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Genomics Gaining Favor for Food Safety Applications

By Andrea Anderson

NEW YORK (GenomeWeb News) – As part of their ongoing effort to curtail foodborne infections, academic researchers and public health agencies are increasingly exploring the use of genomics-based approaches to complement existing food safety and surveillance methods.

The microbes that can make their way into the food supply range from innocuous to very dangerous to humans. Common foodborne infection culprits include E. coli 0157:H7, Salmonella, Listeria, and Campylobacter, though other bacteria and even some viruses can cause foodborne illness. As such, having effective ways to distinguish between these bugs — combined with strategies for tracking and controlling potential pathogens — is crucial for food safety.

Indeed, researchers and public health agencies have developed methods both for testing certain foods prior to consumption and for hunting down contamination sources when foodborne illness does occur.

Although existing classification methods have proven useful for these purposes, the prospect of getting even more information about possible pathogens, their sources, and relationships to one another has some experts exploring genomics methods — ranging from SNP genotyping to whole-genome sequencing — to further improve food safety.

"If you can assess [a food bacteria isolate's] relationship to the other isolates that you've characterized in more detail, then you can make accurate predictions about the biology and the risk associated with that particular unknown strain," Todd Ward, a research geneticist with the US Department of Agriculture's Agricultural Research Service, told GenomeWeb Daily News.

One of the most widely used methods for distinguishing between potential foodborne pathogens is pulse-field gel electrophoresis, which involves using restriction enzymes to chop up bacterial DNA and comparing patterns formed when these fragments move through a gel in response to pulsed electrical field signals.

The PFGE method has been standardized by networks such as PulseNet and PulseNet Canada, allowing for consistent PFGE comparisons between labs.

"For the majority of foodborne pathogens, pulse field gel electrophoresis — or PFGE — is still the gold standard sub-typing method," Matthew Gilmour, a researcher with the Public Health Agency of Canada's National Microbiology Laboratory who is also affiliated with the University of Manitoba, told GWDN.

Researchers use PFGE both for traceback — finding the source of contamination during an outbreak involving foodborne pathogens — and in routine surveillance aimed at keeping dangerous bugs out of the food supply.

The USDA's Food Service and Inspection Service, for instance, processes more than 80,000 samples per year — testing for the presence of pathogens such as Escherichia coli, Salmonella, and Listeria.

"Our programs are intended to verify that the plants are doing a good job of controlling for pathogens," USDA-FSIS Senior Microbiologist Peter Evans told GWDN.

For its surveillance programs, he noted, USDA-FSIS strives to use methods that support — and are consistent with — those employed by the US Centers for Disease Control and Prevention and state and local health departments. That, in turn, makes it possible to compare isolates in the food system with those found in sick individuals if outbreaks do occur.

For current surveillance purposes, methods such as PFGE and multi-locus variable nucleotide repeat analysis (MLVA) are usually quite effective, Evans said, though he explained that SNP-typing assays may be incorporated in the future.

To that end, he explained, regulatory agencies in the US have been working to develop SNP-based approaches for distinguishing foodborne pathogens that might pose public health risks.

"The idea of sequencing the entire genome every time you have an isolate doesn't make a lot of sense because most positions will be the same," he explained. "In contrast, focusing on a specific set of SNPs of interest could be useful."

At the USDA-ARS, for example, Ward is collaborating with the USDA-FSIS on projects that might help inform future risk-based surveillance strategies and with the CDC to develop SNP-based assays for augmenting PFGE data during traceback and epidemiological studies.

"I think that's the general trend," Ward said. "These DNA sequence-based approaches will eventually either supplement or supplant pulse-field."

In the case of Listeria, for instance, Ward explained that PFGE has tremendous power for differentiating between strains but is less useful for assessing differences in risk posed by one strain compared to another — or for determining the biological cause of these differences.

In a paper appearing in the Journal of Food Protection in May, he and his colleagues reported on their findings from a multi-locus genotyping study of more than 500 Listeria monocytogenes isolates collected by the USDA-FSIS from a variety of ready-to-eat foods.

While MLGT alone is not usually considered as effective as PFGE for differentiating between strains, the team noted, they found that MLGT and sequencing of a virulence gene called inlA did provide additional information about the isolates.

"[I]ntegration of PFGE and DNA-sequence-based sub-typing provides an improved framework for prediction of relative risk associated with L. monocytogenes strains from [ready-to-eat] foods," they wrote.

And in another recent paper, Ward and collaborators from Colorado State University used genotyping to show that a virulence-decreasing inlA mutation in L. monocytogenes was more common in isolates from ready-to-eat than from isolates from actual human listeriosis cases.

"At this stage, we're still looking at targeting individual sites — many of them, all at once — but not whole genomes," Ward said.

SNP-typing is being used for distinguishing between Salmonella strains as well, particularly for serotypes that are difficult to characterize by PFGE. For example, Sequenom researcher Christiane Honisch and her colleagues are working on a Salmonella molecular typing system that relies on mass spectrometry-based nucleic acid analysis.

Honisch presented a poster outlining work done with collaborators from London's Health Protection Agency at the American Society for Microbiology annual meeting in San Diego this May, describing how the team used the Sequenom MassArray platform to do multi-locus sequence typing, or MLST, on hundreds of Salmonella isolates. Honisch told GWDN that the approach is promising, in part, because mass spec is high-throughput and generates very reproducible data.

A Variety of Approaches to Consider

Despite the range of approaches being developed and tested, though, experts say it's important to consider the sorts of information each method provides and ways of interpreting this data.

For instance, Ward said, researchers still have a fairly basic understanding of the relationships between certain strains and how subtle variations between strains relate to differences in how they persist in food processing environments.

Given such hurdles, Evans argued that it would be difficult to know what to do with all of the information provided by whole-genome sequencing in a surveillance setting, even if sequencing were cheaper or more readily available.

"For surveillance, I don't see whole-genome sequences coming for quite a while," he said. "I don't see how it's going to help us that much more than these other technologies that are very powerful."

Still, there are some who are already starting to use whole-genome sequencing to characterize outbreak strains.

During a session at the recent ASM meeting, Eric Brown, a microbiologist with the US Food and Drug Administration, explained that the FDA has been exploring the use of Roche 454 sequencing to characterize Salmonella isolates and to find markers for tracing outbreak strains back to their source.

And in Canada, the NML's Gilmour was lead author on a paper appearing in BMC Genomics this February in which researchers used the Roche 454 GS FLX platform to sequence the genomes of two L. monocytogenes strains isolated during a 2008 outbreak of listeriosis in Canada that killed 22 people and caused serious illness in dozens more.

"With pulse field gel electrophoresis or the other sub-typing methods like MLST or MLVA, you're capturing a fraction of the genetic content, whereas with whole genomes you're getting the entirety of it," Gilmour said. "There is tremendous value in getting that definitive characterization."

For instance, using the sequencing approach, he and his colleagues detected a 33,000 base pair prophage that explained subtle PFGE profile differences in L. monocytogenes isolates from the same serotype. They also found dozens of SNPs and indels that could be used to compare the 2008 outbreak strains with other Listeria isolates, including some from prior outbreaks.

"This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events," Gilmour and his co-authors wrote, "and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole-genome sequences in the context of epidemiology."

Even so, Gilmour said it will take time for whole-genome sequencing to become a standard traceback method — largely due to remaining bioinformatics challenges.

"It's our job to learn how to use those [sequencing] technologies and glean the interesting information or the informative information," Gilmour said. "That's kind of the bottleneck we're at right now, is developing those bioinformatics tools to take that raw data and quickly parse through it and find relevant information."

Moreover, Ward emphasized that each new approach will need to be appropriately assessed and standardized before becoming widely applicable for food safety.

"When you're looking at a situation as important as food safety — especially in a regulatory or a public health context — it's really about validating that the method is appropriate, is being run appropriately, and that the lab staff are trained appropriately," Ward said. "And all of that is going to take a fair amount of time to get a system up and running."