FDA has created and applied in real time public health use, a U.S.-based open-source whole genome sequencing (WGS) integrated network of state, federal, and commercial partners. The network, known as “GenomeTrakr,” represents the first distributed genomic food shield for identifying and tracing foodborne outbreak pathogens back to their source. In only its third year, GenomeTrakr is already enhancing investigations of outbreaks of foodborne illnesses and compliance actions, enabling more accurate and rapid recalls of contaminated foods, and monitoring of preventive controls effectively in the food manufacturing environment. The resulting public genomic database of foodborne pathogens can support dramatically investigators ability to link specific food products, processing sites, and farms, providing valuable insight into the origin of the contaminated food. GenomeTrakr essentially creates a searchable, digital high-resolution fingerprint of the complete genetic make-up of individual pathogens, permitting otherwise indistinguishable bacteria to be easily separated and identified.
“This [database] is clearly the most powerful approach yet developed for tracking and tracing pathogens, and we expect it to have a very significant positive impact on food safety,” says Steven Musser, PhD, deputy director for scientific operations at FDA’s Center for Food Safety and Applied Nutrition (CFSAN). Considering the limited number of FDA food inspectors and the global nature of the food supply, the development and continual building (adding sequences and metadata) of GenomeTrakr is essential.
The food safety impacts of this network are impressive. Its current membership includes 30 public health, food safety, and academic laboratories from federal, state, and international stakeholders. The network has already shown great promise in enhancing the traceability of food and feed supply contamination events at the national level including Salmonella and Listeria monocytogenes. Moreover, WGS of microbial pathogens is now supplanting traditional microbiological analytics with rapid single data output summaries for antimicrobial resistance profiling, detection of high risk virulence profiles, and general identification strategies that supersede serological, phenotypic, and classical culture testing making the technology critical for an effective public health response to bacterial outbreaks.
An Important Role for WGS
Recent devastating outbreaks associated with consumption of fresh-cut produce have reinforced the notion that foodborne disease remains a substantial global challenge to public health. Mitigating foodborne illness, at times, seems an intractable challenge. One longstanding problem is the ability to rapidly identify the food associated with the outbreak being investigated. Despite the best efforts of food safety experts, the tools available for tracking foodborne outbreaks were sometimes too slow or uninformative to effectively pinpoint the source of the outbreaks. With the limitations of traditional subtyping methods, federal public health and food safety laboratories are exploiting WGS to delimit outbreak scope, traceback to point source, and make early predictions about important traits that a pathogen may harbor such as antimicrobial resistance. Highly parallel robotic genomic sequencers can sequence the DNA of a bacterial pathogen in a matter of hours. When coupled with validated, analytical bioinformatic pipelines such as the one established by FDA’s CFSAN, accurate and stable genetic changes can be identified that can distinguish foodborne outbreak strains down to the source level including specific farms, food types, and geographic regions.
Eric Brown, PhD, director of the division of microbiology at CFSAN, compared the application of WGS for delimiting foodborne outbreaks to the impact that the Hubble Space Telescope had on our understanding of galaxies. “Can you imagine how astronomers felt the day the Hubble sent back its first pictures of the universe? This is exactly how we felt in 2009 when we applied WGS for the first time to a Salmonella-induced foodborne outbreak.”
Numerous recent published examples illustrate the ability of WGS to discern high-resolution genetic relatedness of otherwise indistinguishable isolates. Proof of principle studies have been undertaken using the technology at numerous public health institutes both nationally and internationally. So much so, the success of WGS for rapid source tracking of pathogens is now well documented. In 2012, 425 individuals in the U.S. became sick from ingesting food that contained either Salmonella Bareilly or Salmonella Nchanga. Through traditional epidemiology methods, the illnesses were ultimately linked to a frozen raw yellowfin tuna product known as Nakaochi Scrape, which had been imported from India. (Nakaochi Scrape is tuna backmeat that is scraped from the bones of tuna and may be used in sushi, sashimi, ceviche, and other similar dishes.)
As part of the outbreak investigation FDA performed WGS on Salmonella isolated from product samples and from clinical samples to determine their DNA makeup. This data helped to more accurately determine which illnesses were part of the outbreak and which illnesses were similar but unrelated. However, FDA also did something else—a retrospective analysis. It performed WGS on about a dozen Salmonella Bareilly isolates stored in freezers from previous Salmonella Bareilly food contamination events. What FDA found was that the Salmonella Bareilly DNA for the samples tied to the 2012 outbreak was very similar to the Salmonella Bareilly DNA isolated from shrimp that came from a processing plant in southwest India several years earlier. In fact, the plant that processed the Nakaochi Scrape was only about five miles away from the plant that processed the shrimp. This observation was significant as it indicated that the paring of genomic information with geographic information might have the potential to be a powerful tool for traceback investigations. This event provided the impetus for creating the GenomeTrakr network and the increased use of genomic information in foodborne outbreak investigations.
As noted by Marc Allard, PhD, the genomics-area coordinator for CFSAN’s microbiology program, “Our freezers are virtually full of Salmonella, Listeria, and other enteric pathogens collected as part of FDA’s own inspection and sampling work. These collections represent a virtual treasure trove of genomic diversity and are invaluable as reference strains in an ever-expanding whole genome sequencing database.”
GenomeTrakr: The Basics
In late 2012, FDA launched its GenomeTrakr network, the first distributed WGS network focused on the development of a highly informative, metadata rich, and fully transparent WGS database of environmental and food-associated enteric pathogens. In short, this database was launched to enhance our ability to use WGS to track foodborne pathogens. The network has created a publicly accessible global database containing the genetic makeup of thousands of foodborne disease-causing bacteria. CFSAN and the National Center for Biotechnology Information (NCBI) at the National Institutes of Health (NIH) collaboratively developed the necessary database and associated software tools. Much of this development continues to make genomic analysis more fully accessible to end-users from federal, state, academic, and industry sectors. Many of the state labs also are members of the Food Emergency Response Network putting them directly into many investigations of food contamination events. Our goal is to further enhance the network by growing the database while also adding more partners from public health, clinical, and regulatory agencies around the country as well as internationally.
The GenomeTrakr was originally comprised of labs in FDA/CFSAN, nine FDA Office of Regulatory Affairs field labs, and public health or agricultural labs from four states including New York, Florida, Washington, and Arizona. Data curation and bioinformatics support was provided by NCBI at NIH. In 2013, GenomeTrakr added labs from Minnesota and Virginia, and in 2014, brought onboard labs in New Mexico, Maryland, and Texas, and another New York lab. In addition, the CDC, USDA’s Food Safety and Inspection Service, academic departments of veterinary science and agriculture, public health laboratories, and several other state-related groups have now acquired WGS technology and are actively collaborating with FDA in the sequencing of food and environmental isolates of Salmonella, Listeria monocytogenes, and Shiga toxin-producing E. coli. Most of these laboratories are equipped with Miseq desktop sequencers, and CFSAN provides technical support for wet-lab and bioinformatic methods and a web-based communication tool for real time data sharing. Currently, sequences are streamed from individual state laboratory Miseqs to a CFSAN computer where they are quality checked and formatted for upload to the GenomeTrakr database. CFSAN is working with NCBI and commercial software vendors to develop simpler tools that will allow individual laboratories to add sequences to the public database directly.
A Paradigm Shift on Two Fronts
Everyone who has seen the potential of WGS applied to food safety microbiology realizes that the technology brings huge paradigm shifts for how enteric pathogens will be tested and how they are tracked back to their source. The first paradigm shift includes using the increased resolution from WGS to intervene earlier in investigations. The second paradigm shift involves the new ability to link isolates across multiple years, whereby low-level contamination events can be linked across geographic time and space. However, there is a third paradigm shift and it relates to how information is shared. The GenomeTrakr database is public, meaning that anyone in the world can freely contribute and obtain information from it.
“People like to focus on the technology as the paradigm shift but, in my opinion, a really important advance is the open data-sharing model,” exclaims Ruth Timme, PhD, FDA senior scientist and GenomeTrakr network principal coordinator. Of course, this approach would not prevent some aspects of the metadata to be held by individual organizations because of concerns about public release of proprietary information. The open database also allows FDA to go beyond the development of a source-tracking scheme. Several additional applications and benefits of the technology include: readily available antimicrobial resistance profiling to 98 percent accuracy; serological characterization without a need for classical antibody testing; virulence pathogenicity assessment for emerging bacterial pathogens; and, of course, high resolution subtyping, which has been its most widespread application to date.
“WGS is good because it can dig down deeper and identify the specific isolate and tell investigators what area or producer it could have come from,” says Capt. Palmer Orlandi, PhD, senior science advisor in FDA’s Office of Foods and Veterinary Medicine and member of the Commissioned Corps of the U.S. Public Health Service. Sample collection and sequence cataloging from food production sites can help monitor compliance with FDA’s rules on safe food handling practices and enhance preventive controls for food safety.
Ultimately, sequencing capability should be distributed to as many sites as possible so that public health laboratories can move sequences from their collections and current surveillance and inspection activities into the database as quickly as possible. Dr. Allard emphasizes that “this public approach provides useful data to industry and academic partners, as well as to any federal or international agency that wishes to add value to the collected data.” The current GenomeTrakr database contains sequences from roughly 14,000 Salmonella isolates and more than 3,300 Listeria isolates, and is growing by more than 700 new draft genomes per month. New phylogenetic trees showing emerging linkages and relatedness are produced daily by NCBI and are publicly accessible.
Casting a Broad Net
The GenomeTrakr has already expanded and benefitted from other important WGS projects being carried out by public health experts in the U.S. and abroad. The CDC’s Real-Time Listeria monocytogenes WGS pilot, which is sequencing all clinical cases of L. monocytogenes reported by the states since the fall of 2013 to enhance surveillance, is an example. FDA and other GenomeTrakr sites are working with CDC by contributing genomes of all food and environmental L. monocytogenes to the database. The work is making great strides in public health officials’ efforts to delimit illness clusters and sources of contamination caused by this dangerous pathogen.
Errol Strain, PhD, CFSAN’s lead bioinformaticist, puts a finer point on the importance of the collaboration with CDC. “To be able to go beyond what we once thought was a typical Listeria outbreak and now detect the outlying and more subtle contamination events caused by this pathogen is hugely impactful to food safety and public health.” This real time collaboration has increased the number of Listeria outbreaks discovered and characterized, and has reduced the time to detection and increased regulatory activity for this pathogen in a significant way.
While tracking and tracing foodborne outbreaks is a primary application of the GenomeTrakr network, it is essential to note the broad important uses of such a database to food safety stakeholders. For instance, academic and environmental microbiology partners are using the database to accumulate broad amounts of genomic information on enteric pathogens that thrive in and around agricultural environments. Technology partners are mining these data for novel genetic targets to incorporate into assay design for improved pathogen detection systems, and industry partners are using the technology to mitigate safe food production and processing systems. Effective monitoring of supply chain ingredients means downstream cost and material savings for industry if they catch problems earlier and understand the root cause of the contamination event so that they can fix the problem and prevent it from happening in the future. Moreover, being able to distinguish between resident, facility contaminations versus a reintroduction of a pathogen strain from raw materials is a hugely beneficial application of the technology as the preventative solutions are different depending on where the contamination is coming from. Finally, the cost savings potential through monitoring with high certainty and with multi-analytes in one test cannot be overstated.
Through numerous earlier case studies gathered from 2009 to the present and now weekly regulatory decision making, it is clear that WGS is validated and reproducible. Moreover, WGS will be adopted globally as the new method for foodborne pathogen surveillance and characterization. To be universal and comprehensive, more states and countries need to be added to the database and there needs to be a harmonization of the different networks being built both nationally and internationally. More work still is needed for successful implementation of a global food shield including: increased funding for instrumentation and training; issues surrounding data and metadata release into the public domain; harmonization among different authorities with sometimes distinct mandates and conflicting missions; and finally issues regarding validating alternative informatics approaches to interpreting the data. None of these barriers are insurmountable, and many believe it is only a matter of time until we will see a global food shield.
The years 2014 and 2015 are watershed years for the use of WGS for food safety and public health in that many firsts were encountered as WGS left the research arena and entered into regular production and use across many state, federal, and international food safety agencies. Numerous successful applications of pathogen surveillance and characterization among academic, industry, and government partners has made WGS more prominent than ever and it has never been more apparent that the future lies with this technology. One can expect additional applications and greater impact as more partners join these efforts. These initial investments into the GenomeTrakr network, the new WGS technologies, and the hard work of many public health professionals are truly transforming the public health paradigm and these improvements will have long lasting benefits for the public and food safety.
Dr. Allard is a research microbiologist in the molecular methods and subtyping branch within the division of microbiology at the FDA’s Office of Regulatory Science. To reach him or to get in contact with the other authors, email firstname.lastname@example.org.
Genetic Resistance to Wheat Disease
A recent study co-authored by University of Nebraska-Lincoln (UNL) researchers has unearthed the genetic roots of resistance to a wheat disease that has recently devastated crop yields from southern Africa through the Middle East.
Though reports of stem rust date back to biblical plagues and ancient Greece, plant breeders successfully combated the disease by introducing rust-resistant cultivars in the mid-20th century. Stem rust epidemics largely faded until 1999, when a mutated strain—Ug99—emerged in the east African country of Uganda.
Ug99 and its recent variants have toppled nearly all previously resistant genes. The rare holdouts include Sr2, found in an especially hardy wheat variety named Gage that was co-released by the University of Nebraska and the USDA in 1963.
The study isolated and examined DNA sequences of Gage to ascertain why it enjoys greater resistance to stem rust, including Ug99, than other cultivars featuring the Sr2 gene. The authors concluded that Gage’s rust-resistance during adulthood likely owes to a combination of Sr2 and an additional gene, which the team believes also contributes to the wheat’s resistance in the seedling stage of its development.
The researchers have narrowed down the location and potential identity of this additional gene, which they said they hope to soon verify through further study.
“It so happens that the source of Sr2 that was used to create Gage—the variety Hope—actually had a number of other stem rust resistance genes in it,” reports P. Stephen Baenziger, PhD, a co-author and the Nebraska Wheat Growers Presidential Chair at UNL. “Our results would say that it looks like Gage got the lucky straw, so to speak, from Hope.”
Drawing a genetic map to that level of resistance could prove extremely valuable against Ug99.
“It’s important to understand the resistance to stem rust, because with the mutations that are coming out of Africa, we’re losing genes all the time,” says Dr. Baenziger. “But Sr2 is still resistant to it, and now that we can associate parts of the genome with the resistance, we’re making good progress.”—FQ&S