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.

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.

Emergence and global spread of Listeria monocytogenes main clinical clonal complex.

The bacterial foodborne pathogen Listeria monocytogenes clonal complex 1 (Lm-CC1) is the most prevalent clonal group associated with human listeriosis and is strongly associated with cattle and dairy products. Here, we analyze 2021 isolates collected from 40 countries, covering Lm-CC1 first isolation to present days, to define its evolutionary history and population dynamics. We show that Lm-CC1 spread worldwide from North America following the Industrial Revolution through two waves of expansion, coinciding with the transatlantic livestock trade in the second half of the 19th century and the rapid growth of cattle farming and food industrialization in the 20th century. In sharp contrast to its global spread over the past century, transmission chains are now mostly local, with limited inter- and intra-country spread. This study provides an unprecedented insight into L. monocytogenes phylogeography and population dynamics and highlights the importance of genome analyses for a better control of pathogen transmission.

Free-Living Aquatic Turtles as Sentinels of Salmonella spp. for Water Bodies.

Reptile-associated human salmonellosis cases have increased recently in the United States. It is not uncommon to find healthy chelonians shedding Salmonella enterica. The rate and frequency of bacterial shedding are not fully understood, and most studies have focused on captive vs. free-living chelonians and often in relation to an outbreak. Their ecology and significance as sentinels are important to understanding Salmonella transmission. In 2012-2013, Salmonella prevalence was determined for free-living aquatic turtles in man-made ponds in Clarke and Oconee Counties, in northern Georgia (USA) and the correlation between species, basking ecology, demographics (age/sex), season, or landcover with prevalence was assessed. The genetic relatedness between turtle and archived, human isolates, as well as, other archived animal and water isolates reported from this study area was examined. Salmonella was isolated from 45 of 194 turtles (23.2%, range 14-100%) across six species. Prevalence was higher in juveniles (36%) than adults (20%), higher in females (33%) than males (18%), and higher in bottom-dwelling species (31%; common and loggerhead musk turtles, common snapping turtles) than basking species (15%; sliders, painted turtles). Salmonella prevalence decreased as forest cover, canopy cover, and distance from roads increased. Prevalence was also higher in low-density, residential areas that have 20-49% impervious surface. A total of 9 different serovars of two subspecies were isolated including 3 S. enterica subsp. arizonae and 44 S. enterica subsp. enterica (two turtles had two serotypes isolated from each). Among the S. enterica serovars, Montevideo (n = 13) and Rubislaw (n = 11) were predominant. Salmonella serovars Muenchen, Newport, Mississippi, Inverness, Brazil, and Paratyphi B. var L(+) tartrate positive (Java) were also isolated. Importantly, 85% of the turtle isolates matched pulsed-field gel electrophoresis patterns of human isolates, including those reported from Georgia. Collectively, these results suggest that turtles accumulate Salmonella present in water bodies, and they may be effective sentinels of environmental contamination. Ultimately, the Salmonella prevalence rates in wild aquatic turtles, especially those strains shared with humans, highlight a significant public health concern.

Corrigendum: Whole Genome Sequencing: Bridging One-Health Surveillance of Foodborne Diseases.

[This corrects the article DOI: 10.3389/fpubh.2019.00172.].

PulseNet and the Changing Paradigm of Laboratory-Based Surveillance for Foodborne Diseases.

PulseNet, the National Molecular Subtyping Network for Foodborne Disease Surveillance, was established in 1996 through a collaboration with the Centers for Disease Control and Prevention; the US Department of Agriculture, Food Safety and Inspection Service; the US Food and Drug Administration; 4 state public health laboratories; and the Association of Public Health Laboratories. The network has since expanded to include 83 state, local, and food regulatory public health laboratories. In 2016, PulseNet was estimated to be helping prevent an estimated 270 000 foodborne illnesses annually. PulseNet is undergoing a transformation toward whole-genome sequencing (WGS), which provides better discriminatory power and precision than pulsed-field gel electrophoresis (PFGE). WGS improves the detection of outbreak clusters and could replace many traditional reference identification and characterization methods. This article highlights the contributions made by public health laboratories in transforming PulseNet's surveillance and describes how the transformation is changing local and national surveillance practices. Our data show that WGS is better at identifying clusters than PFGE, especially for clonal organisms such as Salmonella Enteritidis. The need to develop prioritization schemes for cluster follow-up and additional resources for both public health laboratory and epidemiology departments will be critical as PulseNet implements WGS for foodborne disease surveillance in the United States.

Whole Genome Sequencing: Bridging One-Health Surveillance of Foodborne Diseases.

Infections caused by pathogens commonly acquired from consumption of food are not always transmitted by that route. They may also be transmitted through contact to animals, other humans or the environment. Additionally, many outbreaks are associated with food contaminated from these non-food sources. For this reason, such presumed foodborne outbreaks are best investigated through a One Health approach working across human, animal and environmental sectors and disciplines. Outbreak strains or clones that have propagated and continue to evolve in non-human sources and environments often show more sequence variation than observed in typical monoclonal point-source outbreaks. This represents a challenge when using whole genome sequencing (WGS), the new gold standard for molecular surveillance of foodborne pathogens, for outbreak detection and investigation. In this review, using recent examples from outbreaks investigated in the United States (US) some aspects of One Health approaches that have been used successfully to solve such outbreaks are presented. These include using different combinations of flexible WGS based case definition, efficient epidemiological follow-up, traceback, surveillance, and testing of potential food and environmental sources and animal hosts.

PulseNet: Entering the Age of Next-Generation Sequencing.

Since 1996, PulseNet has served as the national laboratory-based surveillance system for the rapid detection of outbreaks caused by foodborne bacterial pathogens in the United States. For the past two decades, pulsed-field gel electrophoresis was the gold standard subtyping method for the pathogens tracked by PulseNet. A new gold standard is now being implemented with the introduction of cost-effective whole genome sequencing (WGS) for analysis of all the organisms tracked by PulseNet. This transformation is a major undertaking that touches every functional aspect of PulseNet, including laboratory workflows, data storage, analysis management and data interpretation, and language used to communicate information (sequence profile nomenclature system). The benefits of implementing WGS go beyond improved discrimination and precision of the data; it provides an opportunity to determine strain characteristics typically obtained through resource-intensive traditional methodologies, for example, species identification, serotyping, virulence, and antimicrobial resistance profiling, all of which can be consolidated into a single WGS workflow. Such a strategy represents a major shift in the workflows currently practiced in most public health laboratories, but one that brings opportunities for streamlining surveillance activities for the network as a whole. In this study, we provide a brief summary of PulseNet's evolution the past decade along with a general description of the challenges and opportunities that lie ahead.

Use of Whole-Genome Sequencing for Food Safety and Public Health in the United States.

Whole-genome sequencing (WGS) is increasingly used by food regulatory and public health agencies in the United States to facilitate the detection, investigation, and control of foodborne bacterial outbreaks, and food regulatory and other activities in support of food safety. WGS has added a level of precision to the surveillance leading to faster and more efficient decision making in the preparedness and response to foodborne infections. In this review, we report the history of WGS technology at the Centers for Disease Control & Prevention (CDC), the Food and Drug Administration (FDA), and the United States Department of Agriculture's Food Safety and Inspection Service (USDA/FSIS) as it applies to food safety. The basic principle of the method, the analysis, and interpretation of the data are explained as is its major strengths and limitations. We also describe the benefits and possibilities of the WGS technology to the food industry throughout the farm-to-fork continuum and the prospects of metagenomic sequencing applied directly to the sample specimen with or without pre-enrichment culture.

Interpretation of Whole-Genome Sequencing for Enteric Disease Surveillance and Outbreak Investigation.

The routine use of whole-genome sequencing (WGS) as part of enteric disease surveillance is substantially enhancing our ability to detect and investigate outbreaks and to monitor disease trends. At the same time, it is revealing as never before the vast complexity of microbial and human interactions that contribute to outbreak ecology. Since WGS analysis is primarily used to characterize and compare microbial genomes with the goal of addressing epidemiological questions, it must be interpreted in an epidemiological context. In this article, we identify common challenges and pitfalls encountered when interpreting sequence data in an enteric disease surveillance and investigation context, and explain how to address them.

Metagenomic Approaches for Public Health Surveillance of Foodborne Infections: Opportunities and Challenges.

Foodborne disease surveillance in the United States is at a critical point. Clinical and diagnostic laboratories are using culture-independent diagnostic tests (CIDTs) to identify the pathogen causing foodborne illness from patient specimens. CIDTs are molecular tests that allow doctors to rapidly identify the bacteria causing illness within hours. CIDTs, unlike previous gold standard methods such as bacterial culture, do not produce an isolate that can be subtyped as part of the national molecular subtyping network for foodborne disease surveillance, PulseNet. Without subtype information, cases can no longer be linked using molecular data to identify potentially related cases that are part of an outbreak. In this review, we discuss the public health needs for a molecular subtyping approach directly from patient specimen and highlight different approaches, including amplicon and shotgun metagenomic sequencing.

An Overview of PulseNet USA Databases.

PulseNet USA is the molecular surveillance network for foodborne disease in the United States. The network consists of state and local public health laboratories, as well as food regulatory agencies, that follow PulseNet's standardized protocols to perform pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) and analyze the results using standardized software. The raw sequences are uploaded to the GenomeTrakr or PulseNet bioprojects at the National Center for Biotechnology Information. The PFGE patterns and analyzed sequence data are uploaded in real time with associated demographic data to the PulseNet national databases managed at the Centers for Disease Control and Prevention. The PulseNet databases are organism specific and provide a central storage location for molecular and demographic data related to an isolate. Sequences are compared in the databases, thereby facilitating the rapid detection of clusters of foodborne diseases that may represent widespread outbreaks. WGS genotyping data, for example, antibiotic resistance and virulence profiles, are also uploaded in real time to the PulseNet databases to improve food safety surveillance activities.

The use of next generation sequencing for improving food safety: Translation into practice.

Next Generation Sequencing (NGS) combined with powerful bioinformatic approaches are revolutionising food microbiology. Whole genome sequencing (WGS) of single isolates allows the most detailed comparison possible hitherto of individual strains. The two principle approaches for strain discrimination, single nucleotide polymorphism (SNP) analysis and genomic multi-locus sequence typing (MLST) are showing concordant results for phylogenetic clustering and are complementary to each other. Metabarcoding and metagenomics, applied to total DNA isolated from either food materials or the production environment, allows the identification of complete microbial populations. Metagenomics identifies the entire gene content and when coupled to transcriptomics or proteomics, allows the identification of functional capacity and biochemical activity of microbial populations. The focus of this review is on the recent use and future potential of NGS in food microbiology and on current challenges. Guidance is provided for new users, such as public health departments and the food industry, on the implementation of NGS and how to critically interpret results and place them in a broader context. The review aims to promote the broader application of NGS technologies within the food industry as well as highlight knowledge gaps and novel applications of NGS with the aim of driving future research and increasing food safety outputs from its wider use.

Advancing biological hazards risk assessment.

This paper focusses on biological hazards at the global level and considers the challenges to risk assessment (RA) from a One Health perspective. Two topics - vector-borne diseases (VBD) and antimicrobial resistance (AMR) - are used to illustrate the challenges ahead and to explore the opportunities that new methodologies such as next-generation sequencing can offer. Globalisation brings complexity and introduces drivers for infectious diseases. Cooperation and the application of an integrated RA approach - one that takes into consideration food farming and production systems including social and environmental factors - are recommended. Also needed are methodologies to identify emerging risks at a global level and propose prevention strategies. AMR is one of the biggest threats to human health in the infectious disease environment. Whereas new genomic typing techniques such as whole genome sequencing (WGS) provide further insights into the mechanisms of spread of resistance, the role of the environment is not fully elucidated, nor is the role of plants as potential vehicles for spread of resistance. Historical trends and recent experience indicate that (re)-emergence and/or further spread of VBD within the EU is a matter of when rather than if. Standardised and validated vector monitoring programs are required to be implemented at an international level for continuous surveillance and assessment of potential threats. There are benefits to using WGS - such as a quicker and better response to outbreaks and additional evidence for source attribution. However, significant challenges need to be addressed, including method standardisation and validation to fully realise these benefits; barriers to data sharing; and establishing epidemiological capacity for cluster triage and response.

An Assessment of Different Genomic Approaches for Inferring Phylogeny of Listeria monocytogenes.

Background/objectives: Whole genome sequencing (WGS) has proven to be a powerful subtyping tool for foodborne pathogenic bacteria like L. monocytogenes. The interests of genome-scale analysis for national surveillance, outbreak detection or source tracking has been largely documented. The genomic data however can be exploited with many different bioinformatics methods like single nucleotide polymorphism (SNP), core-genome multi locus sequence typing (cgMLST), whole-genome multi locus sequence typing (wgMLST) or multi locus predicted protein sequence typing (MLPPST) on either core-genome (cgMLPPST) or pan-genome (wgMLPPST). Currently, there are little comparisons studies of these different analytical approaches. Our objective was to assess and compare different genomic methods that can be implemented in order to cluster isolates of L. monocytogenes. Methods: The clustering methods were evaluated on a collection of 207 L. monocytogenes genomes of food origin representative of the genetic diversity of the Anses collection. The trees were then compared using robust statistical analyses. Results: The backward comparability between conventional typing methods and genomic methods revealed a near-perfect concordance. The importance of selecting a proper reference when calling SNPs was highlighted, although distances between strains remained identical. The analysis also revealed that the topology of the phylogenetic trees between wgMLST and cgMLST were remarkably similar. The comparison between SNP and cgMLST or SNP and wgMLST approaches showed that the topologies of phylogenic trees were statistically similar with an almost equivalent clustering. Conclusion: Our study revealed high concordance between wgMLST, cgMLST, and SNP approaches which are all suitable for typing of L. monocytogenes. The comparable clustering is an important observation considering that the two approaches have been variously implemented among reference laboratories.

PulseNet International: Vision for the implementation of whole genome sequencing (WGS) for global food-borne disease surveillance.

PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease.

Whole genome-based population biology and epidemiological surveillance of Listeria monocytogenes.

Listeria monocytogenes (Lm) is a major human foodborne pathogen. Numerous Lm outbreaks have been reported worldwide and associated with a high case fatality rate, reinforcing the need for strongly coordinated surveillance and outbreak control. We developed a universally applicable genome-wide strain genotyping approach and investigated the population diversity of Lm using 1,696 isolates from diverse sources and geographical locations. We define, with unprecedented precision, the population structure of Lm, demonstrate the occurrence of international circulation of strains and reveal the extent of heterogeneity in virulence and stress resistance genomic features among clinical and food isolates. Using historical isolates, we show that the evolutionary rate of Lm from lineage I and lineage II is low (∼2.5 × 10-7 substitutions per site per year, as inferred from the core genome) and that major sublineages (corresponding to so-called 'epidemic clones') are estimated to be at least 50-150 years old. This work demonstrates the urgent need to monitor Lm strains at the global level and provides the unified approach needed for global harmonization of Lm genome-based typing and population biology.

Urbanized White Ibises (Eudocimus albus) as Carriers of Salmonella enterica of Significance to Public Health and Wildlife.

Worldwide, Salmonella spp. is a significant cause of disease for both humans and wildlife, with wild birds adapted to urban environments having different opportunities for pathogen exposure, infection, and transmission compared to their natural conspecifics. Food provisioning by people may influence these factors, especially when high-density mixed species flocks aggregate. White Ibises (Eudocimus albus), an iconic Everglades species in decline in Florida, are becoming increasingly common in urbanized areas of south Florida where most are hand-fed. We examined the prevalence of Salmonella shedding by ibises to determine the role of landscape characteristics where ibis forage and their behavior, on shedding rates. We also compared Salmonella isolated from ibises to human isolates to better understand non-foodborne human salmonellosis. From 2010-2013, 13% (n = 261) adult/subadult ibises and 35% (n = 72) nestlings sampled were shedding Salmonella. The prevalence of Salmonella shedding by ibises significantly decreased as the percent of Palustrine emergent wetlands and herbaceous grasslands increased, and increased as the proportion of open-developed land types (e.g. parks, lawns, golf courses) increased, suggesting that natural ecosystem land cover types supported birds with a lower prevalence of infection. A high diversity of Salmonella serotypes (n = 24) and strain types (43 PFGE types) were shed by ibises, of which 33% of the serotypes ranked in the top 20 of high significance for people in the years of the study. Importantly, 44% of the Salmonella Pulsed-Field Gel Electrophoresis patterns for ibis isolates (n = 43) matched profiles in the CDC PulseNet USA database. Of these, 20% came from Florida in the same three years we sampled ibis. Importantly, there was a negative relationship between the amount of Palustrine emergent wetland and the number of Salmonella isolates from ibises that matched human cases in the PulseNet database (p = 0.056). Together, our results indicate that ibises are good indicators of salmonellae strains circulating in their environment and they have both the potential and opportunity to transmit salmonellae to people. Finally, they may act as salmonellae carriers to natural environments where other more highly-susceptible groups (nestlings) may be detrimentally affected.

Multiple-locus variable-number tandem repeat analysis for strain discrimination of non-O157 Shiga toxin-producing Escherichia coli.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens of growing concern worldwide that have been associated with several recent multistate and multinational outbreaks of foodborne illness. Rapid and sensitive molecular-based bacterial strain discrimination methods are critical for timely outbreak identification and contaminated food source traceback. One such method, multiple-locus variable-number tandem repeat analysis (MLVA), is being used with increasing frequency in foodborne illness outbreak investigations to augment the current gold standard bacterial subtyping technique, pulsed-field gel electrophoresis (PFGE). The objective of this study was to develop a MLVA assay for intra- and inter-serogroup discrimination of six major non-O157 STEC serogroups-O26, O111, O103, O121, O45, and O145-and perform a preliminary internal validation of the method on a limited number of clinical isolates. The resultant MLVA scheme consists of ten variable number tandem repeat (VNTR) loci amplified in three multiplex PCR reactions. Sixty-five unique MLVA types were obtained among 84 clinical non-O157 STEC strains comprised of geographically diverse sporadic and outbreak related isolates. Compared to PFGE, the developed MLVA scheme allowed similar discrimination among serogroups O26, O111, O103, and O121 but not among O145 and O45. To more fully compare the discriminatory power of this preliminary MLVA method to PFGE and to determine its epidemiological congruence, a thorough internal and external validation needs to be performed on a carefully selected large panel of strains, including multiple isolates from single outbreaks.

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.