SAN FRANCISCO (GenomeWeb) – In the summer of 2014, more than 100 people across 24 US states came down with a Salmonella enterica infection. While standard methods for identifying the cause of the outbreak — surveys of patients to see if they had any common exposures and pulsed field gel electrophoresis (PFGE) — identified contaminated chicken as a likely cause, it wasn't until researchers included whole-genome sequencing that they were able to be more confident in their conclusion and even pinpoint the specific company that had the contaminated chicken.
"Sequencing gives us a lot more precision" than PFGE, said Samuel Crowe, team leader for the National Outbreak Reporting System in the Enteric Diseases Epidemiology Branch at the US Centers for Disease Control and Prevention. "We had some support that it was contaminated chicken, but the whole-genome sequencing data gave us a lot more confidence," he added.
In general, the CDC and the US Food and Drug Administration are increasingly turning to next-generation sequencing to track foodborne pathogen outbreaks in the US. The agencies are implementing the technology in surveillance programs due to its unbiased nature and high resolution. Sequencing can help trace pathogens to their source, even in complex food networks, and researchers are building up databases of pathogen genomes and antimicrobial resistance genes to more quickly identify the causative agent and its resistance profile.
While sequencing may replace PFGE for outbreak tracking, it's unclear whether it will also eventually replace technologies that are used for specific testing. For instance, a number of companies currently market PCR-based products in the food testing space, including Thermo Fisher Scientific and BioMerieux. These tests are marketed primarily to companies that want to test food products for the presence of a specific pathogen, such as Salmonella in poultry. As such, they serve a different market than the sequencing-based surveillance studies that public health labs are performing.
Foodborne outbreaks that cross state borders typically rely on PulseNet, a network of more than 80 public health and food regulatory laboratories in the US that usually use PFGE to identify the responsible pathogen. At the time of the Salmonella outbreak that Crowe and his team described recently in the Journal of Food Protection, sequencing was just starting to be used, he said. Now though, the PulseNet network is increasingly using sequencing for multi-state outbreaks.
Crowe said laboratories first began adopting NGS for Listeria cases, followed by Salmonella, then shiga-toxin producing Escherichia coli. Next up will be Campylobacter.
The CDC has also pushed the use of NGS in foodborne disease monitoring under the Advanced Molecular Detection (AMD) Initiative and the National Antimicrobial Resistance Monitoring for Enteric Bacteria (NARMS) project, a collaboration between the CDC, FDA, and the US Department of Agriculture to track antibacterial resistance. Last year, a NARMS report included whole-genome sequence data for the first time.
Foodborne illnesses can be particularly tricky to trace, in part because of how global our food system has become. In addition, identifying the source of an outbreak associated with chicken is especially hard in the US because of how many Americans eat chicken. According to the study, around 65 percent of Americans eat chicken at home during a given week. Also, Salmonella contamination can occur not just in chicken but many other foods as well. In addition, symptoms may not immediately present, so public health officials have to rely on people's memories about what they ate.
In the recent study, PulseNet laboratories identified 146 Salmonella infections in 24 states between May and December of 2014. Following initial survey data about the foods that patients ate prior to becoming sick, epidemiologists began to narrow their focus on chicken, since 60 percent of patients said they had eaten chicken outside of their home, a significantly higher number than would be expected. Although a large proportion of patients also reported eating chicken and tomatoes in their homes, those numbers were not significantly higher than would be expected. Researchers also identified a sub-cluster of 34 patients who had all attended a catered birthday party, which enabled researchers to focus first on the restaurant that had prepared the chicken.
Researchers at the CDC and the USDA's Food Safety and Inspection Services performed whole-genome sequencing and phylogenetic analyses, which enabled them to connect the patients at the catered party with the other patients in the outbreak. In addition, the analysis helped them to unravel the complex food supply chain to trace the source of contamination to a large poultry company.
They sequenced isolates from a subset of 27 patients in different states, as well as 24 food or animal isolates collected from 10 different companies that slaughter or process chicken.
They then created a phylogenetic map from the sequence data. The researchers were able to trace back the chicken from the catered party as well as other cases whose isolate genomes closely matched those of the cases from the party to two related companies, one of which is a major national poultry producer. The researchers hypothesized that the chicken became contaminated at a processing facility or a slaughterhouse owned by that company.
For the other cases, which were not as closely related and traced back to smaller poultry producers, the researchers hypothesized that they were unrelated outbreaks.
Crowe said that since this study, the CDC has increasingly used NGS to identify the source of outbreaks. For instance, this past year he was involved in a study that used NGS to trace back an outbreak to contaminated flour. "The sequence evidence provided pivotal data that was needed in drawing that conclusion," he said. Relying on patients to recall exactly what they ate and being able to link sick individuals in different states is challenging, he said, so sequencing provides a good tool to help get around that.
Crowe said that while public health labs still use PFGE in outbreak investigations, and sequencing is used primarily in multi-state and complex situations to bolster the PFGE and other epidemiological data, he thinks that eventually, sequencing will replace PFGE. "It provides a lot more and clearer data," he said.
The CDC efforts don't happen in a silo — the FDA has increasingly been using sequencing in food safety applications, too. One main project has been its GenomeTrakr database where state public health laboratories upload whole-genome sequence data from foodborne pathogens. There are currently more than 113,000 foodborne pathogens in the database, according to Marc Allard, a microbiologist and research area coordinator for genomics at the FDA. The National Center for Biotechnology Information houses the GenomeTrakr data as well as clinical data from the PulseNet labs.
The FDA initially began the project in 2014. Since then, it has grown from working with four state labs to providing support for more than 20, Allard said, not including the CDC and USDA labs.
"There's a lot of effort going into harmonizing the data," he said, and "enabling states and labs to share the data in a timely way and in a similar way."
The GenomeTrakr database is currently growing at a rate of about 5,000 genomes per month. Researchers then work to build phylogenetic trees to connect the isolates, identify new isolates, and determine whether there is an outbreak.
If isolates are closely related, say zero to 1 SNP differences between them, they would be presumed to be connected in some way, Allard said. Investigators would then be notified.
This investigation process is not different from previous processes, Allard said, but what's changed is the addition of genomic data. "Outbreaks are often identified earlier because we're making the connections earlier," he said. That often leads to fewer people ultimately becoming infected and the contamination being removed from the food supply sooner.
Going forward, Allard said it would be important to extend collaborations internationally. Already, he said, Public Health England is contributing to the database. "The food we eat is globally distributed, so the things you buy could come from almost anywhere," he said. For example, there was a recall of apples in the US, but apples are shipped all over the world. When data from a patient in Australia was included, researchers realized that the event was part of the same contamination in the US and recalled the apples in Australia as well.
Aside from growing sequencing internationally, Allard thinks that laboratories at the county levels in certain locations may start adopting sequencing. There have been discussions with some labs in the major food-producing counties in California, for example, to start including genomic data to GenomeTrakr.