Food safety fingerprinting

by Joel Crews
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Food safety
 
Whole genome sequencing (WGS) has been around for many years, however it has evolved from a costly, time-consuming and labor-intensive technology that previously was not a practical option for food safety professionals. Its adoption and application by regulators and public health officials in the US has gained momentum in recent years and it is part of plans to create a global database to enhance food safety and traceability when foodborne illness outbreaks occur. The technology allows public health officials to determine the complete DNA sequence of individual bacterium, creating what is comparable to a fingerprint for each one.

While not a new technology, WGS has evolved over the years and more advanced equipment has been developed that has led to new generation sequencing, which facilitates more efficient, faster sample preparation that is less costly than in the past.

“It used to cost thousands of dollars to run one test and now we’re down to a hundred dollars,” says Michael Doyle, who recently retired as regents’ professor of microbiology and director of the Center for Food Safety at the Univ. of Georgia.

Expediency is another benefit both domestically and globally, according to Robert Tauxe, Ph.D., director of the division of foodborne, waterborne and environmental diseases at the Centers for Disease Control (CDC). “We are finding and solving more outbreaks, but finding them when they are smaller,” Tauxe said, during a panel discussion about WGS technology at the Global Food Safety Conference in Houston this year. “Whole genome sequencing is a major step forward in outbreak detection and investigation.”

Michael Doyle
Michael Doyle, Ph.D., says today's whole genome sequencing technology is facilitating the development of a cloud-based database to facilitate global tractability of foodborne illness outbreaks.
 

Agencies in aciton

The US Dept. of Agriculture (USDA), Food and Drug Administration (FDA) and the Centers for Disease Control (CDC) all collect samples from food manufacturers and, in the case of the CDC, from patients suffering from foodborne illnesses. The data from the samples are stored using cloud technology that is maintained by the National Institute of Health (NIH).

“The advantage of that is you can go back to it; it can be queried globally, it’s publicly available information,” Doyle says. “The beauty of that is that it gives us a global network so that if there is a match between certain strains that come up during an outbreak, sequences that are related to an outbreak in this country can be cross-referenced with the global database to see if there are other isolates of the same type of bacteria that had been submitted to the NIH database to establish a connection, if that were the case.”

In mid-2014, the USDA’s Food Safety and Inspection Service (FSIS) began sampling and using WGS for Salmonella and Listeria monocytogenes and later that year added Shiga toxin-producing E. coli (STEC). In 2015 Campylobacter was added. That same year it began to upload WGS data directly to the National Center for Biotechnology Information (NCBI) Genomic Database. The number of isolates sequenced each year by FSIS have gone from about 200 in 2015 to just under 2,000 in 2017. The WGS system used by the FSIS is made up of 12 sequencers and three FSIS labs.

In 2013, the CDC started a pilot project that used WGS to track Listeria. In 2018, the program will be expanded to identify Salmonella, Campylobacter and E. coli.

In 2012, the FDA launched the GenomeTrakr, which consists of a network of laboratories sequencing foodborne pathogens and uploading them to a public database.

As more labs are added to the public health network and the WGS-derived fingerprints of pathogens continue growing, the time required to identify outbreaks will continue to decrease and fewer cases will be required to confirm an outbreak. Building a database that is universally accessible and sharing data globally is vital in today’s food industry.

“We are certain that the public health benefit of WGS will only become more evident with every foodborne pathogen’s genomic sequence that is shared,” said a recent blog post on the FDA’s website. Steven Musser, Ph.D., deputy director for scientific operations in the Center for Food Safety and Applied Nutrition wrote, “Already, GenomeTrakr has collected more than 142,000 sequenced strains, has made them freely available to anyone in the world, and continues to demonstrate how a database of this kind is being used effectively for food safety within the United States, and throughout the world.”

Musser says, “As the food supply becomes increasingly global, the use of WGS in a way that crosses national borders will ultimately help keep us all safe from foodborne illness.”

According to Doyle, “How do you analyze the data to make it useful for PulseNet system,” was the puzzle researchers and public health officials grappled with in the past. PulseNet is used to investigate bacterial isolates from victims of foodborne illnesses and to collect environmental samples from processing plants. PulseNet is the food-safety and traceability system utilized by the CDC, FDA and USDA.

Establishing links between foodborne illnesses and the plant where the food was manufactured before WGS, was achieved using pulsed-field gel electrophoresis (PFGE), which has become somewhat obsolete. According to the CDC, PFGE as well as WGS, are laboratory techniques used by scientists to produce a DNA fingerprint for a bacterial isolate, a group of the same type of bacteria.

Both techniques are used, Doyle says, “to identify clusters of cases of foodborne illness that can be used to identify sources of outbreaks.” By 2018, CDC officials plan to do away with pulsed-field gel electrophoresis (the molecular subtyping technology used prior to the advent of WGS) for Listeria, E. coli, Salmonella and Campylobacter.

WGS technology can be conducted in different manners to achieve the same result: analyzing the sequence of the nucleotides in the genome of bacteria. In the early days of the technology this was made more challenging because accurately testing the entire genome wasn’t possible.

“But we’re there now,” Doyle says, and improvements continue.

As an example, CDC is utilizing a modified whole genome technique known as multilocus sequence typing (MLST). This technology allows CDC researchers to assign a number to a specific group of isolates and group them into clusters.

Another method of WGS is known as single nucleotide polymorphism (SNP, or “SNIP”). “A nucleotide is the backbone of a genome,” Doyle says, and WGS technology can detect when there is a different sequence between samples. “When they have less than a certain number of SNIP differences, they are identified as being similar or identical strains.” When that occurs, and there are a certain number of SNIPS that are associated with different isolates, researchers would identify these as being the same whole genome sequence, “and that’s kind of like a fingerprint,” Doyle says.

Food Safety
Data from positive samples taken at food manufacturing companies and from foodborne illness victims are cross referenced using a global database of what is the equivalent of a fingerprint for every pathogen. 
 

Culture collection

Regardless of the WGS technique, the goal is to both detect and traceback pathogens. “Right now, I’d say it’s largely used in public health for identifying outbreaks and being able to not just identify the outbreak, but even trace it back to its source.” Listeria was the initial model used by FDA, CDC and USDA to determine if WGS could be used to identify clusters of cases of foodborne illnesses that could be categorized as outbreaks. The technology facilitated faster detection of outbreaks while reducing the number of cases required to confirm the occurance of a foodborne illness outbreak.

Doyle says it is not sufficient to conclude that isolates from a patient that match the isolates in a food product or in a food plant establish the source of the illness outbreak. “You have to have epidemiology to connect the two.” There can be, for example, a strain of Listeria that was detected in food and there might be some cases in the PulseNet database that match it, but if the ill patients didn’t ingest the food in question, the food or food company cannot and should not be implicated.

This aspect has proven to be a point of contention. “If you don’t have a direct connection showing that they ate that food or were exposed to the source, whether it’s the food-processing plant or whatever, you can’t say that person became ill from exposure to that food,” Doyle says.

Today, FDA officials routinely go to food processing plants and conduct what Doyle says are known as “swab-a-thons” and take swab samples throughout the processing plant environment. If any of the swabs test positive a whole genome sequence is conducted, and that information is put into the growing cloud-based database. If there are cases or outbreaks in the future, that database is queried to find out if there is a match. If a match is found, further testing of the processing plant and testing of the food manufactured there can be done to determine if there is an epidemiologic link. “Their database is now able to guide them in terms of where they should start looking if there is an outbreak or several clusters of a certain fingerprint of a Listeria or a Salmonella, Campylobacter or an E. coli associated with human cases,” Doyle says.

In the public health realm, there are outbreak cases of foodborne illness and sporadic cases. Sporadic cases are those that cannot be connected or linked to an outbreak. Thanks to WGS technology and the FDA and FSIS maintaining and adding to the database of all cases, CDC officials have begun tracing sporadic cases and linking them to specific food plants. Solving the puzzle of sporadic cases also includes querying the people who became ill to find out if they were exposed to food from that processing plant. This facilitates connecting the outbreak and shortening the time needed to positively identify the source and take the appropriate action, which can include shutting down the plant.


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