Determining the Potential for Phage Based Detection of Escherichia coli O157:H7 in Ground Beef Samples During Shipment

Abstract

Foodborne illnesses caused by pathogens total up to 9.4 million cases annually in the United States. An estimated 73,000 cases were attributed to the specific pathogen Escherichia coli O157, leading to over 2,100 hospitalizations and 60 deaths. From 1982 to 2002, outbreaks associated with Shiga toxin producing E. coliO157:H7 were primarily transmitted by ground beef. During this time (1994) the Food Safety and Inspection Service (FSIS) declared E. coli O157:H7 as an adulterant in raw ground beef with a zero tolerance. This stringent policy requires robust detection methods, which use an enrichment step (15-24 h) to minimize false negatives. Currently FSIS collects samples for their monitoring program and ships them overnight to their testing laboratories. This study assessed the potential of using a recently developed phage based luminescence detection assay during sample shipment, which could reduce current assay times. Key parameters including phage concentrations, temperature, and media to sample ratios were evaluated to determine feasibility. Initial experiments were performed in liquid media to determine the relationship between phage concentrations (10²-10⁵ pfu/mL) and time to detection with E. coli O157:H7 concentrations ranging from approximately 2 to 2x10 ⁵ cells. Results showed that the titer of 1.73x 10³ pfu/mL of ΦV10nluc was able to detect 2 cells in 10 hours and was used in subsequent experiments. The detection of E. coli O157:H7 was further evaluated in kinetic studies using 1:3, 1:2, and 1:1 ground beef sample to enrichment media ratios at 37°C. Optimum results were observed with all sample to media ratios resulting in a positive result for approximately 1 cell in 325 grams ground beef in an average of 15 hours. These results suggest this approach is feasible and could allow the rapid detection of a presumptive positive upon arrival, assuming the sample was shipped at 5pm and arrived at 8am the following day (15h). However, the requirement of 37°C for optimal performance of the assay poses an engineering hurdle, as the shipping container would need to maintain temperature. This limitation should be easily addressed, as the current shipping conditions require cargo holds (FedEx B777) to be maintained at Controlled Room Temperature (15-25°C). If successful, this approach could be expanded to other food samples that require enrichment for detection.