Genetic Diversity in Salmonella enterica in Outbreaks of Foodborne and Zoonotic Origin in the USA in 2006-2017.

Whole genome sequencing is replacing traditional laboratory surveillance methods as the primary tool to track and characterize clusters and outbreaks of the foodborne and zoonotic pathogen Salmonella enterica (S. enterica). In this study, 438 S. enterica isolates representing 35 serovars and 13 broad vehicle categories from one hundred epidemiologically confirmed outbreaks were evaluated for genetic variation to develop epidemiologically relevant interpretation guidelines for Salmonella disease cluster detection. The Illumina sequences were analyzed by core genome multi-locus sequence typing (cgMLST) and screened for antimicrobial resistance (AR) determinants and plasmids. Ninety-three of the one hundred outbreaks exhibited a close allele range (less than 10 allele differences with a subset closer than 5). The remaining seven outbreaks showed increased variation, of which three were considered polyclonal. A total of 16 and 28 outbreaks, respectively, showed variations in the AR and plasmid profiles. The serovars Newport and I 4,[5],12:i:-, as well as the zoonotic and poultry product vehicles, were overrepresented among the outbreaks, showing increased variation. A close allele range in cgMLST profiles can be considered a reliable proxy for epidemiological relatedness for the vast majority of S. enterica outbreak investigations. Variations associated with mobile elements happen relatively frequently during outbreaks and could be reflective of changing selective pressures.

Antimicrobial resistance in multistate outbreaks of nontyphoidal Salmonella infections linked to animal contact-United States, 2015-2018.

Animal contact is an established risk factor for nontyphoidal Salmonella infections and outbreaks. During 2015-2018, the U.S. Centers for Disease Control and Prevention (CDC) and other U.S. public health laboratories began implementing whole-genome sequencing (WGS) of Salmonella isolates. WGS was used to supplement the traditional methods of pulsed-field gel electrophoresis for isolate subtyping, outbreak detection, and antimicrobial susceptibility testing (AST) for the detection of resistance. We characterized the epidemiology and antimicrobial resistance (AMR) of multistate salmonellosis outbreaks linked to animal contact during this time period. An isolate was considered resistant if AST yielded a resistant (or intermediate, for ciprofloxacin) interpretation to any antimicrobial tested by the CDC or if WGS showed a resistance determinant in its genome for one of these agents. We identified 31 outbreaks linked to contact with poultry (n = 23), reptiles (n = 6), dairy calves (n = 1), and guinea pigs (n = 1). Of the 26 outbreaks with resistance data available, we identified antimicrobial resistance in at least one isolate from 20 outbreaks (77%). Of 1,309 isolates with resistance information, 247 (19%) were resistant to ≥1 antimicrobial, and 134 (10%) were multidrug-resistant to antimicrobials from ≥3 antimicrobial classes. The use of resistance data predicted from WGS increased the number of isolates with resistance information available fivefold compared with AST, and 28 of 43 total resistance patterns were identified exclusively by WGS; concordance was high (>99%) for resistance determined by AST and WGS. The use of predicted resistance from WGS enhanced the characterization of the resistance profiles of outbreaks linked to animal contact by providing resistance information for more isolates.

Evaluation of the Illumina iSeq whole genome sequencing system for enteric disease surveillance and outbreak detection.

The Illumina iSeq low-capacity sequencing platform was evaluated for use in foodborne disease surveillance and outbreak detection. The platform produced high quality sequence data comparable to that of the Illumina MiSeq and was cost-effective with fast turn-around time in low sample volume environments.

Sequencing of Enteric Bacteria: Library Preparation Procedure Matters for Accurate Identification and Characterization.

Enzymatic library preparation kits are increasingly used for bacterial whole genome sequencing. While they offer a rapid workflow, the transposases used in the kits are recognized to be somewhat biased. The aim of this study was to optimize and validate a protocol for the Illumina DNA Prep kit (formerly Nextera DNA Flex) for sequencing enteric pathogens and compare its performance against the Nextera XT kit. One hundred forty-three strains of Campylobacter, Escherichia, Listeria, Salmonella, Shigella, and Vibrio were prepared with both methods and sequenced on the Illumina MiSeq using 300 and/or 500 cycle chemistries. Sequences were compared using core genome multilocus sequence typing (cgMLST), 7-gene multilocus sequence typing (MLST), and detection of markers encoding serotype, virulence, and antimicrobial resistance. Sequences for one Escherichia strain were downsampled to determine the minimum coverage required for the analyses. While organism-specific differences were observed, the Prep libraries generated longer average read lengths and less fragmented assemblies compared to the XT libraries. In downstream analysis, the most notable difference between the kits was observed for Escherichia, particularly for the 300 cycle sequences. The O group was not predicted in 32% and 4% of XT sequences when using blast and kmer algorithms, respectively, while the O group was predicted from all Prep sequences regardless of the algorithm. In addition, the ehxA gene was not detected in 6% of XT sequences and 34% were missing one or more of the type III secretion systems and/or plasmid-associated genes, which were detected in the Prep sequences. The coverage downsampling revealed that acceptable assembly quality and allele detection was achieved at 30 × coverage with the Prep libraries, whereas 40-50 × coverage was required for the XT libraries. The better performance of the Prep libraries was attributed to more even coverage, particularly in genome regions low in GC content.

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.

Outbreak of Multidrug-Resistant Salmonella Heidelberg Infections Linked to Dairy Calf Exposure, United States, 2015-2018.

In August 2016, the Wisconsin Department of Health Services notified the U.S. Centers for Disease Control and Prevention of multidrug-resistant (MDR) Salmonella enterica serovar Heidelberg infections in people who reported contact with dairy calves. Federal and state partners investigated this to identify the source and scope of the outbreak and to prevent further illnesses. Cases were defined as human Salmonella Heidelberg infection caused by a strain that had one of seven pulsed-field gel electrophoresis (PFGE) patterns or was related by whole genome sequencing (WGS), with illness onset from January 1, 2015, through July 2, 2018. Patient exposure and calf purchase information was collected and analyzed; calves were traced back from the point of purchase. Isolates obtained from animal and environmental samples collected on-farm were supplied by veterinary diagnostic laboratories and compared with patient isolates using PFGE and WGS. Antimicrobial susceptibility testing by standardized broth microdilution was performed. Sixty-eight patients from 17 states were identified. Forty (63%) of 64 patients noted cattle contact before illness. Thirteen (33%) of 40 patients with exposure to calves reported that calves were sick or had died. Seven individuals purchased calves from a single Wisconsin livestock market. One hundred forty cattle from 14 states were infected with the outbreak strain. WGS indicated that human, cattle, and environmental isolates from the livestock market were genetically closely related. Most isolates (88%) had resistance or reduced susceptibility to antibiotics of ≥5 antibiotic classes. This resistance profile included first-line antibiotic treatments for patients with severe salmonellosis, including ampicillin, ceftriaxone, and ciprofloxacin. In this outbreak, MDR Salmonella Heidelberg likely spread from sick calves to humans, emphasizing the importance of illness surveillance in animal populations to prevent future spillover of this zoonotic disease.

Evaluating whole-genome sequencing quality metrics for enteric pathogen outbreaks.

BACKGROUND: Whole genome sequencing (WGS) has gained increasing importance in responses to enteric bacterial outbreaks. Common analysis procedures for WGS, single nucleotide polymorphisms (SNPs) and genome assembly, are highly dependent upon WGS data quality. METHODS: Raw, unprocessed WGS reads from Escherichia coli, Salmonella enterica, and Shigella sonnei outbreak clusters were characterized for four quality metrics: PHRED score, read length, library insert size, and ambiguous nucleotide composition. PHRED scores were strongly correlated with improved SNPs analysis results in E. coli and S. enterica clusters. RESULTS: Assembly quality showed only moderate correlations with PHRED scores and library insert size, and then only for Salmonella. To improve SNP analyses and assemblies, we compared seven read-healing pipelines to improve these four quality metrics and to see how well they improved SNP analysis and genome assembly. The most effective read healing pipelines for SNPs analysis incorporated quality-based trimming, fixed-width trimming, or both. The Lyve-SET SNPs pipeline showed a more marked improvement than the CFSAN SNP Pipeline, but the latter performed better on raw, unhealed reads. For genome assembly, SPAdes enabled significant improvements in healed E. coli reads only, while Skesa yielded no significant improvements on healed reads. CONCLUSIONS: PHRED scores will continue to be a crucial quality metric albeit not of equal impact across all types of analyses for all enteric bacteria. While trimming-based read healing performed well for SNPs analyses, different read healing approaches are likely needed for genome assembly or other, emerging WGS analysis methodologies.

Comparison of four enzymatic library preparation kits for sequencing Shiga toxin-producing Escherichia coli for surveillance and outbreak detection.

Four enzymatic DNA library preparation kits were compared for sequencing Shiga toxin-producing E. coli. All kits produced high quality sequence data which performed equally well in the downstream analyses for surveillance and outbreak detection. Important differences were noted in the workflow user-friendliness and per sample cost.

Assessment of Genetic Stability During Serial In Vitro Passage and In Vivo Carriage.

In this study, our objective was to evaluate the genetic stability of foodborne bacterial pathogens during serial passage in vitro and persistent in vivo carriage. Six strains of Listeria, Campylobacter, Escherichia, Salmonella, and Vibrio were serially passaged 20 times. Three colonies were picked for whole-genome sequencing (WGS) from passes P0, P5, P10, P15, and P20. In addition, isolates of Salmonella and Escherichia from three patients with persistent infections were sequenced. Genetic stability was evaluated in terms of variations detected in high-quality single-nucleotide polymorphism (hqSNP), core genome multilocus sequence typing (cgMLST), seven-gene MLST, and determinants encoding serotype, antimicrobial resistance (AMR), and virulence. During serial passage, increasing diversity was observed in Listeria, Salmonella, and Vibrio as measured by hqSNPs (from median of 0 SNPs to median of 3-5 SNPs, depending on the organism) and to a lesser extent with cgMLST (from median of 0 alleles to median of 0-5 alleles), while Escherichia and Campylobacter genomes showed minimal variation. The serotype, AMR, and virulence markers remained stable in all organisms. Isolates from persistent infections lasting up to 10 weeks remained genetically stable. However, isolates from a persistent Salmonella enterica ser. Montevideo infection spanning 9 years showed early heterogeneity leading to the emergence of one predominant genotype that continued to evolve over the years, including gains and losses of AMR markers. While the hqSNP and cgMLST variation observed during the serial passage was minimal, culture passages should be limited to as few times as possible before WGS. Our WGS data show that in vivo carriage lasting for a few weeks did not appear to alter the genotype. Longer persistent infections spanning for years, particularly in the presence of selective pressure, may cause changes in the genotype making it challenging to differentiate persistent infections from reinfections.

Gen-FS coordinated proficiency test data for genomic foodborne pathogen surveillance, 2017 and 2018 exercises.

The US PulseNet and GenomeTrakr laboratory networks work together within the Genomics for Food Safety (Gen-FS) consortium to collect and analyze genomic data for foodborne pathogen surveillance (species include Salmonella enterica, Listeria monocytogenes, Escherichia coli (STECs), and Campylobactor). In 2017 these two laboratory networks started harmonizing their respective proficiency test exercises, agreeing on distributing a single strain-set and following the same standard operating procedure (SOP) for genomic data collection, running a jointly coordinated annual proficiency test exercise. In this data release we are publishing the reference genomes and raw data submissions for the 2017 and 2018 proficiency test exercises.

Shigella sonnei Outbreak Investigation During a Municipal Water Crisis-Genesee and Saginaw Counties, Michigan, 2016.

Objectives. To investigate a shigellosis outbreak in Genesee County, Michigan (including the City of Flint), and Saginaw County, Michigan, in 2016 and address community concerns about the role of the Flint water system.Methods. We met frequently with community members to understand concerns and develop the investigation. We surveyed households affected by the outbreak, analyzed Shigella isolate data, examined the geospatial distribution of cases, and reviewed available water quality data.Results. We surveyed 83 households containing 158 cases; median age was 10 years. Index case-patients from 55 of 83 households (66%) reported contact with a person outside their household who wore diapers or who had diarrhea in the week before becoming ill; results were similar regardless of household drinking water source. Genomic diversity was not consistent with a point source. In Flint, no space-time clustering was identified, and average free chlorine residual values remained above recommended levels throughout the outbreak period.Conclusions. The outbreak was most likely caused by person-to-person contact and not by the Flint water system. Consistent community engagement was essential to the design and implementation of the investigation.

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.

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.

Zoonotic Source Attribution of Salmonella enterica Serotype Typhimurium Using Genomic Surveillance Data, United States.

Increasingly, routine surveillance and monitoring of foodborne pathogens using whole-genome sequencing is creating opportunities to study foodborne illness epidemiology beyond routine outbreak investigations and case-control studies. Using a global phylogeny of Salmonella enterica serotype Typhimurium, we found that major livestock sources of the pathogen in the United States can be predicted through whole-genome sequencing data. Relatively steady rates of sequence divergence in livestock lineages enabled the inference of their recent origins. Elevated accumulation of lineage-specific pseudogenes after divergence from generalist populations and possible metabolic acclimation in a representative swine isolate indicates possible emergence of host adaptation. We developed and retrospectively applied a machine learning Random Forest classifier for genomic source prediction of Salmonella Typhimurium that correctly attributed 7 of 8 major zoonotic outbreaks in the United States during 1998-2013. We further identified 50 key genetic features that were sufficient for robust livestock source prediction.

Protracted Outbreak of Salmonella Newport Infections Linked to Ground Beef: Possible Role of Dairy Cows - 21 States, 2016-2017.

In January 2017, CDC identified a cluster of Salmonella enterica serotype Newport infections with isolates sharing an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern, JJPX01.0010 (pattern 10), through PulseNet, the national molecular subtyping network for foodborne disease surveillance. This report summarizes the investigation by CDC, state and local health and agriculture departments, and the U.S. Department of Agriculture's Food Safety and Inspection Service (USDA-FSIS) and discusses the possible role of dairy cows as a reservoir for strains of Salmonella that persistently cause human illness. This investigation combined epidemiologic and whole genome sequencing (WGS) data to link the outbreak to contaminated ground beef; dairy cows were hypothesized to be the ultimate source of Salmonella contamination.

Genome wide characterization of enterotoxigenic Escherichia coli serogroup O6 isolates from multiple outbreaks and sporadic infections from 1975-2016.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea globally, particularly among children under the age of five in developing countries. ETEC O6 is the most common ETEC serogroup, yet the genome wide population structure of isolates of this serogroup is yet to be determined. In this study, we have characterized 40 ETEC O6 isolates collected between 1975-2016 by whole genome sequencing (WGS) and by phenotypic antimicrobial susceptibility testing. To determine the relatedness of isolates, we evaluated two methods-whole genome high-quality single nucleotide polymorphism (whole genome-hqSNP) and core genome SNP analyses using Lyve-SET and Parsnp respectively. All isolates were tested for antimicrobial susceptibility using a panel of 14 antibiotics. ResFinder 2.1 and a custom quinolone resistance determinants workflow were used for resistance determinant detection. VirulenceFinder 1.5 was used for prediction of the virulence genes. Thirty-seven isolates clustered into three major clades (I, II, III) by whole genome-hqSNP and core genome SNP analyses, while three isolates included in the whole genome-hqSNP analysis only did not cluster with clades I-III by both analyses and formed a distantly related outgroup, designated clade IV. Median number of pairwise whole genome-hqSNPs in clonal ETEC O6 outbreaks ranged from 0 to 5. Of the 40 isolates tested for antimicrobial susceptibility, 18 isolates were pansusceptible. Twenty-two isolates were resistant to at least one antibiotic, nine of which were multidrug resistant. Phenotypic antimicrobial resistance (AR) correlated with AR determinants in 22 isolates. Thirty-two isolates harbored both enterotoxin virulence genes while the remaining 8 isolates had only one of the two virulence genes. In summary, whole genome-hqSNP and core genome SNP analyses from this study revealed similar evolutionary relationships and an overall diversity of ETEC O6 isolates independent of time of isolation. Less than 5 pairwise hqSNPs between ETEC O6 isolates is circumstantially indicative of an outbreak cluster. Findings from this study will be a basis for quicker outbreak detection and control by efficient subtyping by WGS.

Shiga Toxin-Producing E. coli Infections Associated with Flour.

BACKGROUND: In 2016, a multijurisdictional team investigated an outbreak of Shiga toxin-producing Escherichia coli (STEC) serogroup O121 and O26 infections linked to contaminated flour from a large domestic producer. METHODS: A case was defined as infection with an outbreak strain in which illness onset was between December 21, 2015, and September 5, 2016. To identify exposures associated with the outbreak, outbreak cases were compared with non-STEC enteric illness cases, matched according to age group, sex, and state of residence. Products suspected to be related to the outbreak were collected for STEC testing, and a common point of contamination was sought. Whole-genome sequencing was performed on isolates from clinical and food samples. RESULTS: A total of 56 cases were identified in 24 states. Univariable exact conditional logistic-regression models of 22 matched sets showed that infection was significantly associated with the use of one brand of flour (odds ratio, 21.04; 95% confidence interval [CI], 4.69 to 94.37) and with tasting unbaked homemade dough or batter (odds ratio, 36.02; 95% CI, 4.63 to 280.17). Laboratory testing isolated the outbreak strains from flour samples, and whole-genome sequencing revealed that the isolates from clinical and food samples were closely related to one another genetically. Trace-back investigation identified a common flour-production facility. CONCLUSIONS: This investigation implicated raw flour as the source of an outbreak of STEC infections. Although it is a low-moisture food, raw flour can be a vehicle for foodborne pathogens.

High-Quality Draft Genome Sequences for Four Drug-Resistant or Outbreak-Associated Shigella sonnei Strains Generated with PacBio Sequencing and Whole-Genome Maps.

Drug-resistant Shigella sonnei poses a clinical and public health challenge. We report here the high-quality draft whole-genome sequences of four outbreak-associated S. sonnei isolates; three were resistant to two or more antibiotics, and one was resistant to streptomycin only.