|EDLIND, TOM - Microbitype Llc|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/15/2016
Publication Date: 8/1/2016
Citation: Edlind, T., Paoli, G., Brewster, J.D. 2016. An enrichment, amplification, and sequence-based typing (EAST) approach for foodborne pathogen detection and surveillance. Meeting Abstract. [abstract]International Association for Food Protection.
Technical Abstract: Introduction: Detection of foodborne pathogens typically involves microbiological enrichment with subsequent isolation and identification of a pure culture. This is typically followed by strain typing, which provides information critical to outbreak and source investigations. In the early 1990’s pulsed-field gel electrophoresis (PFGE) was considered the gold standard for strain typing and, since the establishment of the PulseNet laboratory consortium in 1996, PFGE has been the primary typing method employed to identify and track outbreaks of foodborne illness. Nevertheless, multiple limitations of PFGE 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 culture enrichment 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 Listeria monocytogenes, STEC, or S. enterica demonstrated sensitivity (inoculum of = 1 CFU/g) and specificity (unique or nearly unique alleles relative to databases of > 300 strains). Using EAST analysis with unspiked retail chicken parts, 3 of 11 samples yielded S. enterica-specific PCR products with sequence similarities to strains of serotypes Schwarzengrund, Montevideo, and Typhimurium/Infantis (2 strains in 1 sample). 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.