoplastic elastomers being compatible with thermoforming fabrication techniques leading to low-cost single-use devices. Microfluidic chip design and pumping protocols are optimized by investigating diffusive losses numerically with coupled Navier-Stokes and convective-diffusion theoretical models. Stability of inertial lateral migration and separation of debris is assessed through fluorescence microscopy measurements with labelled particles serving as a model system. Efficiency of debris cleaning is experimentally investigated by monitoring microchip outlets with in situ optical turbidity sensors, while retention of targeted pathogens (i.e., Listeria monocytogenes) within the sample stream is assessed through bacterial culture techniques. Optimized pumping protocols can remove up to 50 % of debris from ground beef samples while percentage for preserved microorganisms can account for 95 % in relatively clean samples. However, comparison between inoculated turbid and clean samples (i.e., with and without ground beef debris) indicate some degree of interference between debris inertial lateral migration and hydrodynamic focusing of small microorganisms. Although this interference can lead to significant decrease in chip performance through loss of target bacteria, it remains possible to reach 70 % for sample recovery and more than 50 % for debris removal even in the most turbid samples tested. Due to the relatively simple design, the robustness of the inertial migration effect itself, the high operational flow rates and fabrication methods that leverage low-cost materials, the proposed device can have an impact on a wide range of applications where high-throughput separation of particles and biological species is of interest.

Big Data Impacting Dynamic Food Safety Risk Management in the Food Chain.

Foodborne pathogens are a major contributor to foodborne illness worldwide. The adaptation of a more quantitative risk-based approach, with metrics such as Food safety Objectives (FSO) and Performance Objectives (PO) necessitates quantitative inputs from all stages of the food value chain. The potential exists for utilization of big data, generated through digital transformational technologies, as inputs to a dynamic risk management concept for food safety microbiology. The industrial revolution in Internet of Things (IoT) will leverage data inputs from precision agriculture, connected factories/logistics, precision healthcare, and precision food safety, to improve the dynamism of microbial risk management. Furthermore, interconnectivity of public health databases, social media, and e-commerce tools as well as technologies such as blockchain will enhance traceability for retrospective and real-time management of foodborne cases. Despite the enormous potential of data volume and velocity, some challenges remain, including data ownership, interoperability, and accessibility. This paper gives insight to the prospective use of big data for dynamic risk management from a microbiological safety perspective in the context of the International Commission on Microbiological Specifications for Foods (ICMSF) conceptual equation, and describes examples of how a dynamic risk management system (DRMS) could be used in real-time to identify hazards and control Shiga toxin-producing Escherichia coli risks related to leafy greens.

Rotaviruses from Canadian farm samples.

Animal rotavirus (RoV) strains detected in Canadian swine and dairy cattle farms were characterized by sequence analysis of viral protein 4 (VP4), VP6, VP7 and non-structural protein 4 segments from 15 RoV strains. Some porcine strains were found to contain a mixture of segments typical of human and animal viruses. One strain represented a novel VP6 genotype "I14", G2-P[27]-I14. Other strains detected in porcine samples represented multiple different segment types. These results illustrate the active evolution of animal RoV strains and underline the need for surveillance of both animal and human strains in public health-monitoring programs.

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.

Outbreak of Escherichia coli O157:H7 Infections Linked to Mechanically Tenderized Beef and the Largest Beef Recall in Canada, 2012.

Contaminated beef is a known vehicle of Escherichia coli O157:H7 infection, although more attention is given to the control of E. coli O157:H7 in ground, rather than whole-cut, beef products. In September 2012, an investigation was initiated at an Alberta, Canada, beef plant after the detection of E. coli O157:H7 in two samples of trim cut from beef originating from this plant. Later in September 2012, Alberta Health Services identified five laboratory-confirmed infections of E. coli O157:H7, and case patients reported eating needle-tenderized beef steaks purchased at a store in Edmonton, Alberta, produced with beef from the Alberta plant. In total, 18 laboratory-confirmed illnesses in Canada in September and October 2012 were linked to beef from the Alberta plant, including the five individuals who ate needle-tenderized steaks purchased at the Edmonton store. A unique strain of E. coli O157:H7, defined by molecular subtyping and whole genome sequencing, was detected in clinical isolates, four samples of leftover beef from case patient homes, and eight samples of Alberta plant beef tested by industry and food safety partners. Investigators identified several deficiencies in the control of E. coli O157:H7 at the plant; in particular, the evaluation of, and response to, the detection of E. coli O157 in beef samples during routine testing were inadequate. To control the outbreak, 4,000 tons of beef products were recalled, making it the largest beef recall in Canadian history. This outbreak, in combination with similar outbreaks in the United States and research demonstrating that mechanical tenderization can transfer foodborne pathogens present on the surface into the interior of beef cuts, prompted amendments to Canada's Food and Drug Regulations requiring mechanically tenderized beef to be labeled as such and to provide safe cooking instructions to consumers. A detailed review of this event also led to recommendations and action to improve the safety of Canada's beef supply.

Myths of the high medical cost of old age and dying.

This report challenges commonly held beliefs about the financial and medical impact of older Americans during their last months of life. Written by physicians specializing in geriatrics, the report offers a wealth of data to refute seven misconceptions that currently influence U.S. health care policies: (1) that the growing number of older people has been the primary factor driving the rise in U.S. health care costs; (2) that as the population ages, health care costs for older Americans will necessarily overwhelm and bankrupt the nation; (3) that putting limits on health care for the very old at the end of life would save Medicare significant amounts of money; (4) that aggressive hospital care for the aged is futile and the money spent is wasted; (5) that it is common for older people to receive heroic, high-tech treatments at the end of life; (6) that Medicare covers everything that older adults need in terms of their health care; (7) that if older patients had living wills or other kinds of advance directives, it would resolve dilemmas of how aggressively to provide care.

Outbreak of Escherichia coli O157:H7 Infections Linked to Aged Raw Milk Gouda Cheese, Canada, 2013.

Between 12 July and 29 September 2013, 29 individuals in five Canadian provinces became ill following infection with the same strain of Escherichia coli O157:H7 as defined by molecular typing results. Five case patients were hospitalized, and one died. Twenty-six case patients (90%) reported eating Gouda cheese originating from a dairy plant in British Columbia. All of the 22 case patients with sufficient product details available reported consuming Gouda cheese made with raw milk; this cheese had been produced between March and July 2013 and was aged for a minimum of 60 days. The outbreak strain was isolated from the implicated Gouda cheese, including one core sample obtained from an intact cheese wheel 83 days after production. The findings indicate that raw milk was the primary source of the E. coli O157:H7, which persisted through production and the minimum 60-day aging period. This outbreak is the third caused by E. coli O157:H7 traced to Gouda cheese made with raw milk in North America. These findings provide further evidence that a 60-day ripening period cannot ensure die-off of pathogens that might be present in raw milk Gouda cheese after production and have triggered an evaluation of processing conditions, physicochemical parameters, and options to mitigate the risk of E. coli O157:H7 infection associated with raw milk Gouda cheese produced in Canada.

An overview of microbial food safety programs in beef, pork, and poultry from farm to processing in Canada.

Canada's vision for the agri-food industry in the 21st century is the establishment of a national food safety system employing hazard analysis and critical control point (HACCP) principles and microbiological verification tools, with traceability throughout the gate-to-plate continuum. Voluntary on-farm food safety (OFFS) programs, based in part on HACCP principles, provide producers with guidelines for good production practices focused on general hygiene and biosecurity. OFFS programs in beef cattle, swine, and poultry are currently being evaluated through a national recognition program of the Canadian Food Inspection Agency. Mandatory HACCP programs in federal meat facilities include microbial testing for generic Escherichia coli to verify effectiveness of the processor's dressing procedure, specific testing of ground meat for E. coli O157:H7, with zero tolerance for this organism in the tested lot, and Salmonella testing of raw products. Health Canada's policy on Listeria monocytogenes divides ready-to-eat products into three risk categories, with products previously implicated as the source of an outbreak receiving the highest priority for inspection and compliance. A national mandatory identification program to track livestock from the herd of origin to carcass inspection has been established. Can-Trace, a data standard for all food commodities, has been designed to facilitate tracking foods from the point of origin to the consumer. Although much work has already been done, a coherent national food safety strategy and concerted efforts by all stakeholders are needed to realize this vision. Cooperation of many government agencies with shared responsibility for food safety and public health will be essential.

Evidence for the benefits of food chain interventions on E. coli O157:H7/NM prevalence in retail ground beef and human disease incidence: A success story.

OBJECTIVES: Human infection with Escherichia coli O157:H7/NM has historically been associated with consumption of undercooked ground beef. The purpose of this paper is to investigate the correlation of the decline in E. coli O157:H7/NM infections in Canada with the introduction of control efforts in ground beef by industry. METHODS: The human incidence of E. coli O157:H7/NM, prevalence in ground beef and interventions from 1996 to 2014 were analyzed. Pathogen prevalence data were obtained from federal government and industry surveillance and inspection/compliance programs. A survey of the largest ground beef producers in Canada was conducted to identify when interventions were implemented. RESULTS: The incidence of E. coli O157:H7/NM infections in Canada declined from ≈4 cases/100 000 to ≈1 case/100000 from 2000 to 2010. Verotoxigenic Escherichia coli (VTEC) prevalence in ground beef sold at retail declined from about 30% around the year 2000 to <2% since 2012. Other measures of the prevalence of E. coli, VTEC, and E. coli O157:H7/NM in beef and ground beef also declined. The number and types of interventions implemented in the major beef processing establishments in Canada increased from 1996 to 2016. CONCLUSION: The observed decline in human illnesses and pathogen levels in relation to retail meats was associated with the introduction of control efforts by industry, federal and provincial/territorial governments, and the general population. Industry-led changes in beef processing along with the introduction of food safety policies, regulations, and public education have led to improved food safety in Canada.

Canadian listeriosis reference service.

Listeria monocytogenes, a psychrotrophic organism capable of growing at refrigeration temperatures, is of major concern in extended shelf life, refrigerated foods. Considering that as much as 80-90% of human listeriosis cases are linked to the ingestion of contaminated food, human cases are predominantly seen in high-risk individuals, including organ-transplant recipients, patients with AIDS and HIV-infected individuals, pregnant women, cancer patients, and the elderly. In 2001, the Canadian Listeriosis Reference Service (LRS) was created by the Bureau of Microbial Hazards (Health Canada) and the National Microbiology Laboratory (now part of the Public Health Agency of Canada). Major goals of the LRS include investigation of listeriosis cases and maintenance of a national collection of isolates. The LRS intends to create a comprehensive molecular epidemiological database of all isolates in Canada for use as a resource for outbreak investigations, research and other microbiological investigations. The PFGE profiles are being established and stored for clinical, food, environmental, and possibly animal strains of L. monocytogenes. The LRS pursues research activities for investigation and implementation of other molecular methods for characterizing L. monocytogenes isolates. Ribotyping, Multi-locus Sequence Typing (MLST), Variable Number of Tandem Repeats (VNTR), Multi-locus virulence sequence typing (MLVA), microarray- based technologies and sequence-based typing schemes, are being investigated on selected diversity sets. The LRS has also used PFGE typing for outbreak investigations. The molecular epidemiological data, timely coordination and exchange of information should help to reduce the incidence of listeriosis in Canada. In Canada, listeriosis is not a national notifiable disease, except for the province of Quebec, where it has been since 1999. The LRS, Canadian Public Health Laboratory Network, and federal epidemiologists are currently working on making human listeriosis notifiable throughout Canada.

Assessment of the Microbiological Quality of Ready-To-Use Vegetables for Health-Care Food Services.

The microbiological quality of ready-to-use (RTU) vegetables, including chopped lettuce, salad mix, carrot sticks, cauliflower florets, sliced celery, coleslaw mix, broccoli florets, and sliced green peppers was determined before and after processing. Microbial profiles were obtained 24 h after processing and on days 4, 7, and 11 after storage at 4 and 10°C to simulate temperature abuse. In addition, the microbial profiles of four RTU vegetables, coleslaw mix, salad mix, cauliflower florets, and sliced green peppers were determined 7 days after distribution to a select group of Ontario hospitals. RTU vegetables, with the exception of green peppers, showed up to a 1-log decrease in aerobic colony counts after processing. These counts increased to preprocessing levels after 4 days of storage at both 4 and 10°C. RTU vegetables stored at temperature abuse conditions (10°C)had significantly higher counts (P < 0.001) on days 4 to 11 as compared to those stored at 4°C. Green peppers had the highest bacterial counts while cauliflower and chopped lettuce had the lowest counts at both storage temperatures (P < 0.05). Increased levels of Listeria monocytogenes in RTU vegetables were associated with temperature abuse. Levels of >100 MPN/g for L. monocytogenes were detected in 8 of 120 (6.7%) samples stored at 10°C but not in 175 samples stored at 4°C after 7 days (P < 0.05). Overall, L. monocytogenes was detected in 13 of 120 (10.8%) RTU vegetables stored for up to 11 days at 10°C and 5 of 176 (2.8%) samples stored at 4°C (P < 0.05). E. coli was detected in 2 of the 120 (1.7%) processed RTU vegetables after day 7 of storage at 10°C and 1 of the 65 (1.5%) unprocessed vegetables from the same batches of vegetables used for processing. This indicator organism was not detected in RTU vegetable samples stored at 4°C or in any of the RTU vegetable samples obtained from hospital coolers. Other pathogenic bacteria, such as Salmonella spp., Campylobacter spp., Yersinia enterocolitica (serotype O:3) and verocytotoxigenic E. coli (VTEC) were not detected in any of the RTU vegetables tested, Recommendations regarding processing, distribution, and storage of these products are presented.