Shiga-toxin-producing Escherichia coli (STEC) is a group of pathogenic E.coli that has been found to contaminate leafy greens and other fresh produce items. These contamination events contribute to the overall burden of foodborne illnesses and represent a primary challenge in the goal to improve produce food safety.  STEC is a broad and diverse category of E.coli, with an estimated number of over 400 serotypes considered within the STEC group.  

What’s the definition of STEC positive? As with all complicated answers, it begins with “it depends.” Illness caused by shiga-toxin includes symptoms such as cramping, diarrhea (sometimes bloody), and vomiting, and it can lead to the development of the potentially deadly Hemolytic Uremic Syndrome (HUS). However, while an illness from a STEC infection may be easy to define, STEC-positive samples in the food industry often have a more complicated definition.  

The definition of STEC in molecular diagnostic tools has evolved differently across the globe based on regulatory body/regulatory framework, industry standards and available testing technologies. This variability has often led to unintended confusion between stakeholders on the definition of a positive and has created variability within the food industry on what and when to react. The common denominator for most molecular methods (i.e., Polymerase Chain Reaction (PCR)) is to detect STEC positive E.coli containing at least one Shiga toxin gene. There are two main types of Shiga toxin, Shiga Toxin 1 (Stx1) and Shiga Toxin 2 (Stx2), with most severe illness being attributed to Stx2. As with all pathogens, there are usually numerous pathogenicity genes that contribute to overall virulence. These include but are not limited to genes for attachment and adherence, and those that contribute to the organism’s ability to evade host immune responses. One of these STEC attachment genes, intimin (eae), is often included in detection assays for STEC due to frequent presence within strains associated with illness. The eae attachment gene is an important virulence factor that, when present, provides more confidence that a detected STEC would have public health significance. However, eae is not required to cause illness and some STEC strains associated with illnesses and outbreaks have not harbored the eae gene.  

Molecular testing for STEC, especially if numerous gene targets (stx1, stx2 and eae) are used, leads to questions on whether the necessary virulence genes exist within one organism (as opposed to different targets coming from different organisms in the sample), and as a result, whether that STEC detection is an organism that may likely cause illness if consumed. Initial molecular screening for the virulence genes (stx1, stx2, eae) is often taken to secondary confirmation by either molecular or cultural methods. These confirmation steps work to verify that the virulence genes exist in one living organism (i.e., not from dead cell DNA detection), and/or to identify if the virulence genes are contained within specific highly pathogenic O-serogroups (Top 7: O157, O26, O45, O111, O103, O121, O145). 

Table 1 captures the positive STEC test outcome requirements for commonly used and referenced US food regulatory methods. Top 7 STEC serogroups are part of the required USDA FSIS Methods Laboratory Guide (MLG) STEC testing definition, while the Top 7 serogroups are not considered required elements for a STEC positive within the FDA Bacteriological Analytics Manual (BAM). When testing for O serogroups, secondary molecular testing is often completed utilizing secondary PCR for the O group genes, and/or, by immunomagnetic capture based on antigen-antibody reactions for the Top 7, O serogroups. After using the immunomagnetic beads, only Top 7 STECs will remain and when coupled with additional PCR for the virulence genes, will provide evidence that the virulence genes are also present with Top 7 serogroups.  

Table 1: STEC positive detection results based on common regulatory methods relevant to US fresh produce

Beyond secondary PCR and immunoassay capture, a newer molecular confirmation method is that of droplet digital PCR (ddPCR). ddPCR is a variant of PCR that is able to confirm that the virulence genes (stx1&/or stx2, eae) are located within one bacterial cell. This emerging technology utilizes PCR performed in millions of partitioned droplets that physically exclude more than one cell into one PCR reaction. Using this technology, presumptive samples can rapidly identify from an enrichment culture whether the toxin gene(s) and attachment gene (eae) are co-located within one cell. 

Finally, a familiar method in food microbiology testing includes cultural confirmations, a process that isolates cells and confirms bacterial identity through their traditional biochemical characteristics (e.g., sugar fermentation, antibiotic resistance, growth conditions). Cultural confirmations have been used for many decades, but cultural methods may be challenging for broad and diverse groups such as STEC given that biochemical tests may present differently dependent on the STEC strain that may be present. The large number of STECs (> 400 serotypes) and their potential to have variable biochemical phenotypes on culture medias/tests may lead to STEC organisms being mischaracterized in the culture confirmation process. The chosen cultural confirmation method is often based on the regulation that a food is produced under – FDA regulated foods following FDA BAM Chapter 4a methods for STEC detection and isolation, and USDA regulated foods using USDA MLG Chapter 5C.03 for STEC confirmation. In some cases, FDA regulated foods may perform MLG methods based on laboratory offerings and expertise. 

STEC is an emerging group of pathogenic E.coli that increasingly lead to outbreaks, illnesses and recalls. When incorporating STEC testing into routine and investigatory testing, it is important to evaluate and determine what a STEC positive is within the testing program and remain transparent in the program design to allow for analysis (i.e., shiga toxin alone, shiga toxin + intimin, shiga toxin + intimin + O serogroups, molecular confirmation, cultural confirmation). This clear characterization is important in developing the risk strategy within a testing program and is critically important when analyzing testing program data. For more information on how to optimize testing data for ongoing analysis and learning, see The Western Growers GreenLink® Best Practices for Sharing Tissue and Water Data Guide.