Enrichment, amplification, and sequence-based typing of Salmonella enterica and other foodborne pathogens

Authors: EDLIND, TOM - Microbitype Llc; Brewster, Jeffrey; Paoli, George

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/27/2016
Publication Date: 1/1/2017
Citation: Edlind, T., Brewster, J.D., Paoli, G. 2017. Enrichment, amplification, and sequence-based typing of Salmonella enterica and other foodborne pathogens. Journal of Food Protection. 80(1):15-24.

Interpretive Summary: Food processors, regulatory agencies, and public health officials require sensitive, rapid and accurate methods to detect foodborne pathogens to monitor the safety of foods, prevent the distribution of contaminated foods, institute recalls of contaminated food products, and to identify and track outbreaks of foodborne illness. Current methods for detection and characterization of foodborne pathogens typically take several days to weeks. In this study we report the development of an enrichment, amplification, and sequence-based typing (EAST) method, which can be completed in as little as 48 hours. After overnight culture enrichment, the PCR-detection, targeted DNA sequencing, and comparative DNA analysis can be completed in less than 24 hours. Unique genetic targets for EAST-based detection (PCR) and typing (DNA sequencing) were identified and validated for three important foodborne pathogens, Salmonella enterica, Listeria monocytogenes, and pathogenic Escherichia coli. The EAST method was successful in detecting and typing low levels of pathogens in artificially contaminated foods. The EAST method will be useful for food regulatory and public health agencies to detect and track foodborne pathogens and for the food industry to determine the presence of pathogens and to determine if these bacterial contaminants are persistent or occur sporadically.

Technical Abstract: Detection and characterization 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) is the current gold standard for strain typing, but its multiple limitations have encouraged development of alternative methods including whole genome sequencing (WGS). Both PFGE and WGS require a pure culture, which adds to cost and turnaround time. To address this, an enrichment, amplification, and sequence-based typing (EAST) approach was developed involving: (1) overnight enrichment and total DNA preparation, (2) amplification of polymorphic tandem repeat-containing loci with electrophoretic detection, and (3) dideoxynucleotide sequencing and bioinformatic analysis to identify related strains. EAST required 48 to 72 h to complete and provided strain resolution exceeding serotyping and, for some typing targets, comparable to or better than PFGE. Evaluation with ground beef and turkey samples, unspiked or spiked with strains of Listeria monocytogenes, Shiga toxigenic Escherichia coli (STEC), or Salmonella enterica demonstrated sensitivity (product from starting inoculum of =1 CFU/g) and specificity (unique or nearly unique alleles relative to databases of >300 strains). In tests with unspiked retail chicken parts, 3 of 11 samples yielded S. enterica-specific PCR products, and sequence analysis of three distinct typing targets (SeMT1, SeCRISPR1, and SeCRISPR2) showed consistent similarities to specific serotype Schwarzengrund, Montevideo, and Typhimurium/Infantis (2 strains in 1 sample) database strains. 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.