Mick Bosilevac, Ph.D., a research microbiologist with the USDA Agricultural Research Service, U.S. Meat Animal Research Center, Meat Safety and Quality Research Unit, has a good piece about testing for STECs, or shiga-toxin producing E. coli in the current Food Safety Magazine.

Those are the E. coli that make people really sick.

A few edited highlights are below, but the full piece is available at http://www.foodsafetymagazine.com/magazine-archive1/aprilmay-2013/when-stec-are-your-target-where-do-you-aim/.

Shiga toxin-producing Escherichia coli (STEC) can cause illnesses that range from diarrhea to hemorrhagic colitis and hemolytic-uremic syndrome (HUS). More than 70 serotypes of STEC have been described, and fortunately, only a handful have been associated with these severe diseases. The STEC that can cause these severe diseases are referred to as Enterohemorrhagic E. coli (EHEC). E. coli O157:H7 is the EHEC most often associated with the severest forms of disease as well as outbreaks from contact with or consumption of contaminated foods, animals and water. It should be noted that the food has historically been ground beef; the animals, cattle; and the water presumably contaminated by runoff from a cattle production facility.

Numerous non-O157 EHEC have been linked to illnesses and outbreaks of disease similar to that of E. coli O157:H7, with only one occurrence linked to beef. The U.S. Centers for Disease Control and Prevention has identified the most common non-O157 EHEC and reported that just six serotypes (O26, O45, O103, O111, O121 and O145) are responsible for 71 percent of total infections from all sources. As part of its strategy to focus more on prevention, the U.S. Department of Agriculture Food Safety and Inspection Service (USDA FSIS) in the Federal Register implemented its decision to consider STEC of the six most frequent serogroup adulterants in certain beef products and began testing beef trimmings for these pathogens on June 4, 2012. In response, commercial test kit manufacturers and testing laboratories have rapidly introduced test kits and services that can detect and/or confirm the presence of the “Top Six” EHEC.

Recently, our group completed and submitted a research paper describing the strengths and weaknesses of five commercially available EHEC detection methods with comparison to the FSIS MLG and our own in-house EHEC detection and confirmation assays. In that forthcoming paper, we describe that each method, when used according to the manufacturer’s directions, was able to identify 1–3 CFU EHEC in samples ranging in size from 25 g and 65 g to 375 g. However, since many methods have their own proprietary enrichment media and enrichment conditions, it is difficult if not impossible to directly compare the same sample in a side-by-side fashion. Thus, in our study, we followed up the inoculation experiments with 500 enrichments acquired from a regional service lab and tested each by all the methods. The results of this side-by-side comparison were unexpected: 170 sample enrichments were positive by one or more of the methods used, whereas only 2 samples were identified as positive by all seven methods. Culture results from these two samples identified one containing an EHEC O26, whereas the other contained a STEC, an EPEC (Enteropathogenic E. coli that contain eae and lack stx) and an O26 E. coli. Thus, all methods identified the one sample that contained a Top Six EHEC, and all methods identified a sample containing E. coli that produced a false, potential-positive result.

Delving deeper into the range of discrepancies between methods, we found that a large portion of the difference was due to the sample preparation before PCR. Some used 20 µL of enrichment, some, 50 µL and others, 1 mL for the preparation of DNA. A greater amount of sample going into a preparation did not necessarily agree with more positive tests or greater sensitivity. In many cases, we were able to take discrepant DNA preparations from the same sample and run them through the other assays. When this was done, negative DNA preparations stayed negative and positive preparations stayed positive. No one DNA preparation could be determined to perform better, because each test method had its own unique potential-positive samples.  

Using the most extensive culture isolation protocols to confirm potential positive samples still fails to identify an EHEC much of the time. In fact, even confirming the presence of a STEC has been accomplished only at an approximate rate of 30 percent. In a 2011 study of commercial ground beef, we cultured all stx-positive enrichments. Every stx-positive enrichment was repeatedly plated to washed sheep’s blood agar and up to 50 colonies were picked and individually screened to determine if they each were a STEC. The rate of successfully isolating a STEC ranged from 10 to 50 percent, depending on the sources and batches of ground beef examined. In a more recent study, all samples identified as reactive by any of five EHEC detection methods under comparison were sent to our group for culture resolution of the results. There were 550 samples in this study: 36 were identified as reactive and 21 as potential positive by one or more of the methods. Culture isolation was able to recover two different Top Six EHEC: one from a sample identified as potential positive by all methods examined, and one from a sample that was identified as reactive by only one of the methods examined. In the second case, this was an EHEC O111 that went through all the methods undetected. Even the one method that called the sample containing this EHEC reactive failed to call the sample potential positive. This isolate must have been at a very low concentration for the PCR methods to have not adequately detected it. The culture isolation process took nearly 6 weeks to complete on these 36 enrichments, and even then with 2 samples found to contain an EHEC, 14 containing a STEC, 12 an EPEC and 5 others a generic E. coli of Top Six serotype, there were still 16 samples from which no isolates could be recovered to explain any of the PCR-detected EHEC targets. This raises the question of how useful the results of current EHEC detection methods may be without adequate culture and isolation techniques by which to assess them.