Magnetic Approach to VTEC/STEC

The USDA protocol for the detection and isolation of the proscribed non-O157 STECs sets out the use of real-time PCR for the detection of stx1, stx2, and eae genes followed by the detection of serogroup-specific genes. Any samples testing positive for stx and eae gene sequences as well as any serogroup-specific genes are subjected to serogroup-specific enrichment using IMS. Here beads coated with the appropriate serogroup-specific antibodies, as indicated from the PCR testing, are used and the resulting IMS concentrate is plated to an appropriate selective chromogenic medium. Resulting colonies are subjected to confirmatory serological, PCR, and biochemical testing.

While the USDA protocol includes an immunocapture step alongside PCR, the ISO protocol specifies enrichment broth or serogroup-specific enrichment, e.g. IMS.

Resurgence of IMS

Immunomagnetic separation is an established technique that is in effect a powerful sorting process. It involves the use of antibody-coated super paramagnetic particles and can be used in a number of different biological applications. As part of microbiology test protocols, it is generally there to help concentrate target organisms.

Once mixed with a sample, the antibody-coated beads bind to cell surface antigens forming an antibody-antigen complex between the bead and the target organism, thus capturing the target cell. The beads are then simply pulled out of suspension using a magnetic concentrator. Wash steps remove any nonspecifically bound material and the resulting bead concentrate is plated to a suitable medium or, depending on the application, is subjected to other testing.

A number of factors influence the effectiveness of this process. The robustness of the physical separation system itself is important, but the choice of antibody perhaps more so. Criteria for success include a highly specific and stable antibody (in this case targeted towards the O serogroup-specific antigen) that binds well to the surface of the bead, and that also demonstrates high avidity and affinity for the target antigen.

Enhancing Conventional Culture

Despite the availability of a range of effective culture media for E.coli O157:H7, IMS also has a role in speeding up the isolation of this important organism. When culturing the sorbitol-negative O157 on conventional media, overgrowth of more numerous sorbitol-fermenting E.coli may obscure the colonies of interest. Applying IMS to the sample allows concentration of target organisms while removing non-target cells, so improving the chances of E.coli O157:H7 isolation.

A Look Ahead

It is to be expected that future STEC testing will see a progressive move towards PCR or comparable rapid method technologies. These however will still require efficient enrichment broths to generate sufficient assay target for successful detection. In addition there will remain a need to isolate viable cells from enrichment cultures to enable the confirmation of presumptive positive isolates. It is anticipated that future developments in chromogenic plating media will allow enhanced differentiation of STEC by conventional culture methods and assist in this isolation. The efficient concentration and specificity that IMS can achieve will continue to make this a method of choice for this testing regime.

Dr. Potter, a microbiologist at Lab M with many years’ experience in high level academic research at leading U.K. universities, is head of research and development. Reach him at [email protected].

References

1. Brooks et al. Non-O157 Shiga Toxin–Producing Escherichia coli Infections in the United States, 1983–2002. J Infect Dis. (2005) 192 (8): 1422-1429. http://jid.oxfordjournals.org/content/192/8/1422.abstract?ijkey=2dc0f330a65ad3025ade399040e431342e14f75e&keytype2=tf_ipsecsha (Accessed 7 Jan 2014).
2. USDA. Flow Chart Specific for FSIS Laboratory non-O157 Shiga Toxin-Producing Escherichia coli (STEC) ­Analysis (Oct 2013). www.fsis.usda.gov/wps/wcm/connect/1d61852b-0b71-45e9-8914-8ff95af7aaa8/MLG-5B-Appendix4.pdf?MOD=AJPERES (accessed 7 Jan 2014).
3. COMMISSION REGULATION (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CONSLEG:2005R2073:20071227:EN:PDF (Accessed 7 Jan 2014).
4. PD CEN ISO/TS 13136:2012 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. European Committee for Standardisation www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=53328 (Accessed 7 Jan 2014).