title: Enrichment, Amplification, and Sequence-Based Typing of Salmonella enterica and Other Foodborne Pathogens.
abstract: Detection of Salmonella enterica in foods typically involves microbiological enrichment, molecular-based assay, and subsequent isolation and identification of a pure culture. This is ideally followed by strain typing, which provides information critical to the investigation of outbreaks and the attribution of their sources. Pulsed-field gel electrophoresis is the "gold standard" for S. enterica strain typing, but its limitations have encouraged the search for alternative methods, including whole genome sequencing. Both methods typically require a pure culture, which adds to the cost and turnaround time. A more rapid and cost-effective method with sufficient discriminatory power would benefit food industries, regulatory agencies, and public health laboratories. To address this need, a novel enrichment, amplification, and sequence-based typing (EAST) approach was developed involving (i) overnight enrichment and total DNA preparation, (ii) amplification of polymorphic tandem repeat-containing loci with electrophoretic detection, and (iii) DNA sequencing and bioinformatic analysis to identify related strains. EAST requires 3 days or less and provides a strain resolution that exceeds serotyping and is comparable to pulsed-field gel electrophoresis. Evaluation with spiked ground turkey demonstrated its sensitivity (with a starting inoculum of ≤1 CFU/g) and specificity (with unique or nearly unique alleles relative to databases of >1,000 strains). In tests with unspiked retail chicken parts, 3 of 11 samples yielded S. enterica -specific PCR products. Sequence analysis of three distinct typing targets (SeMT1, SeCRISPR1, and SeCRISPR2) revealed consistent similarities to specific serotype Schwarzengrund, Montevideo, and Typhimurium strains. EAST provides a time-saving and cost-effective approach for detecting and typing foodborne S. enterica , and postenrichment steps can be commercially outsourced to facilitate its implementation. Initial studies with Listeria monocytogenes and Shiga toxigenic Escherichia coli suggest that EAST can be extended to these foodborne pathogens.
authors.affiliation: MicrobiType LLC, 5110 Campus Drive, Plymouth Meeting, Pennsylvania 19462.
authors.forename: Tom
authors.initials: T
authors.lastname: Edlind
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Brewster
authors.affiliation: U.S. Department of Agriculture, Agricultural Research Service, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA.
authors.forename: George C
authors.initials: GC
authors.lastname: Paoli
mesh_terms: D000818:Animals; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D019779:Salmonella enterica / Q000302:isolation & purification*; D012703:Serotyping
keywords: Enrichment; Foodborne outbreak; Foodborne pathogen; Genotype; Listeria; Salmonella