🧬 STEC/VTEC — Verotoxigenic E. coli

Microbiology PN-EN ISO 13136

Overview

STEC (Shiga toxin-producing Escherichia coli), also known as VTEC (Verocytotoxin-producing E. coli), are pathogenic E. coli strains producing Shiga toxins (Stx1 and/or Stx2) — among the most dangerous foodborne pathogens. STEC infection can lead to hemorrhagic colitis (bloody diarrhea) and hemolytic uremic syndrome (HUS) with kidney failure, thrombocytopenia and hemolytic anemia. HUS mortality is 3–5%, and permanent kidney damage occurs in 25% of children with HUS.

The most dangerous serogroups (so-called "Top 5" or "Big 6") are: O157 (most common, best known), O26, O111, O103 and O145. Infection source is most often beef (raw or undercooked), unpasteurized milk, raw sprouts (alfalfa, broccoli), lettuce and water. Infectious dose is extremely low — only 10–100 cells are sufficient.

The PN-EN ISO/TS 13136 standard describes molecular STEC detection method based on real-time PCR. This is the only standardized PCR method for STEC in food. The method includes: sample enrichment (18–24 h), DNA extraction, virulence gene detection (stx1, stx2 — Shiga toxin genes; eae — intimin gene, adhesion protein) and serogroup determination (O157, O111, O26, O103, O145) using real-time PCR.

If stx and eae genes and "Top 5" serotype are detected — strain isolation is required, often using immunomagnetic separation (IMS). Testing is mandatory in EU for sprouts and sprouting seeds (Regulation (EC) 2073/2005) and ground beef in some countries. The STEC O104:H4 epidemic crisis in Germany in 2011 (fenugreek sprouts, >4000 cases, 54 deaths) significantly strengthened testing requirements.

Method principle

Three-stage method: (1) Enrichment — sample is incubated in enrichment broth (e.g., BPW or mTSB with novobiocin) at 37°C/18–24 h. (2) DNA extraction — DNA is isolated from enriched culture (thermolysis or extraction kits). (3) Real-time PCR — real-time PCR reactions with fluorescent probes (TaqMan) are performed to detect: stx1 and stx2 genes (Shiga toxins), eae gene (intimin — adhesion to enterocytes), and serogroup genes (wzgA-O157, wzx-O111, wzx-O26, wzx-O103, wzx-O145). If sample is positive for stx + eae + "Top 5" serogroup — STEC strain is isolated, often using IMS (immunomagnetic separation) with anti-O antibody-coated beads.

Applications

• Sprouts and sprouting seeds control — Regulation (EC) 2073/2005
• Ground beef testing (minced, steaks, burgers)
• Unpasteurized milk and raw milk cheeses control
• Lettuce, leafy vegetables and fruits testing (ready-to-eat)
• Epidemiological investigation in HUS/hemorrhagic colitis outbreaks
• Drinking water and recreational water monitoring
• Imported food control (especially from countries with high endemicity)
• Testing within food safety control programs (RASFF)

Key parameters

Standard

Standard number

PN-EN ISO/TS 13136:2013

Title (PL)

Mikrobiologia żywności i pasz — Metoda reakcji łańcuchowej polimerazy (PCR) w czasie rzeczywistym do wykrywania patogenów przenoszonych przez żywność — Horyzontalna metoda wykrywania Escherichia coli wytwarzających toksynę Shiga (STEC) oraz oznaczania serogrup O157, O111, O26, O103 i O145

Title (EN)

Microbiology of food and animal feed — Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens — Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups

Step-by-step procedure

1. Sample preparation and enrichment

   Weigh 25 g sample, add 225 mL enrichment broth (BPW or mTSB with novobiocin). Homogenize in stomacher. Incubate 37 ± 1°C for 18–24 h.

   ⏱ Time: 18–24 h • 🌡 Temperature: 37°C

2. DNA extraction

   Take 1 mL enriched culture. Extract DNA using extraction kit (silica columns, magnetic beads or thermolysis: 100°C/10 min). Measure DNA concentration and purity (optional).

   ⏱ Time: 30–60 min

3. PCR screening — virulence genes

   Prepare 3 PCR reactions (simplex or multiplex): stx1, stx2, eae. Add DNA, primers, TaqMan probes, Master Mix. Program: 95°C/10 min → 40 cycles (95°C/15 s, 60°C/60 s). Analyze fluorescence curves — Ct < 35 = positive result.

   ⏱ Time: 90 min

4. PCR screening — serogroups (if stx+)

   If stx1 and/or stx2 detected — prepare 5 PCR reactions for serogroups: O157, O111, O26, O103, O145. Same thermal program. Positive result = Ct < 35.

   ⏱ Time: 90 min

5. Screening interpretation

   Variants: (A) stx+ / eae+ / Top5 serogroup+ → potentially positive sample, strain isolation required. (B) stx+ / eae− or serogroup negative → STEC present, but not Top5, report. (C) stx− → negative sample.

6. Strain isolation (IMS)

   For variant (A): add 20 µL immunomagnetic beads (anti-O of appropriate serogroup) to 1 mL enriched culture. Incubate 15–30 min with mixing. Separate on magnet. Wash 3x. Plate on CT-SMAC (O157) or chromogenic agar.

   ⏱ Time: 2 h

7. Isolate confirmation

   From CT-SMAC/chromogenic plate select typical colonies (O157: sorbitol-, colorless). Confirm by PCR — isolate must possess stx1/stx2 + eae + appropriate serogroup gene. Optionally: latex agglutination (anti-O157, etc.).

   ⏱ Time: 24 h

8. Reporting

   Screening result: "STEC (stx+) detected/not detected in 25 g". If strain isolated: "STEC O157:H7 (stx2+, eae+) isolated from 25 g sample". Positive result in food = RASFF notification.

   ⏱ Time: 10 min

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• STEC — BSL-2 pathogen with extremely low infectious dose (10–100 cells!), use highest precautions
• Work with cultures exclusively in class II laminar flow cabinet
• Separate pre-PCR and post-PCR zones — prevent amplicon contamination
• Novobiocin — antibiotic, avoid skin contact
• Autoclave used materials and cultures (121°C/30 min) before disposal
• In case of O157 suspicion — consider notifying reference laboratory

Frequently asked questions