A Q&A on STEC Testing

The presence of Shiga toxin producing E. coli (STEC) in food can cause severe illness that could lead to a critical condition called hemolytic-uremic syndrome (HUS). The USDA FSIS reinforces a test-hold-release practice for meat products to ensure contaminated products are not sold to consumers, but how can we deliver accurate and consistent STEC testing to prevent infections?

QA sat down with Season (Yicheng) Xie, Ph.D., global product manager of ddPCR food applications at Bio‑Rad, to discuss recent trends in STEC testing, the role of digital PCR in the meat industry and opportunities and challenges for the future of STEC testing technologies.

QA: What are the dangers associated with E. coli contamination in the meat industry, and how can accurate testing help to minimize risk?

Season (Yicheng) Xie: Most E. coli are harmless, but certain groups of pathogenic E. coli can cause severe human illnesses that pose significant public health risks. For the meat industry, the most concerning pathogenic E. coli group is the Shiga toxin producing E. coli, which causes infectious gastroenteritis that could lead to a critical condition called hemolytic-uremic syndrome (HUS), affecting the blood, kidneys and central nervous system.

The main source of STEC is cattle, with transmission occurring through consumption of contaminated food or water, contact with a contaminated environment or contact with an infected patient. Most major outbreaks of STEC are foodborne, commonly originating from meat, salads or dairy products.

The USDA FSIS reinforces a test-hold-release practice for STEC, where meat products are held and tested before release to consumers to ensure that the public is not purchasing and ingesting potentially contaminated products. Accurate testing is therefore critical in identifying STEC infection and ensuring the safety of the general population.

QA: What are the latest trends in E. coli testing and prevention technologies?

SX: Pathogen testing has evolved from traditional methods, reliant on time-consuming growth in culture media, to molecular testing and even whole genome sequencing (WGS). The improvement of testing offers the industry methods with a faster time-to-result, enhanced accuracy and different dimensions of information other than prevalence (e.g. risk-based approaches including microbial enumeration, pathogenesis, identification that allows traceability etc.)

WGS allows governing bodies to identify populations with the same strain of STEC to help identify the source of infection. This approach enables agencies such as the FDA to issue quick recalls and minimize infection spread and hospitalization rates.

QA: How are modern regulations influencing the continued improvement of testing technologies?

SX: In 1993-1994, the USDA FSIS first declared E. coli O157:H7 as an adulterant in beef products, which, at the time, were tested specifically targeting just this serogroup as it was required by the regulatory party for the test-hold-release system as part of the HACCP plan. And later in 2012, the USDA FSIS extended their zero-tolerance policy to six additional serogroups (O26, O45, O103, O111, O121, O145) in beef products, which pushed technology developers to come up with new methods that detect these regulated serogroups beyond O157:H7.

In 2018, the USDA FSIS updated the definition and testing scheme of STEC as E. coli isolates that contain the stx and eae genes and which are genetically identified as one or more of the regulated serogroups (O26, O45, O103, O111, O121, O145 and O157). This was the first time that the regulatory agency shifted the focus of primary testing from serotype to virulence, as the virulence genes are responsible for causing human illness. With this new focus on virulence, technology developers once again shifted gears to focus on detecting the stx and eae genes. As you can see from 1993 to 2024, modern epidemiological data helped the regulatory agency shape regulations to protect public health and further influence the evolution of testing technologies.

QA: How is digital PCR enabling accurate STEC detection?

SX: Due to the current regulatory definition of the STEC, which requires both the Shiga toxin gene stx and intimin gene eae to be present, real-time PCR (qPCR)-based methods for screening STEC have common challenges of separating enrichments with linked and unlinked virulence. When using a qPCR-based method, since the whole population of bacteria is being lysed and analyzed as a bulk, it is difficult to differentiate between samples where a single organism contains both stx and eae (a true positive) from samples in which stx and eae reside in different organisms (an unconfirmed positive result). There are qPCR technologies that utilize additional markers commonly linked to stx/eae positive E. coli strains, which can improve the accuracy of detecting true STEC organisms. However, since the detection of these additional markers is conducted in a bulk with lysed DNA, it still has the common challenge of differentiating if these additional markers are coming from a single cell population or from different cells in the bulk.

An alternative approach is the use of droplet-based digital PCR as a screening and molecular confirmation method, where a sample may be partitioned into up to 20 thousand droplets that encapsulate individual intact E. coli cells, and cell lysis and PCR amplification take place independently in each droplet. The droplets can then be individually screened for both stx and eae targets to determine if both virulence genes are coming from the same bacterial population. This unique approach of partitioning and whole cell encapsulation is equivalent to isolating single colonies on a media agar plate during cultural confirmation.

QA: What opportunities are there for improvement of STEC testing technologies, and what challenges are associated with the development of more accurate and faster tests?

SX: In recent years, public health data has indicated the necessity to identify stx subtypes that are most associated with severe human infections (stx2a), finding emerging STEC serogroups that are not the top seven regulated serogroups (O80) and exploring additional pathogenesis such as identifying additional intimin genes besides eae (aggR).

In addition, expanding the testing matrices to include pre-harvest sample types (pre-harvest feedlot environmental samples, fecal sample, irrigation water samples, etc.) to allow the traceability of food safety from farm to fork has become a new trend in both government labs and food manufactures to further control the food safety risks from its origins.

Season (Yicheng) Xie, Ph.D., is the global product manager of ddPCR food applications at Bio‑Rad. Xie obtained her bachelor’s degree in food science from Kansas State University and her doctorate in animal science from Texas A&M University. Before she joined Bio‑Rad, Xie was a research scientist at Food Safety Net Services, where she provided technical support to internal and external clients and carried out contract research projects on process validations, challenge studies and method validations.