Challenges Associated with Investigating Salmonella Enteritidis with Low Genomic Diversity in New York State: The Impact of Adjusting Analytical Methods and Correlation with Epidemiological Data.

Defining investigation-worthy genomic clusters among strains of Salmonella Enteritidis is challenging because of their highly clonal nature. We investigated a cluster identified by core genome multilocus sequence typing (cgMLST) consisting of 265 isolates with isolation dates spanning two and a half years. This cluster experienced chaining, growing to a range of 14 alleles. The volume of isolates and broad allele range of this cluster made it difficult to ascertain whether it represented a common-source outbreak. We explored laboratory-based methods to subdivide and refine this cluster. These methods included using cgMLST with a narrower allele range, whole genome multilocus sequence typing (wgMLST) and high-quality single-nucleotide polymorphism (hqSNP) analysis. At each analysis level, epidemiologists retroactively reviewed exposures, geography, and temporality for potential commonalities. Lowering the threshold to 0 alleles using cgMLST proved an effective method to refine this analysis, resulting in this large cluster being subdivided into 34 smaller clusters. Additional analysis by wgMLST and hqSNP provided enhanced cluster resolution, with the majority of clusters being further refined. These analysis methods combined with more stringent allele thresholds and layering of epidemiologic data proved useful in helping to subdivide this large cluster into actionable subclusters.

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.

PulseNet Lebanon: An Overview of Its Activities, Outbreak Investigations, and Challenges.

Background: Foodborne diseases are still a major health issue in Lebanon, although some steps have been taken forward in food safety. To this purpose, PulseNet Lebanon, a foodborne diseases tracking network, was established in 2009, through the collaboration between the Ministry of Public Health (MoPH) and the American University of Beirut (AUB). Materials and Methods: Three papers published regarding the PulseNet project were summarized. Initially, clinical and food samples, collected within the surveillance network scope, were identified by using the respective API for Salmonella and Listeria spp. Salmonella spp. were further serotyped by using the Kauffman and White method. Campylobacter spp. were determined by the 16 S rRNA sequencing method. Antimicrobial susceptibility to a number of antibiotics was determined by using the disk diffusion method for Samonella and Campylobacter spp. Genomic diversity was determined by using pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD). Results: Results indicated that 290 clinical and 49 food isolates were identified as Salmonella. Serotyping revealed the prevalence of ten and seven serotypes in the clinical and food samples, respectively. Fifty-one isolates from chicken ceca and carcass were identified to be Campylobacter spp. Fifty-nine samples were identified to be Listeria monocytogenes. Antimicrobial susceptibility testing revealed a wide range of resistance among the different samples. PFGE showed a variation in pulsotypes among the Salmonella serotypes. PFGE also linked certain outbreaks to their food sources. This method also demonstrated 13 subtypes with 100% similarity among the L. monocytogenes isolates. Finally, the Camplyobcater spp. were grouped into nine clusters with a minimum similarity of 43.5% using RAPD. Conclusion: This summary of results shows the importance of implementing a "farm-to-fork" approach in the surveillance of foodborne disease outbreaks in Lebanon, allowing the detection of pathogens causing foodborne disease outbreaks in a timely fashion.

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.

Laboratory Review of Foodborne Disease Investigations in Washington State 2007-2017.

The Washington State Department of Health Public Health Laboratories (WAPHL) has tested 11,501 samples between 2007 and 2017 for a foodborne disease using a combination of identification, serotyping, and subtyping tools. During this period there were 8037 total clinical and environmental samples tested by pulsed-field gel electrophoresis (PFGE), including 512 foodborne disease clusters and 2176 PFGE patterns of Salmonella enterica subsp. enterica. There were 2446 Shiga toxin-producing Escherichia coli samples tested by PFGE, which included 158 foodborne disease clusters and 1174 PFGE patterns. There were 332 samples of Listeria monocytogenes tested by PFGE, including 35 foodborne disease clusters and 104 PFGE patterns. Sources linked to outbreaks included raw chicken, unpasteurized dairy products, various produce types, and undercooked beef among others. As next-generation sequencing (NGS) replaces PFGE, the impact of this transition is expected to be significant given the enhanced cluster detection power NGS brings. The measures presented here will be a reference baseline in future years.

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.

Clinical Microbiology Laboratories' Adoption of Culture-Independent Diagnostic Tests Is a Threat to Foodborne-Disease Surveillance in the United States.

INTRODUCTIONIn November 2015, the Centers for Disease Control and Prevention (CDC) sent a letter to state and territorial epidemiologists, state and territorial public health laboratory directors, and state and territorial health officials. In this letter, culture-independent diagnostic tests (CIDTs) for detection of enteric pathogens were characterized as "a serious and current threat to public health surveillance, particularly for Shiga toxin-producing Escherichia coli (STEC) and Salmonella" The document says CDC and its public health partners are approaching this issue, in part, by "reviewing regulatory authority in public health agencies to require culture isolates or specimen submission if CIDTs are used." Large-scale foodborne outbreaks are a continuing threat to public health, and tracking these outbreaks is an important tool in shortening them and developing strategies to prevent them. It is clear that the use of CIDTs for enteric pathogen detection, including both antigen detection and multiplex nucleic acid amplification techniques, is becoming more widespread. Furthermore, some clinical microbiology laboratories will resist the mandate to require submission of culture isolates, since it will likely not improve patient outcomes but may add significant costs. Specimen submission would be less expensive and time-consuming for clinical laboratories; however, this approach would be burdensome for public health laboratories, since those laboratories would need to perform culture isolation prior to typing. Shari Shea and Kristy Kubota from the Association of Public Health Laboratories, along with state public health laboratory officials from Colorado, Missouri, Tennessee, and Utah, will explain the public health laboratories' perspective on why having access to isolates of enteric pathogens is essential for public health surveillance, detection, and tracking of outbreaks and offer potential workable solutions which will allow them to do this. Marc Couturier of ARUP Laboratories and Melissa Miller of the University of North Carolina will explain the advantages of CIDTs for enteric pathogens and discuss practical solutions for clinical microbiology laboratories to address these public health needs.

Most Common Foodborne Pathogens and Mycotoxins on Fresh Produce: A Review of Recent Outbreaks.

Every year millions of people are affected and thousands of them die due to infections and intoxication as a result of foodborne outbreaks, which also cause billions of dollars' worth of damage, public health problems, and agricultural product loss. A considerable portion of these outbreaks is related to fresh produce and caused by foodborne pathogens on fresh produce and mycotoxins. Escherichia coli O104:H4 outbreak, occurred in Germany in 2011, has attracted a great attention on foodborne outbreaks caused by contaminated fresh produce, and especially the vulnerability and gaps in the early warning and notification networks in the surveillance systems in all around the world. In the frame of this paper, we reviewed the most common foodborne pathogens on fresh produce, traceback investigations of the outbreaks caused by these pathogens, and lastly international early warning and notification systems, including PulseNet International and Rapid Alert System for Food and Feed, aiming to detect foodborne outbreaks.

Implementation of a high-throughput whole genome sequencing approach with the goal of maximizing efficiency and cost effectiveness to improve public health.

This manuscript describes the development of a streamlined, cost-effective laboratory workflow to meet the demands of increased whole genome sequence (WGS) capacity while achieving mandated quality metrics. From 2020 to 2021, the Wadsworth Center Bacteriology Laboratory (WCBL) used a streamlined workflow to sequence 5,743 genomes that contributed sequence data to nine different projects. The combined use of the QIAcube HT, Illumina DNA Prep using quarter volume reactions, and the NextSeq allowed the WCBL to process all samples that required WGS while also achieving a median turn-around time of 7 days (range 4 to 10 days) and meeting minimum sequence quality requirements. Public Health Laboratories should consider implementing these methods to aid in meeting testing requirements within budgetary restrictions. IMPORTANCE: Public Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor).

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road-killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed-field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi-Virulence Locus Sequence Typing (CRISPR-MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella.

High-Level Ciprofloxacin-Resistant Campylobacter jejuni Isolates Circulating in Humans and Animals in Incheon, Republic of Korea.

Campylobacter jejuni is one of the major causative pathogens of outbreaks or sporadic cases of diarrhoeal diseases worldwide. In this study, we compared the phenotypic and genetic characteristics of C. jejuni isolates of human and food-producing animal origins in Korea and examined the genetic relatedness between these two groups of isolates. Regardless of isolation source, all C. jejuni isolates harboured four virulence genes, cadF, cdtB, ciaB and racR, whereas the wlaN and virB11 genes were more frequently observed in human isolates. Antimicrobial susceptibility testing showed that the majority of C. jejuni isolates displayed high-level resistance to fluoroquinolone (95.2%) or tetracycline (76.2%) antibiotics, and 12.4% of isolates exhibited multidrug resistance (more than three classes of antibiotics tested). Pulsed-field gel electrophoresis (PFGE) of all Campylobacter isolates revealed 51 different SmaI-PFGE patterns and six major clusters containing both human and animal isolates. These results indicate that genetically diverse strains of C. jejuni with antimicrobial drug-resistance and virulence properties have prevailed in Incheon. Nevertheless, some particular populations continue to circulate within the community, providing the evidence for an epidemiological link of C. jejuni infections between humans and food-producing animals. Therefore, the continued monitoring and surveillance of C. jejuni isolates of human and food-producing animal origins are required for public health and food safety.