In a previous article on Shiga toxin-producing E.coli detection in the food industry, it was discussed how STEC/EHEC is somewhat of a “choose your own adventure” screening test. Since this is a broad group of organisms, variations in the methodology determine what a positive may include regarding both pathogenicity and gene targets. Often, the food industry defaults to the method and approach of their regulator (e.g., FDA, USDA) and/or common offerings at their commercial laboratory. Given that there are differences in assays and end-to-end approaches, screening for STEC/EHEC requires an intentional selection of what a user wants to use and react to within their program. As with all testing, the prudent choice will ensure both acceptability and defensibility as to why those decisions were made.

Unlike many other pathogen detection procedures, the initial molecular detection for STEC/EHEC often includes various gene targets, and in many cases, requires a combination of gene targets of both the Shiga toxin (stx1 or stx2) and attachment gene (eae) to result in a “positive” sample. When a sample has both gene targets, there is an increased likelihood that a pathogenic strain is present. While the presence of both genes increases the chances of knowing that detection is a clinically relevant strain, organisms with shiga-toxin gene presence without intimin can cause (and have) STEC outbreaks. Despite that, most STEC/EHEC food screening in the United States includes multiple gene targets and that makes understanding what a positive means a bit more complicated for the food industry. Transparent design of a testing program is critical to ensure a company knows what they are testing for, and that they realize any caveats or limitations that may exist with the approach. To find out assay options from manufacturers and descriptions on what your primary STEC/EHEC screening may be designed to find, check out AOAC official validation resources here.

Once you have a primary molecular positive/presumptive for a STEC/EHEC screening test, what options do you have next?

What are some common next steps past the primary virulence gene detections?

The initial detection means that there are good reasons to believe that there may be risk from a shiga toxin-producing E.coli in the sample. The next questions asked, and procedures performed, will help inform the food company/grower of the certainty around that initial molecular detection. Ultimately, these questions can help a company determine where in the “choose your own adventure” you want to stop, and what amount of potential risk to remove. It is important to consider what regulatory body you produce food under as their interpretation may differ, and it is advisable to understand how your process aligns with their definition of a positive. See our prior document that goes over the general difference between the FDA vs. USDA method references.

Secondary testing is usually performed on the presumptive positive sample to see if those initial shiga toxin and attachment gene detections come from one living cell as opposed to those genes existing in different cells (living or non-living) within the sample’s microbial enrichment.

Read here for a summary of common options the industry has following a presumptive STEC/EHEC sample, and some general pros and cons of each.

• Secondary Molecular Screening for Serogroups: Many primary molecular detection kits are validated with the use of secondary molecular assays (e.g., PCR, LAMP) that take an aliquot of the initial enrichment media and run a second molecular assay to look for additional gene targets for the Top 7 serogroups of STEC. The Top 7 (O157, O26, O45, O103, O111, O121, O145) serogroups are often screened for since they commonly contribute the most illnesses associated with the STEC/EHEC group. This secondary screening includes genetic targets for each of the serogroups and provides the end user with more information to know that the virulence genes used in the primary screening (stx1, stx2, eae) are also associated with the presence of the Top 7 serogroups.
  • Pros: This is a very fast secondary option, generally requiring less than two hours for the second assay to be completed. This limits STEC/EHEC reactions to a group of STEC/EHEC that are main contributors to clinical illness.
  • Cons: There are over 400 known types of serogroups for STEC and this is only looking at the Top 7. From a regulation perspective, USDA-regulated entities are directed to ensure the absence of Top 7 serogroups, while FDA-regulated foods include non-Top 7 serogroups. Additionally, the second molecular screening is only confirming that the enrichment culture has these O group genes present, but not that these genes are co-located with the other virulence genes (aka: all genes in one organism). Finally, this molecular screening does not confirm that the organism(s) are living since these are molecular detections based on genetic material (DNA) alone. While there are procedures available to help remove DNA targets from non-living cells before PCR/LAMP testing, this must be discussed with your laboratory regarding availability and compatibility with the assay from a validation perspective. If used, this treatment usually adds 2-3 hours of time to result.
• Secondary Molecular Screening for alternative STEC pathogenicity targets: A newer genetic target for pathogenic E.coli (PEC) detection has recently been AOAC validated that can be used as a primary screening, or as a secondary molecular confirmation to increase the confidence that a pathogenic E.coli is present in a sample. The PEC assay uses a single target for the initial PCR screening and has been found to correlate strongly with only clinically relevant/pathogenic E.coli. More information can be found here.
  • Pros: If used as a primary screen, this assay only includes one genetic target leaving far more confidence that a pathogenic E.coli is present, and given the single target, no question that the signal originates from one cell. When the PEC assay is used as a secondary screening following the shiga toxin and intimin detection, it is a rapid molecular method to further support the presence of pathogenic E.coli. This testing takes 1 hour when using the existing enrichment culture.
  • Cons: The PEC assay is less known in the Produce industry as it is a new offering from the more commonly used shiga toxin and eae gene targets. The PEC assay, as with all molecular assays, relies on genetic detection of DNA and will not confirm culturability/viability. As previously mentioned in the bullet point above, additional measures can be taken to inform whether a gene detection is from a living cell vs. genetic material from non-living organisms. This additional 2–3-hour step must be discussed with your laboratory regarding availability and compatibility with the assay from a validation perspective.
• Immunomagnetic separation (IMS) with Secondary Molecular Screening: Another option following an initial screening detection of shiga toxin and eae genes utilizes magnetic beads that are coated with antibodies to attach to surface antigens found on STEC cells. Most commonly in the detection industry, the target STEC surface antigens are the Top 7 O serogroups (O157, O26, O45, O103, O111, O121, O145). When using this approach, an aliquot of the primary enrichment that had tested positive for STEC/EHEC (and often already PCR screened for Top 7 serogroups), is run through an IMS step to capture if those serogroups are present along with the virulence genes in the enrichment. The beads are then separated from the total enrichment culture by using the magnetic nature of the bead, and some preparation/washing leaves the beads separated and, if positive, coated with cells that have the surface O groups. Once separated, these captured cells on the beads have an additional molecular screening that confirms that the initial virulence genes (shiga toxin + eae) are also present with one (or more) of the Top 7 O serogroups. The use of IMS shows that the serogroup is present and that the virulence genes are also present.
  • Pros: This is a relatively quick screening method to separate the Top 7 STEC serogroups from other cells in the enrichment culture. Generally, the whole process can be completed in 3-4 hours. This allows for greater confidence that the presence of the virulence genes is combined with the presence of serogroups of STEC/EHEC that often lead to illness and outbreaks.
  • Cons: The IMS step has the potential to lose target cells in the process, and if a sample is near the limit of detection (10⁴ cells/mL at the end of enrichment) it is possible to fail to capture enough cells for the secondary screening. Additionally, there are not serogroup IMS beads for all STECs, mainly the Top 7 being the only commercially available. Finally, official validations including IMS are usually only addressing these 7 serogroups in their validations.
• Droplet Digital PCR (ddPCR): ddPCR is a newer technology that was recently AOAC validated for the detection of STEC in enrichment cultures/samples. The primary enrichment that screened positive for stx1 and/or stx2 and eae can be used for a secondary process that involves droplet partitioning. In effect, this technology creates millions of droplets that, through size exclusion, partition individual cells into their own PCR reaction. Then, the instrument performs PCR in these droplets and can determine through fluorescence whether the virulence genes co-exist within one cell. This new method is rapid with results able to be returned within 4-6 hours of the initial detection. More information can be found here.
  • Pros: A rapid method that takes hours from an initial detection to confirm whether virulence genes are co-located in one cell. Additionally, ddPCR screens for all STECs, not those limited to Top 7 serogroups. This method provides a high level of confidence that all genes are in one cell and avoids the pitfalls of culture confirmation discussed later in this document.
  • Cons: This is a new approach, and the ddPCR platform is not available in all laboratories. As such, while the approach is fast, shipping of presumptive enrichments may still be needed. The ddPCR test is more costly than a simple secondary PCR and/or IMS combined with secondary PCR testing. As with all PCR, or assays based on molecular detection, a ddPCR detection does not confirm culturability/viability.
• Culture confirmation: A long-considered “gold standard” in food pathogen testing is culture confirmation. When paired with a rapid molecular screening, culture confirmation uses traditional microbiological testing approaches that grow and isolate the target pathogen based on biochemical and phenotypic response – or, more simply, culture confirmation grows cells out onto plates. This method is slow, generally taking at least 3-5 days, and for STEC/EHEC, more commonly takes weeks if isolation proves difficult. As mentioned, STEC is a broad group with over 400 different serogroups and the diversity of the group makes culture confirmation very difficult since a common appearance and reactions do not occur for all organisms within the group. This variability can increase the probability that culture confirmation may fail to detect the target. In these instances, this is a false negative that could potentially release STEC/EHEC into commerce. There are numerous culture enrichment methods, with the most commonly applied in the US being that of the FDA BAM Chapter 4a and the USDA MLG 5.0c methods.
  • Pros: Cultural confirmation, when successful, leads to official confirmation that the organism is present, and living. Additionally, the colonies are isolated which offers additional opportunities for Whole Genome Sequencing (WGS) and isolate characterization.
  • Cons: Culture confirmation is very slow, and at best can be completed in 3-5 days but often takes up to 1-2- weeks for STEC/EHEC. Culture confirmation for any/all organisms is influenced by the state of the organism, and highly stressed or adapted cells may not culture past the primary enrichment (and may not be detected in the primary assay to begin with). Additionally, the diversity of the STEC group leads to challenges in isolation and detection using cultural processes. STEC/EHEC culture confirmation is known to have high false negative rates due to the challenge of identifying them from generic E.coli and resuscitating them for isolation. Finally, during lab culturing, it has also been documented while researching STEC/EHEC that when culturing them, STEC/EHEC cells may lose the virulence genes that were originally found to be within one cell.

Here is a general flow diagram of commonly used STEC/EHEC detection pathways: