EHJ May 2004, pages 142-144
A salmonella outbreak in Wyre DC provided an opportunity to test a new method of detecting the bacteria, saving vital days. EHJ explains how the new technology works and summarises the research that led to the breakthrough

Anyone involved in a food poisoning outbreak knows that time is key to minimising the number of people affected. When David Watson, food safety manager at Wyre DC, discovered that two people in neighbouring villages were suffering from salmonella enteritidis, and they had both bought vanilla slices from a local bakery, he suspected he may have a serious outbreak on his hands. So when a team of microbiologists approached him and asked whether this outbreak could be used to test a new laboratory method for detecting salmonella, he agreed. The new method, known as automated immunomagnetic separation-enzyme immunoassay or AIMS-EIA, was run in parallel to the conventional standard method and produced accurate results 48 hours earlier.  

As a result, the bakery closed voluntarily within two days of the outbreak. The early closure prevented potentially contaminated birthday cakes, being prepared for children's parties, from being sold. Mr Watson believes the new technique prevented a far more serious outbreak from occurring.

"Identifying a bacteria quickly when you have an outbreak could save lives," said Mr Watson. "In this case I was stunned as I have never done environmental swabbing where so many places come up positive. The salmonella was all over the place and the chances were that this outbreak would have carried on for another three or four days, so we were able to put a stop to it far faster than normal."   

This is the first time this technology has been used to detect salmonella pathogens in a real outbreak situation. Over the next two pages EHJ summarises the published research that followed the outbreak and invites Dr David Wareing, one of the paper's authors, to explain how the new technology differs from standard procedures currently being used. We  also outline how new microbiological procedures get to be adopted in UK food laboratories and by organisations like the Health Protection Agency.

RESEARCH SUMMARY

INTRODUCTION

Wyre DC was notified of two laboratory confirmed cases of salmonella enteritidis from neighbouring villages. Cases were visited at home and detailed food histories were taken, identifying vanilla slices from a local bakery as the common source. A relative of one of the cases also contracted salmonella after eating a slice. A fourth family member was ill but did not submit a sample for analysis.   

SAMPLING

As part of the outbreak investigation, EHPs took 30 samples from the suspect bakery, 24 environmental swabs, and six food samples, including raw ingredients and ready-to-eat foods. Food handlers submitted six faecal samples for analysis. Environmental swabs were also taken at the manufacturer of sausage meat supplied to the bakery. Farm investigations were carried out to check for salmonella in eggs used for baking. Two local farms were used and swabs were taken from the grading and packing area and from manure belts from each row of cages.

MICROBIOLOGICAL RESULTS

Using the standard method of analysis (see below) 18 samples came back as positive for salmonella. Three food samples, 14 environmental swabs and one faecal sample submitted by a food handler. The AIMS-EIA system (see overpage) was run in parallel to the conventional method. The results from this method, obtained in two days, showed that 17 of the 18 samples that were positive under conventional analysis also showed positive using AIMS-EIA. One sample, a swab off a shell egg, was positive using a conventional culture but negative using AIMS-EIA. The 18 samples that showed negative using the conventional method also showed negative using AIMS-EIA. These included samples from the farms and from the producer of sausage meat.

CONCLUSION

One of the shortcomings of using conventional culture techniques is the time taken to detect salmonella, which can be anything between 72 and 96 hours. When dealing with an outbreak linked to a retail premises, the outbreak control team needs far quicker results so that any risk to public health can be better managed. During this outbreak the AIMs-EIA method was used to gain a rapid detection of positive results, which were later confirmed by the traditional culture method. The new method was therefore shown to compliment the conventional approach in this investigation to produce more timely results.

Microbiologist Dr David Wareing explains how the new AIMS-EIA procedure differs from the standard method used in UK food laboratories

THE CURRENT STANDARD METHOD EXPLAINED

STAGE 1:

Food samples or surface swabs from the suspect premises are taken for analysis.

STAGE 2:

The salmonella bacteria in the sample may have been damaged through heating, in the case of food, or prolonged exposure in the case of an environmental swab. A recovery or enrichment culture medium is used to encourage the bacteria to multiply to provide enough healthy bacteria to be analysed. This liquid enrichment medium, containing essential nutrients for growth, is incubated for around 24 hours to allow the bacteria to multiply. In this primary enrichment medium one salmonella bacterium can multiply to produce 100,000,000 bacteria.

STAGE 3:

During this incubation period, the primary enrichment medium will also help to multiply many other types of bacteria present in the sample in addition to the salmonella. During the next stage, a small portion of the primary enrichment is transferred to a second "selective" liquid culture medium containing antibiotics and other inhibitory chemicals, which stops the growth of these other bacteria but allows the salmonella bacteria to continue to grow. This can take another 24 hours.

STAGE 4.

A sample of this "selective" medium is inoculated onto two types of solidified culture media. These "agar plates" contain important meat protein and sugars essential for salmonella growth and also more inhibitory agents to stop the growth of any remaining contaminants. Also, in these media are "indicators" that change colour when salmonella grow. After incubation overnight, "colonies" of salmonella bacteria can be seen on the agar plates, which are instantly recognisable by the laboratory scientists.

The laboratory can now confirm the presence of salmonella to the local authority.  The time taken to produce this result can be anything between four-to-six days.

There are many other tests, which now need to be done to "fingerprint" the particular strain of salmonella so that isolates from infected cases can be matched with the implicated source. A sample of the colony is then sent to the National Reference Centre in London to determine its phage type. Similar procedures are used for detecting E.coli O157, Campylobacter and Listeria

HOW DOES AIMS-EIA DIFFER FROM THE STANDARD METHOD CURRENTLY USED IN LABORATORIES?

STAGE 1 AND 2 ARE THE SAME

A sample is provided and then allowed to multiply and regenerate using the primary enrichment medium taking up to 24 hours.

STAGE 3:

Salmonella antibodies attached to microscopic magnetic spheres or particles are added to a sample of the enrichment medium and tumbled around. The antibodies attached to the spheres then "capture" the salmonella bacteria present in the sample.

STAGE 4:

The magnetic spheres, with the salmonella attached, are then drawn out of the enrichment medium using a magnet and other contaminating bacteria are washed away.

The magnetic particles, still with salmonella attached, are automatically transferred and released from the magnet into a second tube that contains a second antibody, which can also attach to the salmonella bacteria. This second antibody is labelled with a molecule, which glows in the dark when other chemicals (reagents) are added. The magnetic particles containing the "labelled" salmonella bacteria are attracted to the magnet once more and automatically transferred into the glow in the dark detection reagents. Within 10 minutes the scientists can tell if the original sample was positive for salmonella bacteria. This saves two days over the standard method.

Following this immuno-magnetic separation (IMS) process, the captured salmonella still attached to the magnetic beads are placed on the agar media to produce a salmonella colony, which can be seen after 24 hours. This allows the laboratory scientists to continue with the strain fingerprinting process to link human cases with the implicated premises. Combining both new methods the local authority receives the results within two days of testing.

Dr David Wareing was the principal clinical scientist at the Public Health Laboratory Service during this investigation and has since become senior scientist at Dynal Biotech, based at the University of Central Lancashire, the company that has developed the AIMS-EIA technology. He would like to acknowledge the contribution made by Dave Watson and Nicola Wilkinson, the two lead investigators.

HOW COULD AIMS-EIA BE ADOPTED BY LABORATORIES AS A STANDARD METHOD TO TEST FOR SALMONELLA IN OUTBREAKS?

For the AIMS-EIA system to be adopted it has to be validated, following an evaluation period. This can be done by many different organisations in the UK and internationally, including the Health Protection Agency. Once a test is validated however, this does not necessarily mean it will be adopted as issues of cost, training and ease of use have to be considered.

It is up to individual laboratories whether to adopt new technology. The United Kingdom Accreditation Service also has to accept a new procedure as the body responsible for accrediting laboratories. As part of this process inspectors will look at methodology, and consider whether scientists are using acceptable techniques.

There are many different types of standard methods used around the country. Some differ according to the types of products being tested. Some differ according to who is doing the testing. For example, most supermarket chains will have their own standard methods for testing foods.

There are also international standard methods. However, individual countries may have testing methods that are unique to them for specific products. The AIMS-EIA system has been accepted as an international standard and by UKAS for testing for E.coli O157. This means that the equipment needed for the AIMS-EIA method is currently available in UK laboratories.

The luminescent substrate used in this experiment to detect the salmonella bacteria is a far newer technique and so is likely to take a number of years to become accepted. This procedure saves an additional 24 hours in testing time.