Pinpointing a Pathogen

Using the latest screening procedures, even the elusive E. coli O157:H7 strain can be detected in six to eight hours.

Imagine getting sick from eating a hamburger from your local fast food restaurant, or from consuming a supposedly healthy salad. Most consumers think that the food they buy from grocery stores or restaurants will be safe, but for the past 25 years this has not always been the case.

E. coli O157:H7 was first recognized as a foodborne pathogen in 1982 during an investigation into an outbreak of hemorrhagic colitis (bloody diarrhea) associated with consumption of contaminated hamburgers. From 1982 until 2002, the Centers for Disease Control and Prevention reported 350 outbreaks, representing 8,598 cases, 1,493 (17%) hospitalizations, 354 (4%) hemolytic uremic syndrome (HUS) cases, and 40 (0.5%) deaths. Because a limited number of sick people actually go to the doctor, and only 10% of infections occur in an outbreak, the rest of the cases occur sporadically. CDC now estimates that there are approximately 73,000 E. coli O157:H7-related illnesses in the United States each year.

Considering that there are close to two million cases of human enteric diseases each year caused by both Salmonella and Campylobacter, why should we be so concerned about E. coli O157:H7? The primary reason is the severity of the disease it causes, especially in young children, the elderly, and immuno-compromised individuals. The spectrum of illnesses it causes includes bloody diarrhea in 30 to 90% of cases, kidney disease (HUS in children) in about 6% of cases, and death in about 1% of cases. In the United States, HUS is the principal cause of acute kidney failure in children, and most of these cases are caused by E. coli O157:H7.With the severity of the disease caused by these infections, and the potentially dramatic effect on children, the U.S. Department of Agriculture issued regulations in 1998 making E. coli O157:H7 a legal adulterant.

Testing for E. coli O157:H7 is more difficult than testing for other pathogens because there are many non-pathogenic strains of E. coli that are part of the normal flora of the intestinal tract of humans and most other animals. Fortunately, the vast majority of the hundreds of different types of E. coli strains do not cause gastrointestinal illnesses, and low levels of these common, non-pathogenic strains are frequently found in most uncooked food products.

Historically, traditional cultural microbiological procedures have been used for the isolation and identification of E. coli O157:H7. Isolating it from foods is like trying to find a needle in a very large haystack. First, the foods to be tested are placed into a growth medium that allows the E. coli to grow for 18 to 24 hours while inhibiting the growth of many competitors. Then, the growth medium is placed on a selective/differential plate that has been developed to take advantage of the inability of E. coli O157:H7 to ferment sorbitol and to express a specific enzyme that will fluoresce blue in the presence of indicators. After 24 hours of growth the O157:H7s can then be differentiated from common E. coli strains. A number of sorbitol- containing media, including most commonly, sorbitol MacConkey Agar (SMAC) and more recently specialty chromogenic media, have been used to isolate E. coli O157:H7 from food and clinical samples. Because SMAC and chromogenic media are not very selective, high levels of normal flora in foods may mask the presence of O157:H7, thus underestimating the number of positive food samples. These traditional procedures take about 48 hours before a tentative identification of a positive food sample is determined.

Speeding Up Detection
Innovative technologies are being developed and used for the microbiological analysis of foods. Unlike conventional methods that rely on specific media to select and grow pathogens, newly developed “rapid methods” use alternative procedures such as immunoassays or molecular biology techniques to detect pathogens in foods. Compared to conventional procedures, alternative methods are generally easier and more rapid to perform. Rapid methods that are available today still require that food samples undergo culture enrichment prior to analysis to grow the pathogen of interest to detectable levels, and these methods usually shorten the analysis time by one to two days compared to conventional methods.

One of the most widely used of the rapid methods is an automated immunoassay. This procedure was developed to detect the antigens of specific bacterial pathogens using an enzyme-linked fluorescent assay (ELFA). The procedure utilizes a solid phase receptacle (SPR) that serves as the solid phase of the immunoassay as well as the pipetting device, thus allowing the automation of the procedure. Reagents for the procedure are ready-to-use and pre-dispensed in the sealed reagent strips. After the food being tested is incubated overnight in a medium designed to enhance the number of the pathogenic bacteria present, the test strip is inoculated with a small portion of the medium. The testing instrument then completes all of the steps of the immunoassay automatically.

The method was developed using highly specific antibodies to different bacterial pathogens including Salmonella, Listeria monocytogenes, and Campylobacter. The sensitivity of the system using the antibodies to the specific pathogens usually requires from 100,000 to 1 million cells of the pathogen per milliliter of enrichment medium before a fluorescent signal from a positive sample can be detected.

In 2008, a new assay for the detection of E. coli O157:H7 was developed for the automated ELFA platform. Instead of specific antibodies that recognize E. coli O157 strains, highly specific proteins from the tail fibers of bacteriophage are being used to recognize, capture, and bind the E. coli O157:H7 bacteria to the SPR. The advantage of the phage protein receptors over antibodies is that the phage proteins are much more specific with very little cross-reactivity and they form a tighter bond with the bacteria with a net effect of increased sensitivity and specificity of the assay.

Survivor Viruses
Why bacteriophage proteins? Bacteriophage are viruses that are thought to be the most abundant “life” form on earth. They live and reproduce exclusively by infecting specific bacteria. Archeological and evolutional studies have demonstrated that bacteriophage have been on earth for millions of years. These unique organisms have only one mechanism for survival. They must recognize, attach, and inject their genetic material into specific bacteria in order to replicate and live.

For the past 10 years, a German biotech company, Profos, has been studying and characterizing bacteriophage. It has identified the specific tail fiber proteins of bacteriophage specific to E. coli O157 that allow these viruses to attach to the bacteria. The company then isolated these specific proteins and cloned large amounts of them. France-based diagnostic company bio- Mérieux has licensed the use of these phage-based proteins and has developed a new assay on the automated ELFA platform. The new assay replaces the antibodies used to capture the E. coli O157:H7 with the specific cloned phage protein. The outcome has been the development of an assay that can detect E. coli O157:H7 within six to eight hours from all meat and produce samples tested to date. Official validation studies are being conducted and were expected to be completed by the end of 2008.

This development of a more sensitive and specific test for E. coli O157:H7 will provide food producers with a new tool to assist them in the assurance that the foods they produce and market will be free of this extremely dangerous pathogen. Significant savings will be gained as a result of a rapid screening procedure that is completed in six to eight hours. This technology will enable processors to confidently release products to the supply chain more rapidly, decreasing their inventory hold times. Similar phage protein assays for the detection of Salmonella and Listeria are currently being developed.

This article originally appeared in the February 2009 issue of Occupational Health & Safety.

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