E. coli O157 and STEC Testing in Food: Methods, Culture Media, and Best Practices

Understanding E. coli O157 and non-O157 STEC:

STEC are E. coli strains that produce Shiga toxins, principally Stx1 or Stx2 and their variants. These toxins can damage intestinal cells and contribute to severe disease. Symptoms may include abdominal cramps, diarrhea, and vomiting. Some infections progress to HUS, a potentially serious condition affecting kidney function.

E. coli O157 is distinguished by its O157 somatic antigen and H7 flagellar antigen. Many typical O157 strains do not ferment sorbitol under conventional screening conditions, making sorbitol MacConkey agar (SMAC) useful for presumptive differentiation.

However, other STEC serogroups may ferment sorbitol and resemble nonpathogenic E. coli. The six non-O157 serogroups commonly referred to as the Big Six are O26, O45, O103, O111, O121, and O145.

Because pathogenic and nonpathogenic strains may share similar culture characteristics, colony appearance alone cannot confirm or exclude STEC.

Why is STEC testing important in the US food industry?

STEC contamination may occur during animal production, slaughter, harvesting, food processing, transportation, or handling.

Foods of particular concern include ground beef, other meat products, leafy greens, fresh produce, unpasteurized dairy products, and foods exposed to contaminated water.

Ground beef requires particular attention because grinding can distribute surface contamination throughout the product. Fresh produce presents different challenges, including contamination through irrigation water, agricultural environments, and processing equipment.

Food microbiology laboratories must therefore select testing procedures appropriate to the sample type, regulatory requirements, and intended use of the results.

FDA BAM Chapter 4A addresses diarrheagenic E. coli in foods, while USDA-FSIS MLG 5C.04 covers specified STEC testing applications involving meat products, carcasses, and environmental sponge samples. These methods should not be considered interchangeable.

STEC testing workflow: From enrichment to confirmation

1. Sample enrichment:

Enrichment supports the recovery and multiplication of target organisms that may be present in low numbers or may have been stressed during food processing. The broth, sample quantity, incubation conditions, and selective supplements depend on the chosen reference method.

HiMedia products relevant for evaluation in appropriate enrichment workflows include:

• Buffered Peptone Water with Pyruvate (M1851)
• ACV Selective Supplement (FD284)
• Modified EC Broth Base (M1285)

Buffered Peptone Water with Pyruvate may be relevant to methods requiring a compatible pyruvate-containing enrichment formulation. The ACV supplement provides acriflavin, cefsulodin, and vancomycin for selective applications where specified. Modified EC Broth Base may be considered for compatible procedures that require this formulation.

Important: Product selection must be verified against the exact reference-method formulation and instructions. A commercially available medium should not automatically be assumed equivalent to a specified reference broth.

2. Molecular screening using PCR:

Following enrichment, laboratories commonly use real-time polymerase chain reaction (PCR) to screen for selected genetic targets. These may include Shiga toxin genes, such as stx1 and stx2, and markers associated with O157 or other relevant STEC serogroups.

PCR offers several advantages:

• Rapid screening of enriched samples
• Detection of specific genetic targets
• Efficient prioritization of samples requiring further investigation

Nevertheless, a positive PCR result does not by itself prove that a viable STEC isolate has been recovered. Results must be interpreted according to the validated assay and reference method, including the requirement for isolation and confirmation where applicable.

3. Isolation using selective and differential media:

Culture media help laboratories recover presumptive target organisms and distinguish them from competing microorganisms. Different formulations may be required for O157 and non-O157 STEC.

Sorbitol MacConkey agar:

SMAC is used for presumptive O157 differentiation because many typical O157 strains do not ferment sorbitol, whereas many other E. coli strains produce pink colonies.

Relevant HiMedia products include:

• MacConkey Sorbitol Agar Base (M298I)
• HiCrome MacConkey Sorbitol Agar Base (M1340)

Where required by the reference method, the specified cefixime-tellurite supplement and preparation instructions must be followed. Chromogenic formulations should be selected according to their documented applications and method compatibility.

Chromogenic agar:

Chromogenic media use enzyme substrates to produce characteristic colony colors, helping distinguish presumptive target organisms from background flora.

HiCrome EC O157 Agar, Modified (M1574A) is a relevant HiMedia product to evaluate for presumptive O157 differentiation.

Chromogenic appearance is a screening characteristic, not definitive proof of pathogenicity. Presumptive colonies require additional testing specified by the selected method.

Levine EMB agar:

Levine eosin methylene blue (EMB) agar differentiates bacteria according to characteristics such as lactose fermentation. It may support selected isolation workflows but is not specific for Shiga toxin production.

EMB Agar, Levine (M022S) can be evaluated for applications where this differential medium is appropriate. It should not replace a method-required STEC-selective or chromogenic medium.

4. Confirmation and identification:

Presumptive isolates undergo further examination to establish their identity and determine whether they meet the relevant STEC criteria.

Depending on the reference method, confirmation may include molecular testing for virulence genes, serogroup-associated markers, biochemical identification, serological testing, and additional characterization.

For non-O157 STEC, isolation may be particularly challenging because some strains resemble background E. coli on conventional differential media. Molecular screening and appropriate confirmatory procedures are therefore important components of the overall workflow.

Table 1. FDA BAM Chapter 4A versus USDA-FSIS MLG 5C.04

The two references differ in their intended applications and procedural requirements. Laboratories should consult the current official versions when selecting sample quantities, enrichment conditions, screening targets, isolation media, and confirmation criteria.

Quality assurance in STEC testing:

Reliable results depend on appropriate media, validated procedures, trained personnel, and a documented quality management system.

Key considerations include:

• Media quality control: Verify growth promotion, selectivity, and differential characteristics using appropriate control organisms and established acceptance criteria.
• Incubation control: Follow the selected method's specified temperatures, durations, and atmospheric conditions.
• PCR controls: Apply the assay's required positive, negative, and inhibition controls, where applicable.
• Colony selection: Follow reference-method instructions for selecting representative presumptive colonies, including atypical colonies when indicated.
• Documentation: Record sample identity, media lots, incubation conditions, screening results, confirmation findings, and procedural deviations.
• Biosafety: Handle presumptive STEC using the laboratory's approved risk assessment, containment procedures, and waste management practices.

Laboratories should also evaluate the compatibility of commercial media with their selected method before implementation, particularly when a product is proposed as an alternative to a specified formulation.

Conclusion

STEC testing is a critical component of food safety verification in the United States. Combining appropriate enrichment, molecular screening, selective isolation, and confirmation enables laboratories to investigate presumptive contamination systematically.

Selecting suitable culture media and maintaining rigorous quality control are equally important. HiMedia's range of enrichment media, selective supplements, and differential and chromogenic agars offers laboratories products to evaluate for relevant food microbiology workflows.

For reliable implementation, laboratories should match each product to its intended application, verify method compatibility, and follow the current FDA BAM or USDA-FSIS MLG procedure appropriate to the sample being tested.

Acknowledgement:

The authors express their sincere gratitude to Dr. Rahul Warke (Director, Microbiology Department) and Dr. Girish Mahajan (Executive Vice President, Microbiology Department) for their exceptional scientific guidance, critical insights, and continuous support throughout the development of this work. The authors also thank Ms. Vrutti Mistry (Scientific Writer) for her support in drafting the blog.