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From: Food Quality & Safety magazine, April/May 2012

Outbreaks Spur Rapid STEC Tests

by Barbara Robleto

First implicated in a 1993 U.S. outbreak caused by undercooked ground beef, the Shiga toxin-producing E. coli (STEC) known as O157:H7 has become a familiar term associated with foodborne illness.

High-profile outbreaks—last year’s deadly crisis in Germany, attributed to O104 in sprouts, and another in Japan linked to O111 in raw beef—have significantly raised public awareness of lesser-known pathogenic types of E. coli called non-O157 STEC.

The CDC estimates that out of 265,000 STEC infections in the U.S. annually, 64% are caused by non-O157 STEC. Although infections that progress to severe illness are typically caused by E. coli O157:H7, life-threatening conditions such as hemolytic uremic syndrome (HUS) have been associated with non-O157 STEC.

FoodNet, a CDC surveillance system for tracking illness, began collecting data on non-O157 infections in 2000. Ten years later, the annual number of cases had risen from 57 to 451, due in part to more frequent testing, increased capability, and better reporting.

Until recently, foods implicated in U.S. outbreaks of non-O157 STEC—milk, salad, punch, apple cider, lettuce, berries, cheese, margarine—did not include beef. But in 2010, one major food business ended up recalling 8,500 pounds of ground beef after three people became ill with E. coli O26 infections.

New Regulations

The CDC estimates that six serogroups are responsible for 70% to 83% of non-O157 STEC illnesses. In September, the USDA’s FSIS determined that pathogenic strains of those serogroups—O26, O111, O121, O45, O103, and O145—would be considered adulterants in beef.

This means that, starting this year, beef companies need to ensure trim and non-intact beef are free of not only O157:H7 but also the “top six” non-O157 STEC. Any product found to contain these adulterants must either be processed further before selling into the market or destroyed.

Food companies are looking for technological advances and reliable new assays that will make food safety testing faster, more accurate, and more convenient. Recognizing the rising concern, DuPont Qualicon collaborated with the USDA Agricultural Research Service in 2010 to develop a new test.

The FSIS based its decision on criteria similar to the standards used in 2002 to determine O157:H7 as an adulterant: These serogroups can be carried in cattle and find their way into the beef supply, they can survive ordinary cooking, and small doses (1-10 cfu) can cause human illness.

One difference between these serogroups and O157:H7, however, is that while all O157 STEC are considered pathogenic, non-O157 STEC must test positive for certain virulence factors in order to be considered pathogenic. The FSIS determined that the critical virulence factors in non-O157 STEC for food testing purposes are the shiga toxins (stx1 and stx2) and the binding factor (eae or intimin).

Need for New Tests

To meet the challenges of these new regulations, food companies are looking for technological advances and reliable new assays that will make food safety testing faster, more accurate, and more convenient.

Recognizing the rising concern about illnesses linked to non-O157 STEC, and based on the success of previous collaborations, DuPont Qualicon entered into a collaborative research agreement with the USDA Agricultural Research Service in 2010 to develop a new test for non-O157 STEC.

By 2011, the BAX System STEC suite of real-time polymerase chain reaction assays—a screener and two panels—was launched. Designed to align well with the FSIS method for STEC testing, the screener can be run first to quickly clear stx/eae-negative samples. The panels can then be run on potential positives to identify which, if any, of

the top six non-O157 STEC are present. These assays also run under identical program parameters as the E. coli O157:H7 assay, giving food companies flexibility in configuring E. coli testing to fit within their HACCP programs.

During development of the STEC suite, DuPont Qualicon considered inserting an immunomagnetic separation step prior to PCR to concentrate the six serogroups. This option was not included in the final protocol due to several inherent risks, including the potential for introducing operator error and the potential for false negative results due to interference from food debris and/or microbial effects.

A challenge for all non-O157 STEC detection methods is reliable confirmation. Isolation of E. coli O157:H7 is readily achievable because most serotypes have common phenotypic characteristics, such as sorbitol fermentation and telluride resistance. Non-O157 STEC, however, do not have specific characteristics in common that distinguish them from each other or from non-pathogenic E. coli.

The FSIS reference method uses an IMS step after PCR to concentrate cells in presumptive positive samples before streaking onto plates. However, at the time of this writing, commercial beads are not readily available for two of the serogroups—O45 and O121—so additional culture steps must be used for these. The confirmation procedure described in the FSIS Microbiology

Laboratory Guidebook, which was revised in November 2011 to include an acid treatment step, shows indications of better recovery.

What’s Next?

Many factors will influence food safety diagnostic needs in the future, including consumer and media pressure, globalization, and economics, to name just a few.

Increasing regulations will certainly have a major impact on how food companies operate. They will also drive the need for new tests that will help make it faster, easier, and more convenient for food companies to meet these new directives.

The evolution of our diet, with an increased emphasis on convenience foods, fresh foods, and “natural” foods, all of which have relatively short shelf lives, is also sure to place new demands on food safety testing.

Barbara Robleto is communications manager at DuPont Qualicon. She can be contacted at Barbara.d.robleto@dupont.com.

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