Detection and isolation of the "Top 7" Shiga toxin-producing Escherichia coli in ground beef: comparison of the Rapidfinder kits to the USDA microbiology laboratory guidebook method

Authors: Fratamico, Pina; Bagi, Lori; Abdulwakeel, Aisha

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/26/2016
Publication Date: 5/1/2017
Publication URL: http://handle.nal.usda.gov/10113/5682496
Citation: Fratamico, P.M., Bagi, L.K., Abdul Wakeel, A.Y. 2017. Detection and isolation of the "Top 7" Shiga toxin-producing Escherichia coli in ground beef: comparison of the Rapidfinder kits to the USDA microbiology laboratory guidebook method. Journal of Food Protection. 80:829-836.

Interpretive Summary: Food-borne pathogenic bacteria known as Shiga toxin-producing Escherichia coli (STEC) and belonging to types O157:H7, O26, O45, O103, O111, O121, and O145 (often referred to as the top 7 STEC) are responsible for the majority of STEC infections. Cattle and other ruminants are reservoirs for these pathogens, thus food of bovine origin may be a vehicle for infection with STEC. Thus, these bacteria have been classified as adulterants in meat by the USDA Food Safety and Inspection Service (FSIS). Rapid and sensitive methods for detection of these pathogens in animal reservoirs and in food are needed to determine their prevalence and to ensure that food contaminated with STEC does not reach the consumer. The purpose of this study was to evaluate a new commercially available real-time polymerase chain reaction- (amplification of specific segments of DNA of the pathogens) based kit to determine the specificity for the target STEC and to compare the performance of this kit to the methods currently used by the FSIS for detection of these pathogens. The pathogens were detected by both methods in ground beef that was artificially contaminated with very low numbers of the STEC strains, and it was possible to recover the target bacteria from the samples after growth in enrichment broth. However, similar to some other reported work, it was more problematic to recover O111 strains since this STEC type does not grow as well as the others on commonly used isolation agars. In summary, the commercial kit was rapid and simple and performed as well as the current method used for regulatory testing. Thus, it can be used for routine and rapid detection of the “top 7” STEC in beef, and potentially also in other types of samples.

Technical Abstract: Shiga toxin-producing E. coli (STEC) O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are often referred to as the “top 7” STEC, and these have been declared as adulterants in beef by the USDA Food Safety and Inspection Service (FSIS). The aim of this work was to compare the methods described in the USDA FSIS Microbiology Laboratory Guidebook (MLG) to a two-stage Applied Biosystems™ RapidFinder™ STEC Screening and Confirmation Real-Time PCR assay to test for the top seven STEC in raw ground beef. The specificity of the RapidFinder workflow that targets non-O157 STEC O-antigen genes, stx1, stx2, and eae, and E. coli O157-specific targets were determined against 132 top 7 STEC strains and 284 exclusion strains. All inclusion strains were positive, and all exclusion strains gave negative results with the RapidFinder assay. Strains carrying all of the known variants of stx1 and stx2, including stx2f and stx2g were also detected. For RapidFinder™ analysis, 375-g ground beef samples spiked with = 4 CFU of representative STEC strains were enriched in 1 L of tryptic soy broth (TSB) for 10 h, and for the MLG method, spiked 325-g samples were enriched in 975 ml of modified TSB for 15 h. Following DNA extraction, real-time PCR was performed using RapidFinder™ Express software, and for the MLG method, the BAX® Real-Time PCR STEC Suite and the BAX® Real-Time E. coli O157:H7 assay were used with the BAX® System Q7 software. Following immunomagnetic separation, presumptive colonies from modified Rainbow® Agar O157 plates were confirmed by the real-time PCR assays. Results of the RapidFinder™ and BAX® assays were similar; all samples were positive after 10 h and 15 h of enrichment, respectively. Isolation and confirmation of isolates was possible on all samples, except that two O111:NM strains could not be isolated from a portion of the inoculated samples. Thus, the RapidFinder™ system can be used for routine and rapid detection of the “top 7” STEC in beef.