Genetically marked strains of Shiga toxin-producing O157:H7 and non-O157 Escherichia coli: Tools for detection and modelling

Authors: Paoli, George; Uhlich, Gaylen; Wijey, Chandi

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/9/2015
Publication Date: 4/30/2015
Publication URL: http://handle.nal.usda.gov/10113/60764
Citation: Paoli, G., Uhlich, G.A., Wijey, C. 2015. Genetically marked strains of Shiga toxin-producing O157:H7 and non-O157 Escherichia coli: Tools for detection and modelling. Journal of Food Protection. 78(5):888-901.

Interpretive Summary: Harmful E. coli bacteria are major foodborne pathogens, causing an estimated 250,000 illnesses in the US each year. The United State Department of Agriculture Food Safety Inspection Service regularly tests beef products for 7 different types of pathogenic E. coli and has a zero-tolerance policy for samples that test positive. During pathogen testing, it is typical to include a positive control sample (i.e., a sample inoculated with a strain of pathogenic E. coli) along with the test samples to ensure that the procedure is working properly. When this is done, there is always a slight risk that the pathogenic E. coli used to inoculate the positive control sample might cross-contaminate the test sample, leading to a false-positive result. In this study a collection of pathogenic E. coli strains, one each of the 7 regulated types, were genetically manipulated to contain a unique segment of DNA that allows them to be distinguished from any naturally occurring E. coli. Thus, a positive test sample can be distinguished from the E. coli in the positive control sample. In addition to being used by regulatory agencies and food testing laboratories as positive control strains for detection of pathogenic E. coli, the E. coli strains constructed in this study could also be used in developing new methods of detection and to model the growth of pathogenic E. coli in foods.

Technical Abstract: Shiga toxin-producing E. coli (STEC) are among the most important foodborne pathogens in the United States and worldwide. Nearly half of all STEC-induced diarrheal disease in the United States is caused by STEC O157:H7 while non-O157 STEC account for the remaining illnesses. Thus, the USDA Food Safety and Inspection Service has instituted regulatory testing of certain beef products and has a zero-tolerance policy for regulatory samples that test positive for STEC O157:H7 and six other serogroups of non-O157 STEC (serogroups O26, O45, O103, O111, O121 and O145). In this study a strain of E. coli O157:H7 and strains of each of the six USDA-regulated serogroups of non-O157 STEC were constructed to serve as positive-controls during STEC detection. It is intended that these strains be used to inoculate positive-controls samples; thus it is important that they are distinguishable from STEC isolated from test samples to ensure that these samples were not cross-contaminated by the positive control sample. The positive control strains were constructed by integrating a unique DNA target sequence and a gene for spectinomycin resistance into the chromosomes of the 7 STEC strains by allelic exchange. End-point and real-time PCR assays were developed for the specific detection of the positive control strains and were tested for specificity against 87 strains of E. coli (including 38 STEC O157:H7, at least 6 strains of each non-O157 STEC and 2 commensal E. coli) and 51 strains of other bacteria including 30 species from different 20 genera. The PCR assays demonstrated high specificity for the unique target sequence. The target sequence was detectable by PCR after 10 culture passages (approximately 100 generations), demonstrating the stability of integrated target sequence. In addition, the strains were tested for their potential use in modeling the growth of STEC. Plating the positive control strains in ground beef enrichment cultures on modified Rainbow Agar containing spectinomycin eliminated the growth of the background flora that grew on modified Rainbow Agar without the spectinomycin. Thus, these stains could be used to enumerate and model the growth of STEC in presence of foodborne background flora.