Pathogen detection gets ever faster

Rapid methods for pathogen testing are the focus of increasing effort by the food industry, food regulators and technology providers. Susan Birks looks at recent testing developments

Food safety is a concern worldwide and food regulations are getting ever stricter, which means food testing is a growing business. The global food safety testing market is projected to grow from US$9.262bn in 2012 to $14.030bn by 2018, according to the latest report published by Marketsandmarkets. Research data on food microbiology diagnostics trends and practices over the past 15 years, compiled by Strategic Consulting Inc (SCI) through 450 interviews conducted in 19 countries, found that not only is food microbiology testing growing but that the sampling practices and technologies used also vary in the different geographical regions.

The two main types of testing can be split into routine microbiology, which tests for indicators of contamination in food plants and finished products, and pathogen testing, which looks for specific pathogenic organisms known to cause foodborne illness.

In its research, SCI found that test volumes were similar in North America (NA), Europe (EU) and Asia, with routine microbiology accounting for the major volume – 76% of test volume in NA, 81% in the EU and 72% in Asia. But there were major differences in the microbiological methods used for analysis of food safety tests.

Uptake of rapid methods is likely to rise around the world as methods become more affordable, easier to use and acceptable to the regulators

For routine testing, NA uses more easy-to-use ‘convenience methods’ (such as Petrifilm count plates), which account for 52% of all routine testing. The EU uses more culture-based methods (63% of routine test analysis). For pathogen testing, NA also is highly orientated towards rapid methods, with 94% of tests conducted using molecular and antibody-based methods while the EU still relies heavily on traditional or convenience culture methods for pathogen tests (61% of tests analysed). Asia relies most heavily on culture methods, for both routine and pathogen testing.

Uptake of rapid methods is likely to rise around the world, however, as methods become more affordable, easier to use and acceptable to the regulators.

Testing in the food sector focuses mainly on pathogens such as Salmonella, Listeria and Escherichia coli. In some cases it is important to distinguish between relatively harmless strains and more pathogenic versions. When dealing with Salmonella it is usually enough to detect the genus with a presence or absence test; with Listeria, it is the species L.monocytogenes that is important and with E.coli, it is the serotype or sub-variety within the species that is key.

STEC has become a key area of concern to the European food industry

Campden BRI, one of the UK’s leading food research organisations, has been working on the identification of E.coli strains. As Campden’s Microbiological Analytical Services Manager, Julie Archer, explains, most harmful strains responsible for haemorrhagic diarrhoea and Haemolytic Uraemic Syndrome are known as Shiga toxin-producing E.coli (STEC) or Verocytotoxin-producing E.coli (VTEC).

STEC were first identified as foodborne pathogens in 1982, following outbreaks associated with E.coli O157:H7. In May 2011, a novel strain of STEC, E.coli O104:H4, caused a serious outbreak of foodborne illness focused in northern Germany. It affected some 4,000 people with, for some, fatal consequences; this outbreak was associated with imported Fenugreek seeds. Since this outbreak, STEC has become a key area of concern to the European food industry.

Another high risk food for STEC contamination is raw meat, especially raw ground meat. Each year numerous reports of STEC contamination of raw meat are noted in Europe through Rapid Alert System for Food and Feed (RASFF).

EU legislation now requires the analysis of sprouted seeds for six STEC serotypes (O157, O145, O111, O103, O26 and O104:H4) prior to release onto the market. There is also new draft guidance on actions to take if foods are found to contain STEC; this guidance includes (in ready-to-eat foods and some raw meat products) recall of all of the products concerned.

Because STEC is highly pathogenic, testing labs require an enhanced level of containment to handle these organisms

Because STEC is highly pathogenic, testing labs require an enhanced level of containment to handle these organisms. There are few facilities in the UK able to undertake STEC testing, of which Campden BRI is one. As part of a member-funded research programme, the company has spent two years developing, optimising and further validating a polymerase chain reaction (PCR) method of testing for STEC types.

PCR provides a means of synthesising multiple copies of (i.e. amplifying) a specific piece of DNA. The regions of DNA targeted by this assay are Shiga-toxin producing genes (stx1 and/or stx2) and the attachment intimin gene (eae). An advantage of PCR testing is the ability to detect low numbers (2–40 colony forming units per 25g). The specific nature of the method means that when it is performed properly, it gives a high level of confidence in the results, says Campden BRI.

New EU draft guidance covers what actions to take if foods are found to contain STEC

PCR is useful as a detection method where defined DNA targets are used to detect the pathogen of interest. Pulsed field gel electrophoresis, meanwhile, is considered to be the gold standard method for typing, especially in investigating foodborne outbreaks of pathogens, such as Salmonella. More recently, DNA sequencing is being used for identification of unknown pure cultures, and it has also been developed to differentiate between isolates of the same pathogen species for tracking and typing.

DNA-based tests are more sensitive and accurate, allowing closely-related strains to be distinguished from one another

Thermo Fisher Scientific has announced a collaboration with the US Department of Agriculture’s Agricultural Research Service (USDA-ARS) and Pennsylvania State University (Penn State) on the development of rapid methods for detecting the bacterium E.coli in meat products. The extensive collections of E.coli strains held by USDA-ARS and Penn State will be made available to Thermo Fisher for genetic analysis. The company’s scientists will sequence around 200 strains using Life Technologies’ Ion Personal Genome Machine and then use this information to develop rapid DNA-based kits for detecting E.coli in food and identifying specific strains. These commercial kits will be developed on multiple analytical platforms, employing either next generation sequencing or capillary electrophoresis-based sequencing.

Traditionally, the food industry has used serological tests to type bacterial strains, based on detection of surface markers, but DNA-based tests are more sensitive and accurate, allowing closely-related strains to be distinguished from one another, according to Thermo Fisher.

‘Molecular technologies for rapid detection, typing, identification and characterisation of pathogens represent a paradigm shift for the food industry,’ says Pina Fratamico, a research leader with USDA-ARS. ‘The current collaboration will lead us to a much higher resolution understanding of E.coli genetics, as well as highly specific and sensitive methods for detecting pathogenic strains.’

Complex food samples

Testing complex food samples has always been more difficult than testing pure cultures. US-based PathoGenetix is using genome sequence scanning (GSS) technology to confirm and strain type pathogenic E.coli and Listeria bacteria directly from complex food samples, and to reliably determine if the most dangerous serotypes of these foodborne pathogens are present.

The developer of automated systems for rapid bacterial identification recently presented research at the American Society for Microbiology General Meeting, in Boston, demonstrating the ability of its GSS technology to confirm and strain type pathogenic bacteria. The research shows how GSS could be used to identify foodborne pathogens, with one study identifying STEC serotypes and another study not only differentiating L.monocytogenes among the general Listeria species, but even identifying its strains. The company says its GSS technology will be commercially available by early 2015 for use in the food industry.

Because the GSS technology scans microbial DNA directly from a mixed culture, and does not require a pure culture isolate, it can identify multiple bacteria strain types in one sample, and greatly reduce the time, complexity and skill level required to confirm the presence of dangerous pathogens. The level of discrimination provided by GSS is comparable to pulsed field gel electrophoresis (PFGE), says the company, and could enable quicker decisions affecting food safety and public health.

Alternative technologies

Such advanced techniques come at a high cost. PCR, for example, is expensive partly because of the thermocycling required for the amplification step, which in turn requires accurate and precise temperature control throughout the cycle; PCR also involves expensive fluorescent probes. Alternative solutions for DNA amplification under isothermal conditions without the need for a thermocycler have been developed. 3M Food Safety’s Molecular Detection System, for example, integrates two innovative technologies – isothermal DNA amplification and bioluminescence detection – to provide a reliable, rapid qualitative method of pathogen detection.

Alternative solutions for DNA amplification under isothermal conditions without the need for a thermocycler have been developed

With the 3M Molecular Detection System, the amplification and detection processes are completed within 75 minutes with real-time positive results available as early as 15 minutes. However, an overnight single enrichment step is still required at present. Test kits for E.coli O157 including H7, Salmonella and Listeria spp. detection in food and environmental samples are currently available for use with the system.

Life Technologies’ Pathatrix, meanwhile, is a high volume recirculating immuno magnetic-capture system for the detection of pathogens in food and environmental samples, which allows cost-effective screening of large numbers of food samples for specific pathogens such as E.coli O157, Salmonella or L.monocytogenes. It is a microbial detection system that allows for the entire sample to be analysed. It will selectively bind and purify the target organism from a comprehensive range of complex food matrices (including raw ground beef, chocolate, peanut butter and leafy greens).

Pathatrix allows the entire pre-enriched sample or large pooled samples to be recirculated over antibody-coated paramagnetic beads. It can specifically isolate pathogens directly from food samples and in conjunction with quantitative PCR can provide results within hours.

Faster sample preparation

Developing faster methods for separating microbial contamination from food, and ultimately faster detection, is something that a US-based research team at the University of Massachusetts Amherst is looking at. Lead food scientist Sam Nugen, fellow food scientist Amanda Kinchla, doctoral student Juhong Chen and nanochemist Vincent Rotello, are developing a technique to separate bacteria from a food sample in minutes, so that existing technologies for testing a clean sample can be used.

The researchers have created beads made of magnetically-charged cobalt nanoparticles coupled with microbe-specific phages or viruses, each of which binds to a specific, disease-causing bacteria such as Listeria, E.coli or Salmonella. The magnetic virus-bacteria beads can then be quickly removed from liquid samples with a magnet.

Nugen says: ‘The cobalt nanoparticle beads bind to the phages much more strongly than antibodies, and cobalt is very strongly magnetic so the method works quickly, much faster than other magnetic nanoparticles such as iron, for example. Using the iron in the retrieval process might take overnight, but the cobalt works in 30 seconds.’

So far the researchers have shown that their methods work in non-food liquids and they are now about to test actual food samples.

Nugen and colleagues are also working on a method to detect microbes in dry food samples, such as cereals and grains, by sampling air sucked from spaces between individual bits as they pass by on a conveyor belt. Particles collected this way will be pulled into a ‘wetted cyclone’ filter from which a clean sample can be prepared for microbial testing.

Interestingly, each of the specific viruses the researchers introduce to target one the ‘big three’ harmful microbes continues to multiply inside the bacteria, killing them all in 25 minutes in the case of E.coli. Nugen says: ‘If I develop a technique for detecting the signature of a replicated virus that had been growing in E.coli, I can also tell whether there ever was E.coli in that sample.’

He adds: ‘This study is all about separation but we’re setting it up so that this strategy of rapid detection can be an end product of our research.’

New methods for sampling in food factories

A new device designed to sample and detect foodborne bacteria is being trialled by scientists at the University of Southampton. The Biolisme project – started in 2009 by a consortium of six partners from four countries funded by the EU – is using research from the University to develop a sensor capable of collecting and detecting L.monocytogenes on food industry surfaces, to prevent contaminated products from entering the market. L.monocytogenes has the highest hospitalisation (92%) and death rate (18%) among all foodborne pathogens.

Current lab cultivation techniques to detect the bacteria take days, but the new device aims to collect and detect the pathogen on location within 3–4 hours.

Traditional methods of testing, where sample cells are cultivated in labs, are also flawed

Traditional methods of testing, where sample cells are cultivated in labs, are also flawed, as ‘stressed’ cells will not grow in cultures and can therefore produce negative results despite the bacteria being present and potentially harmful. Alternative techniques, based on molecular methods, will detect all cell types, but don’t differentiate between live and harmless dead cells, which can remain after disinfection.

The new device is designed to sample single cells and biofilms. Compressed air and water is used to remove the cells before they are introduced to an antibody. If L.monocytogenes is present, cells react with the antibody to produce a florescent signal, detected by a special camera. Doctor Salomé Gião and Professor Bill Keevil from Southampton’s Centre for Biological Science Unit have been studying L.monocytogenes biofilms under different conditions and will be testing the new prototype.

Dr Gião says: ‘The scientific research we have carried out at the University of Southampton has been used by our Biolisme project partners to develop a device which will have major implications for the food industry. By making the process simpler we hope that testing will be conducted more frequently, thereby reducing the chance of infected food having to be recalled or making its way to the consumer.’

The prototype sensor has been finalised in France and field trials are now underway to test the device before it is demonstrated in food factories.

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