B11004: Development and evaluation of solid media to identify VTEC and detect verotoxin in clinical and food samples

Study Duration: June 1996 to June 1999

Contractor: Laboratory of the Government Chemist (LGC)

Current methods have difficulty in detecting VTEC strains outside specific serogroups. The most common serogroup associated with human infection is E. coli O157:H7, although a number of other serogroups have also been implicated in disease. The majority of identification protocols involve either exploitation of the unique biological characteristics of E. coli O157:H7 or immunological detection of serogroup-specific antigens.

Direct methods of detecting verotoxin are often slow, such as the vero-cell assay, or lack sensitivity and specificity, such as enzyme-linked immunosorbant assay (ELISA) techniques. DNA based methods have been developed to detect VTEC contamination. Although these methods are sensitive and highly specific, they detect the ability to produce toxin rather than the actual presence of the toxin or viable toxin-producing organisms.

In order to increase sensitivity, current techniques rely on an initial enrichment culture to increase the numbers of target organisms. However, the selective media used to isolate the potentially low numbers of VTEC present in mixed populations of bacteria were originally developed for the isolation of O157:H7 strains, and have since been demonstrated to be toxic to stressed cells. In addition, such selective media are not favourable for the growth of some non-O157 verocytotoxin producing serogroups. Standard selective methods also involve growth at 44ºC, and such conditions may be non-permissive for some non-O157 VTECs.

An additional problem with detection of VTEC in clinical samples is the general absence of organisms in samples obtained from patients, 3-5 days after the onset of symptoms. In such samples, being able to detect verotoxin itself would be a significant advantage. With the increasing significance of VTEC other than O157 in foodborne illnesses reported from other countries, there is a need for a rapid yet sensitive, generic detection system able to identify the presence of either verotoxin producing bacteria or their toxins.

The aim of the project was to develop a rapid and low cost assay for the sensitive detection of verotoxin and all verotoxin producing E. coli, by:

• development and evaluation of a method of immunologically detecting viable verotoxin producing bacteria, using solid media containing novel supplements
• development of a system for the in vitro detection of the biological activity of verotoxin 1 and 2

An assay was developed with the aim of detecting viable verotoxin producing bacteria. This would use antibodies raised against verotoxin, to immunologically direct reactive liposomes, to target cells.

Assessment of the sensitivity and specificity of the assay demonstrated that there was no differentiation between live and dead organisms, or between verotoxin producing and non-toxigenic E. coli.

Low level leakage of encapsulated enzyme from the liposomes resulted in high levels of background signal on overnight incubation plates. Improvement of the liposome preparation by trypsin treatment and column purification substantially reduced background signals, but significant improvements to liposome stability would be required to allow sufficient discrimination of signal from noise on overnight incubation.

In addition, the naturally adherent properties of avidin, a vital component of the targeting system, may have contributed to the lack of assay specificity, by causing the biotinylated liposomes to adhere to both target and non-target organisms. Replacement of the biotin-avidin aggregation system with reagents with more specific binding properties may improve the assay�s specificity.

No evidence was found to indicate the occurrence of specific binding of anti-verotoxin antibodies to verotoxigenic strains of E. coli, as assessed by both immuno-magnetic capture and Biosensor studies. It is likely that the majority of verotoxin is either located periplasmically (the space between the plasma and outer membrane) or is secreted from the cell into the surrounding environment. This results in negligible amounts of verotoxin being available for antibody binding on the surface of the target cells.

Consequently, the use of antibodies directed to verotoxin to capture all verotoxin producing organisms may be ineffective. Antibodies directed to serogroup specific antigens could be utilised as an alternative capture mechanism, but would not yield the intended generic assay, capable of detecting any verocytotoxin-producing bacterium.

Final report is available from the Agency's Information centre.
To obtain a copy, please contact the Enquiry Desk, Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: infocentre@foodstandards.gsi.gov.uk).

Contact: For any enquiries concerning this research project, please contact the relevant programme contact or email: science@foodstandards.gsi.gov.uk