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Testing for Non-0157 STECs Leads to Development of New Lab Technology
by Maybelle Cowan-Lincoln
Between the years 2009 and 2012, the food industry—and meat processing in particular—witnessed a wave of activity surrounding certain non-O157 Shiga toxin-producing Escherichia coli. The USDA Food Safety Inspection Service was petitioned in 2009 to declare E. coli strains O26, O45, O103, O111, O121, and O145, collectively known as the “Big Six,” adulterants, banning them from the nation’s food supply. In September 2011, these six strains achieved that status in recognition of the danger they pose to consumer health, given the fact that they produce the same potentially deadly toxins as O157 STEC.
According to the Centers for Disease Control and Prevention, an estimated 175,000 infections are caused by STEC each year. Approximately 36% of these incidences arise from the STEC O157 strain, despite rigorous testing by the meat and produce industries. The remaining 64% of illnesses can be traced to non-O157 strains. Although non-O157 STECs often cause milder cases of illness with less severe diarrhea, fewer hospitalizations, and an estimated single death per year according to CDC statistics, the number of infections significantly overshadows the more familiar O157 serogroup.
Awareness of the prevalence of non-O157 STECs has grown over several years. In April 2006, an outbreak of foodborne illnesses occurred in New York State, and a non-O157 STEC was suspected to be the culprit. This incident spurred the USDA into making inquiries into what kinds of surveillance states were doing and how they were handling the presence of these toxins. The agency discovered that in many cases, non-O157 strains were appearing in higher numbers than the O157 strain. In fact, in 2010, FoodNet’s foodborne illness burden data showed that the prevalence of non-O157 STECs exceeded that of O157.
On June 4, 2012, the FSIS implemented a sampling and testing program for beef trimmings only. To help the food industry and the public health arena comply with the new regulations and ensure that beef trimmings are free of these pathogens, new technologies in user-friendly kits are now commercially available to isolate these toxins in food samples.
One technology is the RapidFinder STEC Detection System developed by LifeTechnologies of Grand Island, N.Y., in cooperation with the USDA. Eric Liu, a product manager with Life Technologies’ food safety business, explained that the company has an ongoing relationship with the USDA that predates its STEC product development.
“That relationship gave us access to key opinion leaders– the scientists involved with designing the regulations–and it was able to give us insight into where the technology is likely to lead. Also, the development of the actual assay requires real world samples of the Big Six STEC, and our interaction with the USDA provided access to a diverse panel of real-world strains to use for testing and development purposes,” he said.
The system has been awarded a “Letter of No Objection” from the USDA, indicating that the company can pursue commercial customers for this product.
The RapidFinder workflow is a real-time polymerase chain reaction system composed of three parts: the STEC Sample Preparation Kit, which uses triptic soy broth to enrich the regulation-specified 375-gram sample of ground beef or beef trimming in as little as eight hours. A small sample of the enriched broth is then transferred to a magnetic particle processing instrument, where chemical and enzymatic lysis (bacterial cell destruction caused by a specific agent) releases DNA from the E. coli. Inside the instrument, the DNA binds to magnetic beads, which are then exposed to a magnet, separating the E. coli DNA out of the sample.
The second part is the screening assay, which tests the prepared sample for STX/EAE, the telltale genes that indicate that the target E. coli bacteria are present. If the sample yields negative results for these virulence factors, the meat is considered clean. If it tests positive for either gene, the next step is to confirm their presence and identify which strains have contaminated the meat. A confirmation assay is run only when the screening assay has detected STX and/or EAE. This second assay confirms the presence of target E. coli DNA and determines if it is O157 or one or more of the Big Six strains.
Finally, the data analysis package provides automated data interpretation so that the user can easily decide how to proceed with a sample. An instrument display shows the presence or absence of a pathogenic STEC and lets the user know when a sample needs to be rerun due to unclear results.
One of the most significant benefits of the workflow is time to results. The system can clear a negative sample in a total of 10 hours—eight hours to enrich the sample plus the two hours it takes to perform the assay. The second assay, to confirm the presence of O157 or a non-O157 STEC, takes only an additional hour.
“Saving time is everything for the end user (producer or service lab). They have beef products, a perishable product, sitting in temperature-controlled storage locations, and the clock is ticking on getting a safe product into their distribution channel,” Liu said. “They also have to make sure they get an accurate answer. We offer a faster detection workflow, but we also have an optimized assay which offers high sensitivity and high accuracy.”
In addition, the hands-free, automated sample prep facilitates a high throughput. Establishments that test a large volume of samples—upwards of 60 at a time—want the speed and efficiency of starting a sample and, after performing a minimal number of steps, moving on to other tasks.
The system is used primarily by private establishments in the food industry, including slaughterhouses and food processing plants, as well as third-party labs that offer testing services to companies lacking this capability.
A popular Big Six testing system for federal and state government testing labs, RapidCheck and RapidChek CONFIRM, supplied by Romer Labs of Newark, Del., includes two test strips that identify all of the Big Six serogroups. Each strip tests for three of the targeted strains, with one strip sensitive to O103, O45, and O26 and the other to O145, O121, and O111. A sample of beef is enriched in media for eight to 18 hours, depending on sample size. After enrichment, 1 mL of the broth is transferred to a test tube, and both strips are inserted. The results can be read in 10 minutes.
The test strip technology offers labs several advantages. The short time to results after enrichment allows for rapid sample clearance and, therefore, rapid product release. The strips are cost effective and easy to use, requiring no special instrumentation or training. The system can be used with the RapidChek proprietary enrichment media or the widely available TSB, modified with the addition of novobiocin, an antibiotic.
Should the test strip screening indicate that a sample is positive, the next step is to confirm the presence of the pathogen with RapidChek CONFIRM. Tim Lawruk, Romer Labs’ senior director of sales and marketing, explained, “Once you have a potential positive from the screening test, you may perform cultural confirmation. RapidChek CONFIRM does that using an immunomagnetic separation kit. This allows you to isolate just the pathogen of interest during cultural confirmation, and not any other organism that may have grown in the enrichment media.”
To definitively identify a pathogen of interest, the technology concentrates target pathogens and washes away competing organisms. It uses antibodies specific to each of the targeted strains. These antibodies are attached to separate, coated magnetic particles. The reagent is added to 1 mL of the potentially positive enrichment and incubated for 15 minutes at room temperature. The sample tubes are then placed in the kit’s magnetic rack for five minutes, allowing the magnetic particles to bind the non-O157 strains using the antibodies. The remaining solution is discarded, and the bound material is washed, reconstituted, and plated on selective agars. The process requires no additional equipment or specialized training.
Lawruk said that the CONFIRM product is listed by the FSIS in its microbiology laboratory guidebook as the process to use to confirm a Big Six pathogen in potentially positive beef samples.
A recent study conducted by the University of Maryland and published in the December 2012 issue of the Journal of Food Microbiology reported finding significant STEC contamination in pork as well as beef. Therefore, the FSIS is expected to expand the testing program in 2013 to include other cuts of beef as well as other meats. Once this becomes a reality, demand for new-technology test kits is expected to rise substantially.
Maybelle Cowan-Lincoln is a science/technical writer based in New Jersey. She is a frequent Wiley-Blackwell contributor who has been featured in numerous publications.