Protecting your food from E. coli

Detecting the more dangerous forms of E.coli in the food chain poses many challenges, but these are driving the continued development of isolation and identification techniques

Escherichia coli is a widespread microorganism that is part of the normal flora in the digestive tracts of many warm blooded animals. While most strains of this Gram-negative, rod-shaped bacterium are generally harmless to human health, some do cause disease, and for certain groups of people infection can have serious consequences. Outside its normal habitat of the lower intestines, E. coli may be found as a causative organism in urinary tract, wound and other infections, but an area of much focus in recent years is its role in outbreaks of diarrhoeal illness, especially where the organism’s transmission is associated with contaminated food. Foodborne outbreaks tend to be linked with a group known as Shiga toxin-producing E. coli (STEC ) also referred to as Verocytotoxin-producing E. coli (VTEC). Their detection and identification in the food chain poses a number of challenges, not least of which is the need for methods that deliver rapid results whilst being able to detect small numbers of organisms and to distinguish these known pathogenic strains from one another and from other E. coli.

According to Public Health England and the Centers for Disease Control in Atlanta, the most commonly identified STEC strain in both the UK and the USA is E. coli O157:H7 – often referred to as O157. Outbreaks of illness have been associated with undercooked beef products such as burgers and with unpasteurised dairy products. In the USA the organism has also been linked to fresh produce and as cattle are the main reservoir for the pathogen it follows that environmental contamination and contamination of watercourses is a risk.

Infected individuals are likely to suffer diarrhoea, stomach cramps and fever.  For most the illness will be self-limiting.  However, in a small proportion of cases, usually children or the elderly, those infected may develop haemolytic uraemic syndrome (HUS), a serious life-threatening condition that results in kidney failure. Some adults may develop a similar condition called thrombotic thrombocytopaenic purpura (TTP). Even when present in very low numbers, the organism can produce symptoms, so its detection and identification is extremely important.

E. coli O157:H7 has been recognised as a foodborne pathogen since the early 1980s, but more recently other STEC serogroups have emerged as important causes of disease. Six of these ‘non-O157 STECs’, identified in a study at the Centers for Disease Control and Prevention as responsible for approximately 70% of non-O157 STEC infections in the USA  from 1983-2002, are now banned from certain meats sold in that country. Since last year, ground beef producers have been required to test  for E. coli O26, O45, O103, O111 O121, and O145 – the ‘Big Six’ – as well as for E. coli O157:H7.  Frequently isolated STEC serotypes vary according to geographical location, i.e. the USDA’s ‘Big Six’ are not all necessarily prevalent in Europe. In 2011 Europe encountered a different problem when a serious outbreak of illness, affecting mainly France and Germany, was attributed to the consumption of fresh sprouted seeds contaminated with E. coli O104:H4. This rare but virulent non-O157 serotype had not previously been associated with foodborne illness.

Escherichia coli is relatively simple to culture and to isolate. Laboratory strains are used in almost every conceivable area of research and E. coli plays a pivotal role in bioengineering and industrial microbiology. Correct use of test protocols and generation of accurate test results to allow the swift and cost-effective identification of organisms of interest is a constant challenge for the food industry.  E. coli is a standard test parameter in many food laboratories. However, rapidly identifying STEC strains can pose extra challenges as not all are biochemically distinct from other E. coli. While there are microbiological culture media that support the improved isolation of E. coli O157, for example, non-O157 serotypes are often indistinguishable from generic E. coli on these media.

[caption id="attachment_34739" align="alignleft" width="200" caption="Figure 1: E. coli O157:H7 Traditional Culture Method v IMS Culture Method"][/caption]

The USDA (United States Department of Agriculture) has devised a method to detect and isolate the ‘Big Six’ non-O157 STEC serogroups in ground beef and beef trim. Following enrichment of the food samples, this method describes real-time PCR detection to screen for both of the shiga toxin gene sequences (stx1 and stx2) and the attachment and effacing intimin gene (eae) sequence, followed by detection of serogroup-specific genes¹. Samples positive for stx and eae gene sequences and any serogroup-specific genes are then subjected to  immunomagnetic separation (IMS) using beads coated with serogroup-specific (as determined in the PCR step) antibodies.  The IMS concentrate is then plated onto a chromogenic medium and, after incubation, undergoes confirmatory testing.

The unique challenge posed by testing for non-O157 STEC strains, whether in beef or sprouting seeds, is driving the development of new methodologies and bringing innovation in both molecular testing and more traditional microbiology techniques. What it has also done is to throw the spotlight on the long-established, but sometimes overlooked, technique of immunomagnetic separation.

Immunomagnetic separation is a powerful sorting process that helps concentrate target microorganisms. It plays a role in the development of rapid methods simply by enabling some of the time-consuming steps in standard microbiological enrichment processes to be eliminated and/or reduced. The principles of the technique are straightforward and involve the use of antibody-coated, microscopic, super paramagnetic particles, which are used to target specific microorganisms in a culture.

When mixed with a sample suspension the antibody-coated beads bind to cell surface antigens to form an antibody-antigen complex between bead and target molecules. The target cell is thus captured. Separation from background organisms and any interfering materials is achieved using a magnetic concentrator and any non-specifically bound material is simply washed away. The resulting beads are plated to appropriate selective media or may be used in further analyses. Successful separation depends on a number of factors which include the quality of the immunomagnetic separation system itself and the choice of antibody, which must be highly specific, stable and easily bound to the bead surface in addition to having high avidity and affinity for the target antigen.

The use of IMS in testing for STECs supports the isolation of specific serogroups.  Take E. coli O121 for example.  Unlike E. coli O157 or O26, which cannot ferment sorbitol and rhamnose respectively, O121 is not biochemically unique and is therefore difficult to isolate from other STEC using conventional culture methods.  Immunomagnetic separation is the only process that can be used for the specific isolation of E. coli O121 in which a viable microbiological culture is obtained.

With E.coli O157 still the most commonly encountered STEC in Europe and the USA, reliable culture and identification methods play an important role in identifying the source of outbreaks and in continued monitoring, whatever the sample type.

The isolation of E. coli O157:H7 itself offers a good example of how IMS can reduce culture time compared with using traditional culture methods only (see Figure 1) . The challenge with E. coli O157:H7 is slightly different from the non-O157 STECs. When culturing O157 on traditional media there is always the risk that the generally sorbitol-negative O157 will be overgrown by more numerous sorbitol-fermenting E. coli, making it difficult to identify the colonies of real interest. By using IMS it is possible to concentrate target cells from the larger samples while removing non-target cells, improving the chances of E. coli O157:H7 isolation. Target organisms are generally obtained with IMS after 6 hours enrichment, although stressed cells may require up to 18 hours.

A range of enrichment media including Buffered Peptone Water and modified Tryptone Soy Broth (mTSB), together  with selective differential media, are used to support the isolation of E. coli O157:H7, while chromogenic media allow improved differentiation of E. coli O157:H7 from other strains, on the basis of colony colour.

As well as its role in accelerating culture and isolation, IMS can be used in parallel with molecular methods, as a means of sample preparation prior to genetic analysis with PCR for example, or after molecular methods to further test samples of interest. These trends in methodology and industry acceptance are reflected in the content of a new technical standard, currently under review, for non-O157 STEC in the EU, which is the first of its kind to recommend molecular methods.

The potentially serious consequences of human infection with E. coli STEC strains and the challenges of tracking down the causative organisms and their sources as new strains have emerged, is driving continued development in isolation and identification techniques. In an area of considerable innovation, traditional microbiology techniques and rapid diagnostic assays all have a role, often working side by side in tackling this continuing public health issue.

References

1. http://www.fsis.usda.gov/wps/wcm/connect/7ffc02b5-3d33-4a79-b50c-81f208893204/MLG-5B.pdf?MOD=AJPERES

Author: Ezzeddine Elmerhebi, Development Scientist and Lisa Green, Product Manager, Lab M

For more information:

When Lab Innovations returns to the NEC, Birmingham on 6 & 7 November 2013 it will feature a special Campden BRI theatre, where experts will deliver a range of talks dedicated to the latest tests and technologies available to food and drink manufacturers.

Included in the packed programme will be a session by Lab M on a magnetic approach to Non-0157 verocytoxin producing E. coli (VTEC)/Shiga toxigenic E. coli (STEC).

More at Lab Innovations 2013

Lab M is just one of the companies taking part in the Campden BRI theatre at Lab Innovations 2013. The show, on the 6 & 7 November 2013 at the NEC, Birmingham, is the UK’s only event dedicated to showcasing the latest laboratory technology & consumables, analytical & biotech equipment.

The show is being supported by Royal Society of Chemistry, Gambica, Campden BRI, the Institute of Measurement and Control, the Society of Biology and the UK Science Park Association.

Not to be missed features at the show include:

• A top flight conference programme, including keynote speakers, designed by the Royal Society of Chemistry and delivered by experts
• A free-to-attend practical workshops programme devised by Campden BRI on micro and analytical topics within food & beverage.
• Over 100 exhibitors showcasing product and service innovations
• UKAS Pavilion featuring the services of the UK’s leading contract laboratories
• A dynamic panel debate hosted by The Royal Society of Chemistry giving industry experts and attendees to the show the opportunity to discuss the critical issues affecting the sector.
• Lions’ Lair where companies will pitch their most innovative products to a panel of experts from the UK’s leading trade bodies.