Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/13/2014
Publication Date: 10/1/2014
Publication URL: http://handle.nal.usda.gov/10113/60022
Citation: Luedtke, B.E., Bono, J.L., Bosilevac, J.M. 2014. Evaluation of real time PCR assays for the detection and enumeration of enterohemorrhagic Escherichia coli directly from cattle feces. Journal of Microbiological Methods. 105:72-79.
Interpretive Summary: Food safety is a continual concern during the processing of beef from farm to fork. Different types of E. coli can vary from causing severe disease to not harming humans. Cattle may excrete disease-causing pathogenic E. coli bacteria in their feces. During cattle harvesting, feces on hides is a source of contamination of raw beef with pathogenic E. coli and cattle shedding high levels in their feces cause most of the contamination. However, the current assays used to detect and enumerate pathogenic E. coli from cattle feces do not cover all the types of pathogenic E. coli, and the protocols require an extensive amount of time. To address these problems, we designed and validated two molecular assays that can rapidly detect and enumerate all types of pathogenic E. coli directly from cattle feces, even at low levels. The significance of this work is that it provides assays that are rapid and economical to identify cattle that are shedding any type of pathogenic E. coli, especially those shedding at high levels, so that focused antimicrobial interventions can be used to decrease the risk of pathogenic E. coli contamination of beef.
Technical Abstract: Shiga toxin–producing Escherichia coli are a growing concern in the area of food safety, and the United States Department of Agriculture Food Safety and Inspection Service has identified the serotypes O26, O45, O103, O111, O121, O145, and O157 as adulterants in certain types of raw beef. The most relevant to human disease are the enterohemorrhagic E. coli (EHEC) strains that possess intimin (eae), Shiga toxin 1 and/or 2 (stx1-2), and in most cases the conserved pO157 or pO157 like virulence plasmid. Contamination of raw beef with EHEC is likely to occur via the transfer of cattle feces on hides to the carcass. To detect EHEC directly from cattle feces, we evaluated the utility of a multiplex real time PCR assay that targets the EHEC associated gene target ecf1 in combination with eae and stx1-2. Our assay had an increased sensitivity and provided a reliable limit of detection (LOD) of 1.25x103 CFUs/mL in an EHEC spiked fecal background. In addition, we evaluated the use of a duplex qPCR assay using ecf1 for the enumeration of total EHEC directly from cattle feces. The reliable limit of quantification (LOQ) was determined to be 1.25x103 CFUs/mL. Our assay requires minimal sample processing and provides a LOD and LOQ of EHEC directly from cattle feces that are the lowest reported. The application of this assay towards the identification of cattle shedding EHEC at a level above 1.25x103 CFUs/mL could be a first line of defense in identifying cattle shedding these pathogens.