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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC AND PROTEOMIC ANALYSIS OF FOODBORNE PATHOGENS

Location: Molecular Characterization of Foodborne Pathogens

Title: Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121 and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment

Authors
item Tillman, Glenn -
item Wasilenko, Jamie
item Simmons, Mustafa -
item Lauze, Todd -
item Miniocozzi, Joseph -
item Oakley, Brian
item Narang, Neelam -
item Fratamico, Pina
item Cray, William -

Submitted to: Journal of Food Protection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 8, 2012
Publication Date: September 1, 2012
Repository URL: http://handle.nal.usda.gov/10113/56448
Citation: Tillman, G., Wasilenko, J.L., Simmons, M., Lauze, T., Miniocozzi, J., Oakley, B., Narang, N., Fratamico, P.M., Cray, W. 2012. Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121 and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment. Journal of Food Protection. 75:1548-1554.

Interpretive Summary: Escherichia coli O157:H7, also known as a Shiga toxin-producing E. coli (STEC), is a major bacterial pathogen that causes many cases and outbreaks of food-borne illness in the U.S. each year. In recent years, it has become evident that other STEC cause similar illnesses linked to contaminated food as illnesses caused by serogroup O157, and at least 70% of the non-O157 STEC cases are caused by the top six serogroups (O26, O45, O103, O111, O121 and O145). Cattle and other ruminants are reservoirs for these pathogens, thus food of bovine origin may be a vehicle for infection with non-O157 STEC. There is a need to develop methods for detection and isolation of these pathogens in animal reservoirs and in food to determine their prevalence and to develop intervention strategies. However, methods used for E. coli O157:H7 must be modified to allow detection and isolation of the non-O157 serogroups since these bacteria have different characteristics compared to O157:H7. To accomplish this, various modifications of two types of agar plating media used for isolation of E. coli O157:H7 were made, and the media were tested using ground beef contaminated with strains belonging to the non-O157 STEC serogroups. Use of one plating agar that was named modified Rainbow Agar O157 (mRBA) in combination with an acid treatment step was found to allow optimal differentiation of the STEC strains from colonies of other bacteria found in ground beef, and the components used to prepare mRBA did not inhibit the growth of the non-O157 STEC strains. Therefore, the combination of acid treatment and mRBA provides a significant improvement over the previous methods for isolation of STEC from raw ground beef.

Technical Abstract: It has been estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by the top six serogroups (O26, O45, O103, O111, O121 and O145). Detection and isolation procedures have been developed for analysis of food products, such as raw ground beef. However, the minimum inhibitory concentrations (MIC) for potassium tellurite, an antimicrobial supplement often used in plating agars, for certain strains of STEC have been reported in the literature to be below the concentration recommended for use in Rainbow Agar O157 (RBA) and Tellurite Cefixime-Sorbitol MacConkey Agar (TC-SMAC). The goal of this study was to identify a plating medium that reduces the strain selection bias due to tellurite concentrations yet provides differentiation of target STEC colonies from confounding organisms. Three plating media (RBA, modified TC-SMAC, and modified RBA) allowed growth of 64.8% (24/37), 91.9% (34/37), and 91.9% (34/37) of strains tested, respectively. However, differentiation of target STEC colonies on modified TC-SMAC was difficult so further experiments were conducted with modified RBA. A post-immunomagnetic acid treatment step at pH 2 was evaluated and found to lethally injure the confounding organisms tested while no deleterious effect was observed for the target STEC strains evaluated. Recovery of target STEC strains from ground beef artificially contaminated with STEC and confounding organisms was then evaluated using a combination of modified RBA and acid treatment versus RBA alone. Using the combined modified RBA and acid treatment, target STEC were isolated from 85.8% (103/120) of the low inoculated samples (1-5 CFU/325 g sample) compared to 56.7% (68/120) of low inoculated samples using no acid treatment and the RBA prepared with the manufacturer’s recommended levels of novobiocin and potassium tellurite. The combination of acid treatment and modified RBA provides a significant improvement over the previous methods for isolation of STEC from raw ground beef.

Last Modified: 7/30/2014
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