Enrichment, Amplification, and Sequence-Based Typing (EAST) of Foodborne Pathogens

Introduction: Detection of foodborne pathogens typically involves microbiological enrichment with subsequent isolation and identification of a pure culture. This is ideally followed by strain typing, which provides information critical to outbreak and source investigations. Pulsed-field gel electrophoresis (PFGE) has for several decades been the gold standard for strain typing. Nevertheless, its multiple limitations have encouraged development of alternative methods including, most recently, whole genome sequencing (WGS).  Both PFGE and WGS are technically challenging and require a pure culture, which adds to cost and time-to-result. 

Purpose: There is a need for a facile, rapid, and robust method for foodborne pathogen detection and typing. To this end, an enrichment, amplification, and sequence-based typing (EAST) approach was developed for STEC, Salmonella enterica, and Listeria monocytogenes.

Methods: The EAST method involves: (1) overnight enrichment from food samples and total DNA preparation, (2) amplification of polymorphic tandem repeat-containing loci with electrophoretic detection, and (3) DNA sequencing and analysis for strain typing.

Results: EAST required <72 h and provided strain resolution better than serotyping and, for some typing targets, exceeding PFGE.  Evaluation with ground beef and turkey samples spiked with strains of L. monocytogenes, STEC, or S. enterica demonstrated sensitivity (inoculum of ≤1 CFU/g) and specificity (unique or nearly unique alleles relative to >300 NCBI database strains).  When EAST was applied to unspiked retail chicken parts, 3 of 11 samples yielded S. enterica-specific PCR products with sequence identities to strains of serotypes Schwarzengrund, Montevideo, and Typhimurium.

Significance: EAST provides a timesaving and cost-effective approach for detecting and tracking specific strains of foodborne pathogens, and post-enrichment steps can be commercially outsourced to facilitate implementation.