Key Points

• VTEC serogroup O157 is the most important but others, such as O26 and O145, should not be overlooked. A major German outbreak in 2011 was caused by O104
• Traditional methods can take up to five days to produce a result.
• A wide range of time-saving rapid methods are commercially available, providing a result within 24 hours.
• Methods for detection of non-O157 VTEC are not widely available.

Storage and preparation of samples

The majority of food samples examined for the presence of VTEC will be raw meat, especially beef, or unpasteurised dairy products. It is unusual for other food categories to be routinely tested for these pathogens.

Samples to be tested for VTEC should be refrigerated after collection and tested as soon as possible. Samples should not be frozen unless there is no alternative, although VTEC cells survive well at low temperatures and detection may still be possible.

Sample preparation typically requires homogenisation in a suitable enrichment broth or diluent.

Detection and isolation: Culture-based methods

O157 VTEC There is a current ISO horizontal method for the detection of VTEC O157 in food and feed. ISO 16654:2001 is recommended for monitoring food and feed in the EU and elsewhere and was reviewed and confirmed in 2012. The FDA Bacteriological Analytical Manual (BAM) includes a similar method, although a real-time PCR assay is now preferred.

For the ISO 16654 method the first stage is an enrichment culture in a modified tryptone soya broth supplemented with 20mg/l of the antimicrobial novobiocin. Typically 25g of sample is homogenised with 225ml of enrichment medium and incubated at 41.5°C for 6 hours.

An immuno-magnetic separation (IMS) procedure is carried out at 6 hours and again at 12-18 hours using commercially available magnetic beads coated with antibodies specific to VTEC O157. The beads are plated out onto two selective agar media. One of these must be sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC). This medium exploits the inability of VTEC O157 to ferment sorbitol within 24 hours to differentiate colonies from those of other E. coli. The second selective agar can be chosen from a range of commercially available chromogenic available media. The plates are incubated for 18-24 hours at 37°C and examined for typical VTEC O157 colonies.

Up to five typical colonies from each medium are then subcultured on a non-selective agar medium and tested for indole formation to confirm that the isolates are E. coli. Presumptive confirmation of VTEC O157 can be done serologically using a slide agglutination test, but conclusive identification and typing of pathogenic VTEC O157 should be done at a reference laboratory. This requires testing presumptive positive isolates using a PCR method to detect the genes necessary for toxin production (eae & vtx).

Non-O157 Currently there are no standard culture-based methods for non-O157 VTEC. Most non-O157 VTEC strains will ferment sorbitol and commercial selective agar media are lacking. However, a few media selective for other VTEC serogroups have been developed, such as a modified CT-SMAC medium for VTEC O26, in which rhamnose is substituted for sorbitol, and a chromogenic O26 medium. The ISO 16654 method has also been adapted by using IMS beads coated with antibodies specific for other important serogroups and modified selective agars. Recently, researchers from the United States Department of Agriculture (USDA) published details of a selective chromogenic medium for the detection of the six non-O157 STEC serogroups (O26, O45, O103, O111, O121 & O145) that must now be routinely screened for in meat samples in the USA.

Detection and isolation: Rapid Methods

O157 Concerns about O157 VTEC contamination in foods have led to the development of a number of rapid methods for their detection and isolation and many of these are now available as commercial test kits and systems. Most are aimed at the meat industry and are designed for testing ground beef and similar products, but can also be applied to other foods where O157 VTEC contamination is suspected.

Immunoassay-based methods Many of the commercial products currently available are based on immunoassay technology. Results are available in 20-26 hours following an 18-24 hour enrichment step. Fully automated immunoassay systems are also available.

Adding an immuno-concentration step to the method can reduce incubation times and improve sensitivity. The concentrated sample can then be tested using an immunoassay or other detection method.

Several manufacturers have developed immunoassay technology to produce very simple-to-use immuno-chromatographic lateral flow assays. These are usually supplied with an enrichment broth and typically claim to provide a clear visual presence or absence result in 10-15 minutes after an enrichment step of only 8 hours, although a standard 20-24 enrichment as described in ISO 16654 can also be used.

Molecular biology-based methods (PCR) A significant number of commercial test kits and systems detecting O157 VTEC using PCR technology have been launched in the last 15 years, many of which include real-time detection. Using proprietary media and/or IMS a preliminary enrichment step can be reduced to as little as 6-8 hours. Results of the PCR assay are then available within a further four hours.

Confirmation Although rapid latex agglutination- based confirmation tests, such as the Remel Wellcolex® test, are available for O157 VTEC, final confirmation should still be done by a reference laboratory as above.

Non-O157

There is a current ISO horizontal method for the detection of STEC and the determination of O157, O111, O26, O103 and O145 serogroups. ISO/TS 13136:2012 is based on a Real-time PCR-based screening method for the stx and eae virulence genes. When one or both genes are detected, the STEC strain present should be isolated if possible for confirmation. The BAM PCR method is also able to detect some non-O157 STEC.

Currently, test kits specifically designed for detection of non-O157 VTEC in food samples are not widely available. However, several PCR-based kits designed to detect the USDA's "big six" serogroups in meat products are now available in the USA. These include the  Hunter® system from Instantlabs Inc. Additionally, in the wake of the 2011 outbreak in Germany, Biotecon developed a foodproof® test kit for serogroup O104.

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