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A Magnetic Approach to VTEC/STEC
by Chris Potter, PhD
Non-O157 verocytotoxin-producing E. coli (VTEC)/Shiga toxin-producing E. coli (STEC) are organisms of significant and growing public health concern because of their ability to cause extremely severe illness and their high potential for foodborne transmission. Increasing regulation in the U.S. and in Europe now requires rigorous testing at various stages of food production processes. It is therefore timely to examine the nature, characterization, and detection of these organisms. Especially noteworthy is the reemergence of immunomagnetic separation (IMS) as a technique of interest. IMS is now written into USDA methodology for the detection and isolation of non-O157 STEC in meat products, alongside real-time polymerase chain reaction (PCR)-based testing, and so we are seeing a renewed emphasis on its use.
Used interchangeably, the terms VTEC and STEC refer to pathogenic strains of the organism that can cause not only diarrhea but also more severe disease in humans, including haemorrhagic colitis and haemolytic uremic syndrome (HUS). These bacteria are of several different E. coli serogroups, a number of which are now firmly associated with the risk of serious illness in vulnerable individuals and populations.
The most commonly identified VTEC/STEC strain is E. coli O157:H7. Often referred to simply as O157, this organism has been recognized as a foodborne pathogen since the early 1980s. It follows that much of what is known about STEC comes from studies of E. coli O157 infection, but over the years other “non-O157” STEC serogroups have continued to emerge as important causes of disease.
The characteristics that are used to define STECs are their serotype, virulence factors, and biochemical profile. This latter relates to their phenotypic expression on diagnostic media and the biochemical similarities of many non-O157 STECs presents challenges when it comes to their isolation and identification.
Serotyping has a key role in the study of E. coli and follows a modified version of a scheme set out by Kauffmann in the 1940s. According to this scheme, E. coli are serotyped on the basis of their O (somatic), H (flagellar), and K (capsular) surface antigen profiles. The O antigen is the O-specific polysaccharride of the cell wall (LPS). A total of 178 different O antigens (O1 – O181), each defining a serogroup, are currently recognized and a further six have been demonstrated. A specific combination of O and H antigens defines the serotype of an isolate.
The main virulence genes for STEC are the Shiga toxin-encoding stx1 and stx2 genes, and the eae gene which encodes the intimin protein. Shiga toxin acts by shutting down cellular protein synthesis in target cells, including vascular epithelium where it can affect small blood vessels, such as those in the gastrointestinal tract and the kidneys, with potentially devastating consequences. The intimin protein is expressed on the bacterial cell surface and has a role in attaching to and effacing cell membranes.
Six non-O157 STECs were identified in a study at the CDC as being responsible for around 70 percent of non-O157 STEC infections in the U.S. over a 19-year period. In the U.S., the presence of these six is now prohibited in certain meats. Producers of ground beef, for example, for use in the U.S., are now required to test for E. coli O26, O45, O103, O111, O121, and O145 (the “big six”), as well as for E. coli O157:H7. As defined within USDA policy, these are classified as adulterants in raw non-intact beef and beef products and the USDA has issued a protocol for testing.
In Europe, an area of major concern is contamination in fresh sprouted seeds. This follows an outbreak of severe illness in 2011 for which the causative organism was found to be E. coli O104:H4, a serotype not previously associated with foodborne illness. Draft amendments to European Commission regulation (EC) No. 2073/2005 on microbiological criteria for foodstuffs will require the absence (in 25 grams of sprouted seeds) of STEC O157, O26, O111, O103, O145, and O104:H4. This regulation references the test protocols described in international standard ISO/TS 1316:2012: Microbiology of food and animal feed—PCR-based method for the detection of foodborne pathogens—Horizontal method for the detection of STEC and the determination of O157, O111, O26, O103, and O145 serogroups.
While routine testing for E. coli may be standard throughout the food industry, rapidly identifying non-O157 STEC strains has brought some new challenges, primarily because many are biochemically indistinct from other E. coli. Although there is a wide choice of both conventional and chromogenic media available for the isolation and culture of E. coli O157:H7, on their own most do not enable non-O157 serotypes to be distinguished. A more targeted approach is therefore needed and this is bringing together molecular methods and more traditional microbiological testing techniques.
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.
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 Chris.Potter@labm.com.
- 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).
- 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).
- 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).
- 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).