A quantitative risk metric to support individual sanitary measure reviews in international trade.

In order for the United States Department of Agriculture's (USDA) Food Safety and Inspection Service (FSIS) to make an equivalence determination for a foreign meat, poultry or egg products inspection procedure that differs from FSIS inspection procedures (an Individual Sanitary Measure or ISM), a country must demonstrate objectively that its food safety inspection system provides the same level of public health protection as the FSIS inspection system. To evaluate microbiological testing data that such countries may submit to this end, we present a possible risk metric to inform FSIS's assessment of whether products produced under an alternative inspection system in another country pose no greater consumer risk of foodborne illness than products produced under FSIS inspection. This metric requires evaluation of prevalence estimates of pathogen occurrence in products for the foreign country and the U.S. and determining what constitutes an unacceptable deviance of another country's prevalence from the U.S. prevalence, i.e., the margin of equivalence. We define the margin of equivalence as a multiple of the standard error of the U.S. prevalence estimate. Minimizing the margin of equivalence ensures the maximum public health protection for U.S. consumers, but an optimum choice must also avoid undue burden for quantitative data from alternative inspection systems in the foreign country. Across a wide range of U.S. prevalence levels and sample sizes, we determine margin of equivalence values that provide high confidence in conclusions as to whether or not the country's product poses no greater risk of foodborne illness from microbiological pathogens. These margins of equivalence can be used to inform FSIS's equivalence determination for an ISM request from a foreign country. Illustrative examples are used to support this definition of margin of equivalence. This approach is consistent with the World Trade Organization's concept of risk equivalence and is transparent and practical to apply in situations when FSIS makes an equivalence determination for an ISM requested by a foreign country.

Use of Whole Genome Sequencing by the Federal Interagency Collaboration for Genomics for Food and Feed Safety in the United States.

ABSTRACT: This multiagency report developed by the Interagency Collaboration for Genomics for Food and Feed Safety provides an overview of the use of and transition to whole genome sequencing (WGS) technology for detection and characterization of pathogens transmitted commonly by food and for identification of their sources. We describe foodborne pathogen analysis, investigation, and harmonization efforts among the following federal agencies: National Institutes of Health; Department of Health and Human Services, Centers for Disease Control and Prevention (CDC) and U.S. Food and Drug Administration (FDA); and the U.S. Department of Agriculture, Food Safety and Inspection Service, Agricultural Research Service, and Animal and Plant Health Inspection Service. We describe single nucleotide polymorphism, core-genome, and whole genome multilocus sequence typing data analysis methods as used in the PulseNet (CDC) and GenomeTrakr (FDA) networks, underscoring the complementary nature of the results for linking genetically related foodborne pathogens during outbreak investigations while allowing flexibility to meet the specific needs of Interagency Collaboration partners. We highlight how we apply WGS to pathogen characterization (virulence and antimicrobial resistance profiles) and source attribution efforts and increase transparency by making the sequences and other data publicly available through the National Center for Biotechnology Information. We also highlight the impact of current trends in the use of culture-independent diagnostic tests for human diagnostic testing on analytical approaches related to food safety and what is next for the use of WGS in the area of food safety.

Multidrug-Resistant Salmonella I 4,[5],12:i:- and Salmonella Infantis Infections Linked to Whole Roasted Pigs from a Single Slaughter and Processing Facility.

We describe two outbreaks of multidrug-resistant (MDR) Salmonella I 4,[5],12:i:- infection, occurring in 2015 to 2016, linked to pork products, including whole roaster pigs sold raw from a single Washington slaughter and processing facility (establishment A). Food histories from 80 ill persons were compared with food histories reported in the FoodNet 2006 to 2007 survey of healthy persons from all 10 U.S. FoodNet sites who reported these exposures in the week before interview. Antimicrobial susceptibility testing and whole genome sequencing were conducted on selected clinical, food, and environmental isolates. During 2015, a total of 192 ill persons were identified from five states; among ill persons with available information, 30 (17%) of 180 were hospitalized, and none died. More ill persons than healthy survey respondents consumed pork (74 versus 43%, P < 0.001). Seventeen (23%) of 73 ill persons for which a response was available reported attending an event where whole roaster pig was served in the 7 days before illness onset. All 25 clinical isolates tested from the 2015 outbreak and a subsequent 2016 smaller outbreak (n = 15) linked to establishment A demonstrated MDR. Whole genome sequencing of clinical, environmental, and food isolates (n = 69) collected in both investigations revealed one clade of highly related isolates, supporting epidemiologic and traceback data that establishment A as the source of both outbreaks. These investigations highlight that whole roaster pigs, an uncommon food vehicle for MDR Salmonella I 4,[5],12:i:- outbreaks, will need further attention from food safety researchers and educators for developing science-based consumer guidelines, specifically with a focus on the preparation process.

Intensified Sampling in Response to a Salmonella Heidelberg Outbreak Associated with Multiple Establishments Within a Single Poultry Corporation.

On June 28, 2013, the Food Safety and Inspection Service (FSIS) was notified by the Centers for Disease Control and Prevention (CDC) of an investigation of a multistate cluster of illnesses of Salmonella enterica serovar Heidelberg. Since case-patients in the cluster reported consumption of a variety of chicken products, FSIS used a simple likelihood-based approach using traceback information to focus on intensified sampling efforts. This article describes the multiphased product sampling approach taken by FSIS when epidemiologic evidence implicated chicken products from multiple establishments operating under one corporation. The objectives of sampling were to (1) assess process control of chicken slaughter and further processing and (2) determine whether outbreak strains were present in products from these implicated establishments. As part of the sample collection process, data collected by FSIS personnel to characterize product included category (whole chicken and type of chicken parts), brand, organic or conventional product, injection with salt solutions or flavorings, and whether product was skinless or skin-on. From the period September 9, 2013, through October 31, 2014, 3164 samples were taken as part of this effort. Salmonella percent positive declined from 19.7% to 5.3% during this timeframe as a result of regulatory and company efforts. The results of intensified sampling for this outbreak investigation informed an FSIS regulatory response and corrective actions taken by the implicated establishments. The company noted that a multihurdle approach to reduce Salmonella in products was taken, including on-farm efforts such as environmental testing, depopulation of affected flocks, disinfection of affected houses, vaccination, and use of various interventions within the establishments over the course of several months.

Utility of Combining Whole Genome Sequencing with Traditional Investigational Methods To Solve Foodborne Outbreaks of Salmonella Infections Associated with Chicken: A New Tool for Tackling This Challenging Food Vehicle.

High consumption rates and a multitude of brands make multistate foodborne outbreaks of Salmonella infections associated with chicken challenging to investigate, but whole genome sequencing is a powerful tool that can be used to assist investigators. Whole genome sequencing of pathogens isolated from clinical, environmental, and food samples is increasingly being used in multistate foodborne outbreak investigations to determine with unprecedented resolution how closely related these isolates are to one another genetically. In 2014, federal and state health officials investigated an outbreak of 146 Salmonella Heidelberg infections in 24 states. A follow-up analysis was conducted after the conclusion of the investigation in which 27 clinical and 24 food isolates from the outbreak underwent whole genome sequencing. These isolates formed seven clades, the largest of which contained clinical isolates from a subcluster of case patients who attended a catered party. One isolate from a chicken processed by a large producer was closely related genetically (zero to three single-nucleotide polymorphism differences) to the clinical isolates from these subcluster case patients. Chicken from this large producer was also present in the kitchen of the caterer on the day before the event, thus providing additional evidence that the chicken from this producer was the outbreak source. This investigation highlights how whole genome sequencing can be used with epidemiologic and traceback evidence to identify chicken sources of foodborne outbreaks.

Benchmark datasets for phylogenomic pipeline validation, applications for foodborne pathogen surveillance.

BACKGROUND: As next generation sequence technology has advanced, there have been parallel advances in genome-scale analysis programs for determining evolutionary relationships as proxies for epidemiological relationship in public health. Most new programs skip traditional steps of ortholog determination and multi-gene alignment, instead identifying variants across a set of genomes, then summarizing results in a matrix of single-nucleotide polymorphisms or alleles for standard phylogenetic analysis. However, public health authorities need to document the performance of these methods with appropriate and comprehensive datasets so they can be validated for specific purposes, e.g., outbreak surveillance. Here we propose a set of benchmark datasets to be used for comparison and validation of phylogenomic pipelines. METHODS: We identified four well-documented foodborne pathogen events in which the epidemiology was concordant with routine phylogenomic analyses (reference-based SNP and wgMLST approaches). These are ideal benchmark datasets, as the trees, WGS data, and epidemiological data for each are all in agreement. We have placed these sequence data, sample metadata, and "known" phylogenetic trees in publicly-accessible databases and developed a standard descriptive spreadsheet format describing each dataset. To facilitate easy downloading of these benchmarks, we developed an automated script that uses the standard descriptive spreadsheet format. RESULTS: Our "outbreak" benchmark datasets represent the four major foodborne bacterial pathogens (Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni) and one simulated dataset where the "known tree" can be accurately called the "true tree". The downloading script and associated table files are available on GitHub: https://github.com/WGS-standards-and-analysis/datasets. DISCUSSION: These five benchmark datasets will help standardize comparison of current and future phylogenomic pipelines, and facilitate important cross-institutional collaborations. Our work is part of a global effort to provide collaborative infrastructure for sequence data and analytic tools-we welcome additional benchmark datasets in our recommended format, and, if relevant, we will add these on our GitHub site. Together, these datasets, dataset format, and the underlying GitHub infrastructure present a recommended path for worldwide standardization of phylogenomic pipelines.

National Outbreak of Multidrug Resistant Salmonella Heidelberg Infections Linked to a Single Poultry Company.

IMPORTANCE: This large outbreak of foodborne salmonellosis demonstrated the complexity of investigating outbreaks linked to poultry products. The outbreak also highlighted the importance of efforts to strengthen food safety policies related to Salmonella in chicken parts and has implications for future changes within the poultry industry. OBJECTIVE: To investigate a large multistate outbreak of multidrug resistant Salmonella Heidelberg infections. DESIGN: Epidemiologic and laboratory investigations of patients infected with the outbreak strains of Salmonella Heidelberg and traceback of possible food exposures. SETTING: United States. Outbreak period was March 1, 2013 through July 11, 2014. PATIENTS: A case was defined as illness in a person infected with a laboratory-confirmed Salmonella Heidelberg with 1 of 7 outbreak pulsed-field gel electrophoresis (PFGE) XbaI patterns with illness onset from March 1, 2013 through July 11, 2014. A total of 634 case-patients were identified through passive surveillance; 200/528 (38%) were hospitalized, none died. RESULTS: Interviews were conducted with 435 case-patients: 371 (85%) reported eating any chicken in the 7 days before becoming ill. Of 273 case-patients interviewed with a focused questionnaire, 201 (74%) reported eating chicken prepared at home. Among case-patients with available brand information, 152 (87%) of 175 patients reported consuming Company A brand chicken. Antimicrobial susceptibility testing was completed on 69 clinical isolates collected from case-patients; 67% were drug resistant, including 24 isolates (35%) that were multidrug resistant. The source of Company A brand chicken consumed by case-patients was traced back to 3 California production establishments from which 6 of 7 outbreak strains were isolated. CONCLUSIONS: Epidemiologic, laboratory, traceback, and environmental investigations conducted by local, state, and federal public health and regulatory officials indicated that consumption of Company A chicken was the cause of this outbreak. The outbreak involved multiple PFGE patterns, a variety of chicken products, and 3 production establishments, suggesting a reservoir for contamination upstream from the production establishments. Sources of bacteria and genes responsible for resistance, such as farms providing birds for slaughter or environmental reservoir on farms that raise chickens, might explain how multiple PFGE patterns were linked to chicken from 3 separate production establishments and many different poultry products.

Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation.

Listeria monocytogenes (Lm) causes severe foodborne illness (listeriosis). Previous molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), were critical in detecting outbreaks that led to food safety improvements and declining incidence, but PFGE provides limited genetic resolution. A multiagency collaboration began performing real-time, whole-genome sequencing (WGS) on all US Lm isolates from patients, food, and the environment in September 2013, posting sequencing data into a public repository. Compared with the year before the project began, WGS, combined with epidemiologic and product trace-back data, detected more listeriosis clusters and solved more outbreaks (2 outbreaks in pre-WGS year, 5 in WGS year 1, and 9 in year 2). Whole-genome multilocus sequence typing and single nucleotide polymorphism analyses provided equivalent phylogenetic relationships relevant to investigations; results were most useful when interpreted in context of epidemiological data. WGS has transformed listeriosis outbreak surveillance and is being implemented for other foodborne pathogens.

Using a Six Sigma Fishbone Analysis Approach To Evaluate the Effect of Extreme Weather Events on Salmonella Positives in Young Chicken Slaughter Establishments.

A six sigma fishbone analysis approach was used to develop a machine learning model in SAS, Version 9.4, by using stepwise linear regression. The model evaluated the effect of a wide variety of variables, including slaughter establishment operational measures, normal (30-year average) weather, and extreme weather events on the rate of Salmonella -positive carcasses in young chicken slaughter establishments. Food Safety and Inspection Service (FSIS) verification carcass sampling data, as well as corresponding data from the National Oceanographic and Atmospheric Administration and the Federal Emergency Management Agency, from September 2011 through April 2015, were included in the model. The results of the modeling show that in addition to basic establishment operations, normal weather patterns, differences from normal and disaster events, including time lag weather and disaster variables, played a role in explaining the Salmonella percent positive that varied by slaughter volume quartile. Findings show that weather and disaster events should be considered as explanatory variables when assessing pathogen-related prevalence analysis or research and slaughter operational controls. The apparent significance of time lag weather variables suggested that at least some of the impact on Salmonella rates occurred after the weather events, which may offer opportunities for FSIS or the poultry industry to implement interventions to mitigate those effects.

Detection and confirmation of Clostridium botulinum in water used for cooling at a plant producing low-acid canned foods.

Our laboratory tested water samples used for cooling low-acid canned foods at a canning facility under investigation by the U.S. Food and Drug Administration. We used an enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies (DIG-ELISA) and real-time PCR as screening methods and confirmed the presence of neurotoxin-producing Clostridium botulinum in the samples by mouse bioassay.