Maldi-Tof confirmation method for campylobacter

12th December 2017

Campylobacter bacteria
The MALDI Biotyper system being used
Close-up view of the metal plate on which samples are placed before they go into the machine

According to the UK’s Food Standards Agency (FSA), Campylobacter bacteria are considered to be responsible for around 280,000 cases of food poisoning each year.

Meanwhile, more than 72,000 laboratory cases of Campylobacter poisoning (campylobacteriosis) are confirmed, with a high proportion of these due to food, and about four in five cases in the UK coming from contaminated poultry.

In fact, the FSA estimates that Campylobacter causes more than 100 deaths a year, and costs the UK economy around £900 million, building a strong case for its continued analysis of the full impact of the bacteria and continuing its concerted effort to bring together the whole food chain to reduce levels of campylobacter in chicken, and therefore the burden of foodborne illness in the UK.

So, a recent announcement that the British Standard Method for the Detection and Enumeration of Campylobacter now includes faster testing options via matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS), has empowered the food industry to more effectively meet strict FSA reporting regulations.

Laboratory process time has been dramatically reduced, meaning confirmation of presumptive Campylobacter results can now be returned within the hour, instead of the days taken using other more traditional methods.

Since 2015, the FSA has led a surveillance project, evaluating interventions at all stages of chicken rearing and processing, with the overall aim to reduce the incidence of chickens containing the highest level of contamination, with testing monitoring how effective the interventions were in reducing the levels of Campylobacter in chickens reaching retailers’ shelves.

Initially the responsibility of commissioning the testing lay with the FSA, performed by Public Health England, breaking results down retailer by retailer. Now retailers commission their own and submit results with contract laboratories carrying out the routine testing.

Recent progress
Catherine Cockcroft, head of microbiology at Eurofins Food and Water Testing UK and Ireland, says there will never be a time when Campylobacter is completely eradicated, but that recent measures have helped to bring down the proportion of chickens found to contain the highest level of contamination.

“Chickens are slaughtered at around 45 days old and from approximately day 28, for reasons not yet fully understood, their intestines become colonised with Campylobacter. In an effort to reduce intestinal colonisation, there has been an emphasis on biosecurity on farms, including around the use of captive footwear and clothing, protection of feed and water from cross contamination, as well as ensuring windows are sealed to prevent transfer by flies. The subsequent process of transport, slaughter, defeathering, evisceration and washing of the birds can then lead to the spreading of the intestinal bacteria over the inner and outer surfaces of the carcass.

“Interventions at the processing plants, including removal of the neck flap skin (the most highly contaminated part of the chicken following evisceration), all contribute to reducing the overall bacterial load on the bird prior to reaching the consumer.”

Cockcroft adds that once received, testing of the sample was time-consuming.

“Traditionally, when testing client samples, confirmation of suspect Campylobacter colonies has required the lab to first take 10 suspect colonies from the selective agar and re-streak them onto another media. For each colony, the lab would then have to prepare a slide to look at cell morphology and motility, then streak onto a third agar to check whether the organism can grow at 25°C in air. Finally, an oxidase test would need to be performed.

“All of this takes considerable time - and a minimum of 72 hours from colonisation on the initial media to a confirmed Campylobacter result. However, this can often be 96 hours and with any lab delays, even longer.”

Given the pressures on the food chain of ensuring the safety of the products destined for retailers’ shelves, despite the lengthy confirmation process, many have been happier to rely on a traditional and trusted technique.

Moreover, the FSA protocol for approved Campylobacter testing methods, which is influenced by British and international testing standards and ratified by Public Health England, and is strongly adhered to by many UK retailers that commission testing from contract laboratories, previously deemed the traditional confirmation method as the only acceptable protocol.

But the lengthy procedure could lead to retailers struggling to collect the required broad data set representative of their sales volumes by bird size and supplier, covering a range of use by dates.

Setting new standards
Now, thanks to the updating of the British Standard in September 2017 (BS EN ISO 10272:2017 Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter spp.), for the first time, retailers and suppliers can have confidence in an alternative testing option that means confirmed results can be made available in as little as 15 minutes.

The new standard specifically mentions the use of MALDI-TOF as an alternative confirmation and identification technique to traditional confirmation methods, and has been recognised as an option by the FSA in its protocol. This is allowed as long as the suitability of the alternative procedure has been verified by the testing laboratory and appropriate accreditation has been obtained.

“We’re UKAS accredited and have demonstrated the suitability of MALDI-TOF for confirmation of Campylobacter in our laboratory,” explains Cockcroft, “but it doesn’t mean the challenges of isolating and growing Campylobacter in the laboratory are removed.

“Whether by traditional methods or MALDI-TOF, we’re keenly aware that Campylobacter bacteria are fastidious in their requirements for growth, particularly regarding the Oxygen concentration in their growing environment.

“Campylobacter is unable to grow or reproduce in atmospheric O2 concentrations and instead requires microaerobic conditions at around 10% O2, so we have to perfectly recreate the atmospheric conditions using gas jars and gas generators.”

Cockcroft says that the benefits of using MALDI-TOF for Campylobacter testing are two-fold. “There are huge benefits for the lab in that it removes the need for lots of confirmatory tests - it’s one dab of the culture on the slide, overlaying with a matrix, placing in the machine and that’s it. More importantly, the timing and speeding up of the delivery of results will really help the clients.

“The British Standard method stipulates that where present a minimum of 10 colonies have to be confirmed in each sample, giving a confidence indicator of the bacterial count. In a situation where even small retailers request the sampling of tens of chickens per month, the huge amount of subsequent confirmations in the context of a lengthy testing process has meant that meeting FSA reporting deadlines has been a tricky challenge.

“With the change to the accepted testing standard, suppliers and retailers have a better chance of meeting their obligations, which will surely positively impact on the fight to not only more accurately reflect but also reduce the levels of Campylobacter in chicken, and therefore the burden of foodborne illness.”

MALDI-TOF MS technologically has been in use in other industry testing for some time, but the food industry has not been speedy to adapt, given the considerable initial outlay for the equipment.

As the pace of advancements in testing technology races ahead, those at the leading edge globally are now moving to whole-genome sequencing. Cockcroft adds that, “In the UK there isn’t yet the mainstream commercial capability to do this but it’s coming down the line. Public Health England has been carrying out sequencing on clinical isolates from food poisoning outbreaks, to determine its true extent.

“It’s expensive at the moment and requires bioinformatics expertise, but certainly something we’re watching closely.”

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