Evaluation of a Multiplex Real-Time PCR Method for Detecting Shiga Toxin–Producing Escherichia coli in Beef and Comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Method3

The ‘‘top-six’’ non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was $1.23 | 10 CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted for additional multiplex assays should regulations expand to include other O serogroups or virulence genes. Escherichia coli O157:H7 and non-O157 Shiga toxin– producing E. coli (STEC) serogroups, including O26, O45, O103, O111, O121, and O145, are major foodborne pathogens that cause hemorrhagic colitis and hemolytic uremic syndrome, which can lead to death. Cattle are a major reservoir for these pathogens, and food of bovine origin has been implicated in many cases of illness and outbreaks caused by E. coli O157:H7 and non-O157 STEC. STEC have been isolated from cattle, beef carcasses, retail beef, cow’s milk, and other foods worldwide (4, 8, 11). E. coli O157:H7 was classified as an adulterant in beef in 1994 after a large outbreak that was associated with undercooked hamburgers served at restaurants. Scallan et al. (15) estimated that non-O157 STEC cause twice the number of foodborne infections in the United States each year than do E. coli O157:H7 strains (112,752 and 63,153, respectively). More than 70% of non-O157 STEC isolates reported to the Centers for Disease Control and Prevention belonged to six E. coli O serogroups: O26, O45, O103, O111, O121, and O145 (6). Disease caused by these STEC serogroups can be as severe as that caused by E. coli O157:H7. Because of the increasing public health impact of these pathogens, in 2011 the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) declared these top six non-O157 STEC serogroups as adulterants in raw beef products (18), and regulatory testing began in June 2012. Non-O157 STEC strains are a heterogeneous group of organisms with no common characteristics that can be utilized for their detection and isolation other than the * Author for correspondence. Tel: 215-233-6525; Fax: 215-233-6581; E-mail: [email protected]. { Present address: U.S. Department of Agriculture, Food Safety and Inspection Service, Eastern Laboratory Outbreaks Section, Athens, GA 30605, USA. { Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 180 Journal of Food Protection, Vol. 77, No. 2, 2014, Pages 180–188 doi:10.4315/0362-028X.JFP-13-248