Culture of Shigatoxigenic E. coli O157:H7 & enumeration fecal indicators SOP

Authors: Condon, Justine; Durso, Lisa

Submitted to: Protocols.io
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2025
Publication Date: 3/20/2025
Citation: Condon, J.C., Durso, L.M. 2025. Culture of Shigatoxigenic E. coli O157:H7 & enumeration fecal indicators SOP. Protocols.io. https://doi.org/10.17504/protocols.io.81wgbrewqlpk/v1.
DOI: https://doi.org/10.17504/protocols.io.81wgbrewqlpk/v1

Interpretive Summary: Shiga toxigenic Escherichia coli O157:H7 (STEC O157) is a dangerous human pathogen that can cause severe food poisoning, kidney failure, and even death.  It is common in the gastrointestinal tract of ruminant animals like cows and goats.  The methods that are used to find it in people do not work for finding and tracking it in the environment.  This protocol provides step-by-step instructions that allow scientists to isolate STEC O157 from the environment, even when it is present in very low numbers, and even when it is hiding among many other bacteria.  This protocol also provides instructions for finding and counting other bacteria associated with fecal contamination. 

Technical Abstract: Purpose To describe the correct procedures on how to set-up culture-based assays for the detection of Shigatoxigenic Escherichia coli O157:H7 (STEC O157) from a variety of animal and environmental sources; and the procedures for enumeration of generic E. coli and total coliforms from those same sources. Scope This protocol describes the procedures for two commonly used culture-based laboratory assays. All culture work must be done on fresh samples that are kept cool and processed within 24 hr of collection (48 hr if sample is collected remotely and shipped). The first procedure applies to immunomagnetic separation (IMS) of different serotypes of STEC. IMS is a technique that will isolate cells from an enriched sample containing background substance matter. IMS is used widely for many organisms; this SOP is specifically for STEC isolation. Dynabeads are used to separate the target cells from the rest of the sample. Target specific antibodies attach to the surface of the magnetic Dynabead sphere, allowing the STEC cells to remain while the rest of the sample is discarded. The isolated cells are then plated onto selective media for incubation to determine the amount of E. coli cells in each sample. These grown colonies can then be saved for freezer isolation. Escherichia coli (E. coli) are a large and diverse group of bacteria. Although most strains of E. coli are harmless, others can make you sick. Some kinds of E. coli can cause diarrhea, urinary tract infections, respiratory illness and pneumonia. Some E. coli cause disease by making a toxin called Shiga toxin. The bacteria that make these toxins are called “Shiga toxin-producing” E. coli, or STEC for short. Still other kinds of E. coli are used as markers for water contamination—so you might hear about E. coli being found in drinking water, which are not themselves harmful, but indicate the water is contaminated. The second procedure applies to the IDEXX Quanti-Tray test. The IDEXX Quanti-Tray Colilert and Enterolert kits count and detect E. coli, total coliforms, and Enterococcus in a 100 mL sample. The Colilert test shows positive total coliforms as a yellow color, and positive E. coli as yellow and fluorescent. The Colilert test uses a proprietary Defined Substrate Technology to detect and quantify both total coliforms and E. coli. This technology uses two nutrient indicators – ONPG and MUG – as the major sources of carbon in the Colilert packets. These nutrients are metabolized by the enzyme ß-galactosidase (coliforms) and ß-glucuronidase (E. coli). Coliforms grown in Colilert metabolize ONPG using ß-galactosidase, which changes it from colorless to yellow. The same is true for MUG. ß-glucuronidase metabolizes MUG and creates a fluorescence. The MPN of E. coli can be found from the table based on the number of large and small wells that are yellow and fluoresce. The MPN for total coliforms can be found based on the number of small and large wells that are yellow with no fluorescence. Enterolert testing involves the same procedure and a different proprietary Defined Substrate Technology. ß-D-glucoside present in Enterococci binds with 4-methyl-umbelliferone in Enterolert to show fluorescence in a positive sample. As with Colilert, the MPN can be found using the table and the number of large and small fluorescent wells.