Development of a Modified USDA FSIS MLG 5B.03 Screen-based Workflow to Detect and Isolate Shiga Toxin-producing Escherichia coli (STEC) Big Six Serogroups and E. coli O157:H7 in Ready-to-Eat Food and Produce

Introduction: Shiga Toxin-producing Escherichia coli (STEC) is an enteric pathogen that causes hemorrhagic diarrhea, which can progress to hemolytic-uremic syndrome (HUS). Foodborne outbreaks causing HUS were initially associated with E. coli O157:H7 (EHEC), but a growing number of incidences were linked to the STEC “Big Six” serogroups (O26, O45, O103, O111, O121, and O145). After observing outbreak and policy trends, BFL-FDACS initiated a study to expand testing into screening and confirmation of STEC Big Six serogroups in Ready-to-Eat (RTE) food and produce.

Purpose: The purpose of this study was to construct a workflow for screening RTE food and produce by optimizing enrichment, DNA extraction, detection, and isolation parameters and apply those to appropriate matrix extensions. This study built upon the USDA MLG 5B.03 method, which only validated meat products.

Methods: The STEC Big Six E. coli serogroups and O157:H7 were spiked into a variety of RTE food matrices and enriched. Extracted DNA was screened for stx1, stx2, and wzy genes, and a secondary screen for Big Six serogroups, eae, and stx was performed using USDA FSIS MLG 5B.03. Screening results steered the cultural isolation and confirmation of the appropriate E. coli.  

Results: The enrichment (mBPW) and extraction (MagNA Pure Compact) were optimized in the initial matrix with 100% detection and confirmation (n = 6). Matrix extensions on RTE sprouts, hot dogs, and cilantro demonstrated the method can detect and confirm STEC in 100% of spiked samples (n = 12 for each commodity).  ChromSTEC and EMB agars most effectively isolated STEC.  

Significance: BFL-FDACS has successfully developed a two-step STEC screening and isolation method for RTE foods and produce that can be expanded to test E. coli isolated from other laboratory food samples. Improved detection and classification of STEC found in food would result in a more effective surveillance program that will enhance public safety.