Comparative Analysis of Subtyping Methods against a Whole-Genome-Sequencing Standard for Salmonella enterica Serotype Enteritidis

Abstract

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variablenumber tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods. Salmonella enterica is currently the most common bacterial foodborne pathogen in the United States, causing over 1 million cases of illnesses annually, including approximately 20,000 hospitalizations and 400 deaths (1). Serotyping is commonly used to subtype strains below the species level for epidemiologic purposes. Salmonella enterica serotype Enteritidis was the serotype most commonly linked to foodborne outbreaks between 1998 and 2008 in the United States, with shell eggs being the major vehicle for foodborne transmission (2). In recent years, S. enterica serotype Enteritidis was also found to cause multistate outbreaks associated with other foods such as ground beef (2012), Turkish pine nuts (2011), and alfalfa and spicy sprouts (2011), in addition to shelled eggs (2010) (3).During outbreak investigations, it is critical to employ subtyping methods capable of distinguishing outbreak isolates from epidemiologically distinct but genetically related bacterial strains. Most S. enterica serotype Enteritidis isolates have been shown to be genetically homogeneous, making it difficult for conventional subtyping methods such as pulsed-field gel electrophoresis (PFGE), the current gold standard for strain-level Salmonella subtyping, to discriminate between strains (4, 5). Among the S. enterica serotype Enteritidis isolates reported to PulseNet (6), approximately 45% display a single PFGE pattern using XbaI (JEGX01.0004), rendering PFGE ineffective in some foodborne outbreak investigations. One strategy to improve subtype resolution is to target hypervariable regions (i.e., regions of the bacterial chromosome with less genetic stability) in the ba...

Usefulness of High-Quality Core Genome Single-Nucleotide Variant Analysis for Subtyping the Highly Clonal and the Most Prevalent Salmonella enterica Serovar Heidelberg Clone in the Context of Outbreak Investigations

“…In other words, outbreaks attributed to food products that are not consumed locally (at a single restaurant or social event, for example) will go undetected, thus preventing strong laboratory evidence to link human cases to each other and to potential sources. Recent studies have demonstrated the strength of WGS subtyping of outbreaks using hqSNV analysis for several foodborne outbreaks or outbreaks attributed to other serovars of Salmonella (4,10,12,14,21), including the previously problematic clonal S. Enteritidis (13). This study yielded very strong laboratory evidence of genetic relatedness between epidemiologically related isolates where previous conventional laboratory evidence obtained by PFGE and phage type was weak at best.…”

“…The increasing readiness of WGS technology combined with the widely hypothesized and previously reported high concordance of WGSbased typing approaches with epidemiological data have resulted in the widespread adoption of WGS surveillance and outbreak response by global contemporaries responsible for public health and food safety, and this method is positioned to replace PFGEbased molecular epidemiology of foodborne pathogens (10,(12)(13)(14). This revolution in global disease surveillance has created the urgency for Canadian public health laboratories to apply WGS technology to routine public health activities.…”

Salmonella Thompson outbreak associated with consumption of chicken shawarma and the usefulness of genome sequencing in the investigation

“…The other strains of S. Thompson analyzed had three or more nucleotide variations and the epidemiological information available regarding the cases with these exposures was different. Genome sequencing has proven to be effective in several outbreaks (19)(20)(21)(22)(23). The use of the whole genome sequencing technique provides additional powers of discrimination, beyond serotyping and PFGE, to delimit and investigate an outbreak (20)(21)(22)(23)(24)(25).…”

Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection

“…For example, PFGE, the gold standard subtyping method implemented in all PulseNet laboratories, and MLVA often do not provide the resolution to differentiate between outbreak and sporadic samples in this serovar (2,10,11). In Minnesota, 74% of S. Enteritidis isolates are comprised of three CDC PulseNet PFGE pattern subtypes: JEGX01.0004, JEGX01.0002, and JEGX01.0005.…”

“…Sequence-based analysis of Salmonella organisms from nextgeneration sequencing data has been used to examine outbreak clusters of Salmonella enterica serovar Montevideo (30)(31)(32) and S. Enteritidis (7,11,29). Outbreak-related organisms in these two highly homologous serovars are distinguished by fewer than 20 pairwise single nucleotide polymorphisms (SNPs) compared to the sequence of a reference strain.…”