Tag Archives: Focus Article

Sasan Amini

NGS in Food Safety: Seeing What Was Never Before Possible

By Sasan Amini
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Sasan Amini

For the past year, Swedish food provider Dafgård has been using a single test to screen each batch of its food for allergens, missing ingredients, and even the unexpected – an unintended ingredient or pathogen. The company extracts DNA from food samples and sends it to a lab for end-to-end sequencing, processing, and analysis. Whether referring to a meatball at a European Ikea or a pre-made pizza at a local grocery store, Dafgård knows exactly what is in its food and can pinpoint potential trouble spots in its supply chains, immediately take steps to remedy issues, and predict future areas of concern.

The power behind the testing is next-generation sequencing (NGS). NGS platforms, like the one my company Clear Labs has developed, consist of the most modern parallel sequencers available in combination with advanced databases and technologies for rapid DNA analysis. These platforms have reduced the cost of DNA sequencing by orders of magnitude, putting the power to sequence genetic material in the hands of scientists and investigators across a range of research disciplines and industries. They have overtaken traditional, first-generation Sanger sequencing in clinical settings over the past several years and are now poised to supplement and likely replace PCR in food safety testing.

For Dafgård, one of the largest food providers in Europe, the switch to NGS has given it the ability to see what was previously impossible with PCR and other technologies. Although Dafgård still uses PCR in select cases, it has run thousands of NGS-based tests over the past year. One of the biggest improvements has been in understanding the supply chain for the spices in its prepared foods. Supply chains for spices can be long and can result in extra or missing ingredients, some of which can affect consumer health. With the NGS platform, Dafgård can pinpoint ingredients down to the original supplier, getting an unparalleled look into its raw ingredients.

Dafgård hopes to soon switch to an entirely NGS-based platform, which will put the company at the forefront of food safety. Embracing this new technology within the broader food industry has been a decade-long process, one that will accelerate in the coming years, with an increased emphasis on food transparency both among consumers and regulators globally.

Transitioning technology

A decade ago, very few people in food safety were talking about NGS technologies. A 2008 paper in Analytical and Bioanalytical Chemistry1 gave an outlook for food safety technology that included nanotechnology, while a 2009 story in Food Safety Magazine2 discussed spectrometric or laser-based diagnostic technologies. Around the same time, Nature magazine named NGS as its “method of the year” for 2007. A decade later, NGS is taking pathogen characterization and food authentication to the next level.

Over the last 30 years, multiple technology transitions have occurred to improve food safety. In the United States, for example, the Hazard Analysis and Critical Control Points (HACCP) came online in the mid-1990s to reduce illness-causing microbial pathogens on raw products. The move came just a few years after a massive outbreak of E. coli in the U.S. Pacific Northwest caused 400 illness and 4 deaths, and it was clear there was a need for change.

Before HACCP, food inspection was largely on the basis of sight, touch, and smell. It was time to take a more science-based approach to meat and poultry safety. This led to the use of PCR, among other technologies, to better measure and address pathogens in the food industry.

HACCP set the stage for modern-era food testing, and since then, efforts have only intensified to combat food-borne pathogens. In 2011, the Food Safety Modernization Act (FSMA) took effect, shifting the focus from responding to pathogens to preventing them. Data from 20153 showed a 30% drop in foodborne-related bacterial and parasitic infections from 2012 to 2014 compared to the same time period in 1996 to 1998.

But despite these vast improvements, work still remains: According to the CDC, foodborne pathogens in the Unites States alone cause 48 million illnesses and 3,000 fatalities every year. And every year, the food safety industry runs hundreds of millions of tests. These tests can mean the difference between potentially crippling business operations and a thriving business that customers trust. Food recalls cost an average of $10M per incident and jeopardize public health. The best way to stay ahead of the regulatory curve and to protect consumers is to take advantage of the new technological tools we now have at our disposal.

Reducing Errors

About 60% of food safety tests currently use rapid methods, while 40% use traditional culturing. Although highly accurate, culturing can take up to five days for results, while PCR and antigen-based tests can be quicker – -one to two days – but have much lower accuracy. So, what about NGS?

NGS platforms have a turnaround of only one day, and can get to a higher level of accuracy and specificity than other sequencing platforms. And unlike some PCR techniques that can only detect up to 5 targets on one sample at a time, the targets for NGS platforms are nearly unlimited, with up to 25 million reads per sample, with 200 or more samples processed at the same time. This results in a major difference in the amount of information yielded.

For PCR, very small segments of DNA are amplified to compare to potential pathogens. But with NGS tools, all the DNA is tested, cutting it into small fragments, with millions of sequences generated – giving many redundant data points for comparing the genome to potential pathogens. This allows for much deeper resolution to determine the exact strain of a pathogen.

Traditional techniques are also rife with false negatives and false positives. In 2015, a study from the American Proficiency Institute4 on about 18,000 testing results from 1999 to 2013 for Salmonella found false negative rates between 2% and 10% and false positive rates between 2% and 6%. Several Food Service Labs claim false positive rates of 5% to 50%.

False positives can create a resource-intensive burden on food companies. Reducing false negatives is important for public health as well as isolating and decontaminating the species within a facility. Research has shown that with robust data analytics and sample preparation, an NGS platform can bring false negative and positive rates down to close to zero for a pathogen test like Salmonella, Listeria, or E.coli.

Expecting the Unexpected

NGS platforms using targeted-amplicon sequencing, also called DNA “barcoding,” represent the next wave of genomic analysis techniques. These barcoding techniques enable companies to match samples against a particular pathogen, allergen, or ingredient. When deeper identification and characterization of a sample is needed, non-targeted whole genome sequencing (WGS) is the best option.

Using NGS for WGS is much more efficient than PCR, for example, at identifying new strains that enter a facility. Many food manufacturing plants have databases, created through WGS, of resident pathogens and standard decontamination steps to handle those resident pathogens. But what happens if something unknown enters the facility?

By looking at all the genomic information in a given sample and comparing it to the resident pathogen database, NGS can rapidly identify strains the facility might not have even known to look for. Indeed, the beauty of these technologies is that you come to expect to find the unexpected.

That may sound overwhelming – like opening Pandora’s box – but I see it as the opposite: NGS offers an unprecedented opportunity to protect against likely threats in food, create the highest quality private databases, and customize internal reporting based on top-of-the-line science and business practices. Knowledge is power, and NGS technologies puts that power directly in food companies’ hands. Brands that adopt NGS platforms can execute on decisions about what to test for more quickly and inexpensively – all the while providing their customers with the safest food possible.

Perhaps the best analogy for this advancement comes from Magnus Dafgård, owner and executive vice president at Gunnar Dafgård AB: “If you have poor eyesight and need glasses, you could be sitting at home surrounded by dirt and not even know it. Then when you get glasses, you will instantly see the dirt. So, do you throw away the glasses or get rid of the dirt?” NGS platforms provide the clarity to see and address problem directly, giving companies like Dafgård confidence that they are using the most modern, sophisticated food safety technologies available.

As NGS platforms continue to mature in the coming months and years, I look forward to participating in the next jump in food safety – ensuring a safe global food system.

Common Acronyms in Food Genomics and Safety

DNA Barcoding: These short, standardized DNA sequences can identify individual organisms, including those previously undescribed. Traditionally, these sequences can come from PCR or Sanger sequencing. With NGS, the barcoding can be developed in parallel and for all gene variants, producing a deeper level of specificity.

ELISA: Enzyme-linked immunosorbent assay. Developed in 1971, ELISA is a rapid substance detection method that can detect a specific protein, like an allergen, in a cell by binding antibody to a specific antigen and creating a color change. It is less effective in food testing for cooked products, in which the protein molecules may be broken down and the allergens thus no longer detectable.

FSMA: Food Safety Modernization Act. Passed in 2011 in the United States, FSMA requires comprehensive, science-based preventive controls across the food supply. Each section of the FSMA consists of specific procedures to prevent consumers from getting sick due to foodborne illness, such as a section to verify safety standards from foreign supply chains.

HACCP: Hazard analysis and critical control points. A food safety management system, HACCP is a preventative approach to quantifying and reducing risk in the food system. It was developed in the 1950s by the Pillsbury Company, the Natick Research Laboratories, and NASA, but did not become as widespread in its use until 1996, when the U.S. FDA passed a new pathogen reduction rule using HACCP across all meat and poultry raw products.

NGS: Next-generation sequencing. NGS is the most modern, parallel, high-throughput DNA sequencing available. It can sequence 200 to 300 samples at a time and generates up to 25 million reads per a single experiment. This level of information can identify pathogens at the strain level and can be used to perform WGS for samples with unknown pathogens or ingredients.

PCR: Polymerase chain reaction. First described in 1985, PCR is a technique to amplify a segment of DNA and generate copies of a DNA sequence. The DNA sequences generated from PCR must be compared to specific, known pathogens. While it can identify pathogens at the species level, PCR cannot provide the strain of a pathogen due to the limited amount of sequencing information generated.

WGS: Whole genome sequencing. WGS uses NGS platforms to look at the entire DNA of an organism. It is non-targeted, which means it is not necessary to know in advance what is being detected. In WGS, the entire genome is cut it into small regions, with adaptors attached to the fragments to sequence each piece in both directions. The generated sequences are then assembled into single long pieces of the whole genome. WGS produces sequences 30 times the size of the genome, providing redundancy that allows for a deeper analysis.

Citations

  1. Nugen, S. R., & Baeumner, A. J. (2008). Trends and opportunities in food pathogen detection. Analytical and Bioanalytical Chemistry, 391(2), 451-454. doi:10.1007/s00216-008-1886-2
  2. Philpott, C. (2009, April 01). A Summary Profile of Pathogen Detection Technologies. Retrieved September 08, 2017, from https://www.foodsafetymagazine.com/magazine-archive1/aprilmay-2009/a-summary-profile-of-pathogen-detection-technologies/?EMID
  3. Ray, L., Barrett, K., Spinelli, A., Huang, J., & Geissler, A. (2009). Foodborne Disease Active Surveillance Network, FoodNet 2015 Surveillance Report (pp. 1-26, Rep.). CDC. Retrieved September 8, 2017, from https://www.cdc.gov/foodnet/pdfs/FoodNet-Annual-Report-2015-508c.pdf.
  4.  Stombler, R. (2014). Salmonella Detection Rates Continue to Fail (Rep.). American Proficiency Institute.
Stanley Rutledge, Stop Foodborne Illness
Food Safety Culture Club

What’s the Point?

By Stanley Rutledge
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Stanley Rutledge, Stop Foodborne Illness

I’m surprised when I meet people who ask me, “What’s the point?”

What’s the point of…contacting people who’ve been impacted by foodborne illness? Sharing those peoples’ stories with industry?

Turns out, most of these people are speaking out of inexperience. For them, foodborne illness is a day or two spent home in bed, or the bathroom. They honestly aren’t aware that every year, for the families and friends of 3000 people*, foodborne illness is a destructive force much like the recent hurricane. It forces many people out of the lives they’re living into dire, and often extreme, situations where they’re required to rely on strangers and others for help. And before they reach a “new normal”—whatever that means—they face a myriad of physical, mental, financial, and social consequences. Unlike the hurricane, however, people who are victims of foodborne illness get no advance warning and are powerless to stop its effects, or even prepare for them.

At Stop Foodborne Illness we know the transforming power of story—of being able to recount an experience so powerful that it set you on a path different from where you started. For us, sharing those stories on an industry level is empowering for everyone involved. I’m always saying that everybody knows they need to wash their hands, but when that knowledge transitions from your head to your heart, then you have habits changing and behavior being modified.

Last month, a constituent from Wisconsin had the opportunity to share her story with about 120 employees of a fruit processing plant also located in Wisconsin. The following is an email we received afterwards that so clearly explains why we do what we do at Stop:

“You did an absolutely wonderful job. The impact on the group was exactly what I had hoped. Rest assured that you are making a difference by telling your story, and I know that was emotional and hard for you. Many people came up to me and said how different it makes them think of things now, having heard someone speak so close to home that almost died.

I can’t thank you enough.”

*The CDC estimates that every year in the United States, 3000 people die from foodborne disease, and that 128,000 people are hospitalized.

Results: FSMA IQ Test on Foreign Supplier Verification Program

By Food Safety Tech Staff
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The results are in! A couple of weeks ago, we asked readers to take a survey to test their knowledge of the Foreign Supplier Verification Program, and most passed with flying colors. If you didn’t take the test yet, you can view it here. The following are the results:

  1. You are only required for your foreign suppliers to meet your company requirements. FALSE
    • 89% answered correctly
  2. All required records for each applicable foreign-supplied product must be maintained for each shipment. TRUE
    • 93% answered correctly
  3. Your FSVP does not require that information for each lot of each product under the program be provided. FALSE
    • 84% answered correctly
  4. Just the location of the manufacturer of the product is required for the entity. FALSE
    • 90% answered correctly
  5. Foreign supplied shipments should include records to comprise the listing of all required information. TRUE
    • 91% answered correctly
  6. A qualified foreign supplier must have a Qualified Individual over the manufacturing of food product that is shipped to the United States. TRUE
    • 83% answered correctly
  7. A foreign supplier does not need to be registered under FDA requirements if the shipment of product is going to a registered facility in the United States. FALSE
    • 78% answered correctly
  8. A foreign supplier of food to the United States must ensure that all the requirements of a FSMA Food Safety Plan under cGMP117.126 be met for the manufacture of the food being exported to the United States. TRUE
    • 94% answered correctly
  9. The product information, including COA compliance, is not required for each lot of a product on a foreign-supplied shipment. FALSE
    • 83% answered correctly
  10. A food broker of foreign-supplied product to the United States does not have any responsibility of meeting the FSMA requirements. FALSE
    • 92% answered correctly
Dollar

Pathogens Drive More Than Half of $12 Billion Global Food Safety Testing Market

By Maria Fontanazza
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Dollar

The importance of food safety testing technologies continues to grow, as companies are increasingly testing their products for GMOs and pesticides, and pathogens and contamination. Last year the global food safety testing market had an estimated value of $12 billion, according to a recent report by Esticast Research & Consulting. Driven by pathogen testing technologies, the global food safety testing market is expected to experience a 7.4% CAGR from 2017–2024, hitting $21.4 billion in revenue in 2024, said Vishal Rawat, research analyst with Esticast.

With a CAGR of 9.3% from 2017–2024, rapid testing technologies are anticipated to lead the market. Testing methods responsible for this growth include immunoassays (ELISA), latex agglutination, impedance microbiology, immune-magnetic separation, and luminescence and gene probes linked to the polymerase chain reaction, said Rawat, who shared further insights about the firm’s market projections with Food Safety Tech.

Food Safety Tech: With the GMO food product testing market expected to experience the highest growth in the upcoming future, can you estimate the projected growth?

Vishal Rawat: The GMO food product testing market is estimated to generate a revenue of approximately $5.2 billion in 2016. The market segment is expected to witness a compound annual growth rate of 8.3% during the forecast period of 2017–2024. This is a global market estimation.

FST: What innovations are occurring in product testing?

Rawat: Nanomaterials and nanobased technologies are attracting interest for rapid pathogen testing. Sustainable technologies such as edible coatings or edible pathogen detection composition can attain a trend in the near future. Also, new rapid allergen testing kits are now emerging out as the latest food testing technology in the market, which are portable and easy to use.

FST: Which rapid pathogen detection testing technologies will experience the most growth from 2017–2024?

Rawat: New and emerging optical, nano-technological, spectroscopic and electrochemical technologies for pathogen detection, including label-free and high-throughput methods would experience the highest growth.

FST: What pathogen testing technologies are leading the way for meat and poultry in the United States?

Rawat: The presence of a microbial hazard, such as pathogenic bacteria or a microbial toxin, in ready-to-eat (RTE) meat or poultry products is one basis on which these products may be found adulterated. The FSIS is especially concerned with the presence of Listeria monocytogenes, Salmonella, Escherichia coli O157: H7, and staphylococcal enterotoxins in RTE meat and poultry products. Rapid pathogen testing for E. coli O157:H7 and Salmonella, for ground beef, steak and pork sausages is going to lead the U.S. market.

An overview of the report, “Food Safety Testing Market By Contaminant Tested (Pathogens, GMOs, Pesticides, Toxins), By Technology (Conventional, Rapid), Industry Trends, Estimation & Forecast, 2015– 2024” is available on Esticast’s website.

Gregory Siragusa, Eurofins
Food Genomics

GenomeTrakr: What Do You Know and What Should You Know?

By Gregory Siragusa, Douglas Marshall, Ph.D.
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Gregory Siragusa, Eurofins

This month we are happy to welcome our guest co-authors and interviewees Eric Brown, Ph.D. and Marc Allard, Ph.D. of CFSAN as we explore the FDA’s GenomeTrakr program in a two-part Food Genomics column. Many of our readers have heard of GenomeTrakr, but are likely to have several questions regarding its core purpose and how it will impact food producers and processors in the United States and globally. In Part I we explore some technical aspects of the topic followed by Part II dealing with practical questions.

Part I: The basics of GenomeTrakr

Greg Siragusa/Doug Marshall: Thank you Dr. Allard and Dr. Brown for joining us in our monthly series, Food Genomics, to inform our readers about GenomeTrakr. Will you begin by telling us about yourselves and your team?

Eric Brown/Marc Allard: Hello, I am Eric, the director of the Division of Microbiology at the U.S. Food and Drug Administration at the Center for Food Safety and Applied Nutrition. Our team is made up of two branches, one that specializes in developing and validating methods for getting foodborne pathogens out of many different food matrices and the other branch conducts numerous tests to subtype and characterized foodborne pathogens. The GenomeTrakr program is in the subtyping branch as Whole Genome Sequencing (WGS) is the ultimate genomic subtyping tool for characterizing a foodborne pathogen at the DNA level.

Hello, my name is Marc, I am a senior biomedical research services officer and a senior advisor in Eric’s division. We are part of the group that conceived, evaluated and deployed the GenomeTrakr database and network.

Siragusa/Marshall: Drs. Allard and Brown, imagine yourself with a group of food safety professionals ranging from vice president for food safety to director, manager and technologists. Would you please give us the ‘elevator speech’ on GenomeTrakr?

Brown/Allard: GenomeTrakr is the first of its kind distributed network for rapidly characterizing bacterial foodborne pathogens using whole genome sequences (WGS). This genomic data can help FDA with many applications, including trace-back to determine the root cause of an outbreak as well providing one work-flow for rapidly characterizing all of the pathogens for which the agency has responsibility. These same methods are also very helpful for antimicrobial resistance monitoring and characterization.

Siragusa/Marshall: From the FDA website, GenomeTrakr is described as “a distributed network of labs to utilize whole genome sequencing for pathogen identification.” We of course have very time-proven methods of microbial identification and subtyping, so why do we need GenomeTrakr for identification and subtyping of microorganisms?

Brown/Allard: If all you want to know is species identification then you are correct, there are existing methods to do this. For some applications you need full characterization through subtyping (i.e., Below the level of species to the actual strain) with WGS. WGS of pathogens provides all of the genetic information about an organism as well as any mobile elements such as phages and plasmids that may be associated with these foodborne pathogens. The GenomeTrakr network and database compiles a large amount of new genetic or DNA sequence data to more fully characterize foodborne pathogens.

GenomeTrakr and WGS are a means to track bacteria based on knowing the sequence of all DNA that comprises that specific bacterium’s genome. It can be called the “ultimate identifier” in that it will show relationships at a very deep level of accuracy.

Siragusa/Marshall: Is it an accurate statement that GenomeTrakr can be considered the new iteration of PulseNet and Pulse field gel electrophoresis (PFGE)? Will PulseNet and PFGE disappear, or will PulseNet and GenomeTrkr merge into a single entity?

Brown/Allard: PulseNet is a network of public health labs run by the CDC, with USDA and FDA as active participants. The network is alive and well and will continue subtyping pathogens for public health. The current and historical subtyping tool used by PulseNet for more than 20 years is PFGE. It is expected that CDC, USDA and FDA’s PFGE data collection will be replaced by WGS data and methods. That transformation has already begun. GenomeTrakr is a network of public health labs run by the FDA to support FDA public health and regulatory activities using WGS methods. Starting in 2012, this network is relatively new and is focused currently on using WGS for trace back to support outbreak investigations and FDA regulatory actions. CDC PulseNet has used WGS data on Listeria and collects draft genomes (i.e., unfinished versions of a final genome are used for quicker assembly) of other foodborne pathogens as well, and USDA’s FSIS has used WGS for the pathogens found on the foods that they regulate. All of the data from GenomeTrakr and Pulsenet are shared at the NCBI Pathogen Detection website (see Figure 1).

Sequences, GenomeTrakr
Figure 1

Siragusa/Marshall: Does an organism have to be classified to the species level before submitting to GenomeTrakr?

Brown/Allard: Yes, species-level identification is part of the minimal metadata (all of the descriptors related to a sample such as geographic origin, lot number, sources, ingredients etc.) required to deposit data in the GenomeTrakr database. This allows initial QA/QC metrics to determine if the new genome is labeled properly.

Siragusa/Marshall: After an isolate is identified to the species level, would you describe to the reader what the basic process is going from an isolated and speciated bacterial colony on an agar plate to a usable whole genome sequence deposited in the GenomeTrakr database?

Brown/Allard: The FDA has a branch of scientists who specialize in ways to isolate foodborne pathogens from food. The detailed methods used ultimately end up in the Bacteriological Analytical Manual (BAM) of approved and validated methods. Once a pathogen is in pure culture then DNA is extracted from the bacterial cells. The DNA is then put into a DNA sequencing library, which modifies the DNA to properly attach and run sequencing reactions depending on the specific sequencing vendor used. The sequence data is downloaded from the sequencing equipment and then uploaded to the National Center for Biotechnology Information (NCBI) Pathogen Detection website. The database is publicly open to allow anyone with foodborne pathogens to upload their data and compare their sequences to what is available in the database.

Siragusa/Marshall: Suppose a specific sequence type of a foodborne bacterial pathogen is found and identified from a processing plant but that the plant has never had a positive assay result for that pathogen in any of its history of product production and ultimate consumption. If an outbreak occurred somewhere in the world and that same specific sequence type were identified as the causative agent, would a company be in anyway liable? Could one even make an association between the two isolates with the same sequence type isolated at great distances from open another?

Brown/Allard: The genetic evidence from WGS supports the hypothesis that the two isolates shared a recent common ancestor. If, for example, the isolate from the processing plant and the outbreak sample where genetically identical across the entire genome, the prediction is that the two samples are connected in some way that is currently not understood. The genetic matches guide the FDA and help point investigations to study the possible connections. This might include additional inspection of the processing plant as well as linking this to the typical epidemiological exposure data. Sometimes due to the indirect nature of how pathogens circulate through the farm to fork continuum and the complex methods of trade, no connection is made. More commonly, these investigative leads from genetic matches help the FDA establish direct links between the two bacterial isolates through a shared ingredient, shared processing, distribution or packaging process. The genetic information and cluster helps the FDA discover new ways that the pathogens are moving from farm to fork. We are unaware of any example where identical genomes somehow independently arose and were unrelated. This is counter to molecular evolutionary theory anyway. Genetic identity equals genetic relatedness and the closer two isolates are genetically to each other, the more recent that they shared a common ancestor. With regard to liability, this is a topic beyond the scope of our group, but genomic data does not by itself prove a direct linkage and that is why additional investigations must follow any close matches.

Siragusa/Marshall: We know that SNPs (Single Nucleotide Polymorphisms or single base pair differences in the same location in a genome) are commonly used to distinguish clonality of bacteria with highly similar genomes. Are there criteria used by GenomeTrakr bioinformaticists that are set to help define what is similar, different or the same?

Brown/Allard: As the database grows with more examples of diverse serotypes or kinds of foodborne pathogens, the FDA WGS group is observing common patterns that can be used as guidance to define what is same or different. For example, closely related for Salmonella and E. coli are usually in the five or fewer SNPs, and closely related for Listeria is 20 or fewer SNPs using the current FDA validated bioinformatics pipeline. These values are not set in stone but should be considered more like guidance for what FDA and GenomeTrakr have observed already from earlier case studies that have already been collected and examined. Often, a greater number (e.g., 21-50) of SNP differences have been observed between strains isolated in some outbreaks. Any close match might support or direct an outbreak investigation if there is evidence that suggests that a particular outbreak looks most closely like an early case from a specific geographic location. WGS data helps investigators focus their efforts toward and international verses domestic exposure or possible country of origin. Even more divergent WGS linkages, when SNPs are greater than 50-100, often connect to different foods or different geographic locations that would lead investigators away from the source of an outbreak as the data provides both inclusivity as well as exclusivity.

When two strains have more than 50–100 SNPs, different food or geographic sources of those strains can be incorrectly linked resulting in investigators pursuing an incorrect source.

Siragusa/Marshall: Can SNPs be identified from different agar-plate clones of the same strain (i.e., Different colonies on the same plate)?

Brown/Allard: Since understanding the natural genetic variation present in foodborne pathogens is the basis to understanding relatedness, the FDA conducted validation experiments on growing then sequencing colonies from the same plate, colonies from frozen inocula, thawing and plating, as well as running the same DNAs on different instruments and with different sequencing technicians. The FDA’s work with Salmonella enterica Montevideo sequencing as well as ongoing proficiency testing among laboratories shows that the same isolate most often has no differences, although some samples have 1-2 SNP differences. Genetic differences observed in isolates collected by FDA inspectors all related to a common outbreak generally have more genetic differences, and this appears to be dependent on the nature of the facility and the length of time that the foodborne pathogen has been resident in the facility and the selective pressure to which the pathogen was exposed to in a range from 0–5 SNPs different.

Siragusa/Marshall: Regarding the use of WGS to track strains in a particular processing plant, is it possible that within that closed microenvironment that strains will evolve sufficiently so that it becomes unique to that source?

Brown/Allard: Yes, we have discovered multiple examples of strains that have evolved in a unique way that they appear to be specific to that source. Hospitals use the same practice to understand hospital-acquired infections and the routes of transmission within a hospitals intensive care unit or surgery. Food industry laboratories as well as FDA investigators could use WGS data in a similar way to determine the root cause of the contamination by combining WGS data with inspection and surveillance. The FDA Office of Compliance uses WGS as one piece of evidence to ask the question: Have we seen this pathogen before?

Siragusa/Marshall: The number of sequences in the GenomeTrakr database is approaching 120,000 (~4,000 per month are added). Are the sequences in the GenomeTrakr database all generated by GenomeTrakr Network labs?

Brown/Allard: The sequences labeled as GenomeTrakr isolates at the NCBI biosample and bioproject databases are the WGS efforts supported by the U.S. FDA and USDA FSIS. GenomeTrakr is a label identifying the FDA, USDA FSIS and collaborative partner’s efforts to sequence food and environmental isolates. Additional laboratories, independent and beyond formal membership in the GT network, upload WGS data to the NCBI pathogen detection website of which GenomeTrakr is one part. CDC shares WGS data on primarily clinical PulseNet isolates and USDA FSIS shares WGS foodborne pathogens for foods that they regulate. Numerous international public health laboratories also upload WGS data to NCBI. The NCBI pathogen detection website includes all publicly released WGS data for the species that they are analyzing, and this might include additional research or public health data. The point of contact for who submitted the data is listed in the biosample data sheet, an example of which can be seen here.

Siragusa/Marshall: Once sequences are deposited into the GenomeTrakr database, are they also part of GenBank?

Brown/Allard: The majority of the GenomeTrakr database is part of the NCBI SRA (sequence read archive) database, which is a less finished version of the data in GenBank. GenBank data is assembled and annotated, which takes more time and analysis to complete. Once automated software is optimized and validated, NCBI likely will place all of the GenomeTrakr data into GenBank. Currently, only the published WGS data from GenomeTrakr is available in GenBank. All of the GenomeTrakr data is available in SRA both at GenomeTrakr bioprojects and in the NCBI pathogen detection website.

Readers, look for the Part II of this column where we continue our exploration with Drs. Brown and Allard and ask some general questions about the logistics surrounding GenomeTrakr. As always, please contact either Greg Siragusa or Doug Marshall with comments, questions or ideas for future Food Genomics columns.

About the Interviewees

Marc W. Allard, Ph.D.

Marc Allard, FDAMarc Allard, Ph.D. is a senior biomedical research services officer specializing in both phylogenetic analysis as well as the biochemical laboratory methods that generate the genetic information in the GenomeTrakr database, which is part of the NCBI Pathogen Detection website. Allard joined the Division of Microbiology in FDA’s Office of Regulatory Science in 2008 where he uses Whole Genome Sequencing of foodborne pathogens to identify and characterize outbreaks of bacterial strains, particularly Salmonella, E. coli, and Listeria. He obtained a B.A. from the University of Vermont, an M.S. from Texas A&M University and his Ph.D. in biology in from Harvard University. Allard was the Louis Weintraub Associate Professor of Biology at George Washington University for 14 years from 1994 to 2008. He is a Fellow of the American Academy of Microbiology.

Eric W. Brown, Ph.D.

Eric Brown, FDAEric W. Brown, Ph.D. currently serves as director of the Division of Microbiology in the Office of Regulatory Science. He oversees a group of 50 researchers and support scientists engaged in a multi-parameter research program to develop and apply microbiological and molecular genetic strategies for detecting, identifying, and differentiating bacterial foodborne pathogens such as Salmonella and shiga-toxin producing E. coli. Brown received his Ph.D. in microbial genetics from The Genetics Program in the Department of Biological Sciences at The George Washington University. He has conducted research in microbial evolution and microbial ecology as a research fellow in the National Cancer Institute, the U.S. Department of Agriculture, and as a tenure-track Professor of Microbiology at Loyola University of Chicago. Brown came to the Food and Drug Administration in 1999 and has since carried out numerous experiments relating to the detection, identification, and discrimination of foodborne pathogens.

Thomas Tarantelli
In the Food Lab

Lead Found in Recalled Ground Cumin

By Thomas Tarantelli
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Thomas Tarantelli

Laboratory reports recently acquired by the Freedom of Information Law from the New York State Department of Agriculture and Markets show the Sol Andino brand ground cumin to contain 1090 ppm lead as well as 259 ppm chromium. The spice was also analyzed by IS:2446, 1980 method, “Detection of Lead Chromate in Chillies, Curry Powder and Turmeric by diphenyl carbizide.” A positive result was given, indicating the presence of hexavalent chromium, which is a component of lead chromate. Lead chromate is a yellow pigment, not allowed in food anywhere in the world as it is toxic, containing both lead and hexavalent chromium. The New York State Department of Agriculture and Markets posted a Class I recall of the Sol Andino ground cumin on July 10, 2017, without mention of the extremely high concentration of lead in the product.

Sol Andino, ground cumin
Sol Andino ground cumin recalled

The author could find no record of an FDA recall for the Sol Andino brand cumin powder containing excessive lead.

Some of us remember the four FDA Class I recalls of Pran brand turmeric for excessive lead in October 2013. These recalls were initiated by the New York State Health Department due to an illness complaint—most likely a child with high blood lead levels. The recalled Pran brand turmeric contained 28–53 ppm lead.

Also worthy of mention is the FDA/Illinois Class I recall of Nabelsi brand Thyme (actually a spice mix containing Thyme) on March 17, 2017.

“There have been two cases of high blood levels of lead associated with this product to date. Both cases have been reported through the Illinois Department of Public Health, Environmental Health Protection.”

According to the recall, the “Thyme” was found to contain 422 ppm lead.

Wondering if the 422 ppm lead was caused by adulteration of the “Thyme” with lead chromate or another lead pigment, a food chemist at the New York State Food Laboratory (a Division of NYS Dept. of Agriculture and Markets) requested from Illinois a sub-sample of the “Thyme” for analysis. Lab analysis of the spice found 323 ppm lead, 109 ppm chromium and a positive result for the chromate test. Thus, this recalled “Thyme” contains lead chromate.

In both cases, Pran turmeric and Nabelsi Thyme, illness complaints led to the recall of lead adulterated spices.

The New York State Department of Agriculture and Markets has a proactive program. Random samples of spices are sampled from retail markets and subsequently analyzed for unallowed colorants, undeclared allergens and heavy metals. In 2016 this resulted in the Oriental Packing Class I recall of 377,000 lb. of turmeric containing spices for excessive lead. (A typo in the FDA recall attributes the recall to the New York State Health Department, instead of the New York State Dept. of Agriculture and Markets.)

Still, it’s even better to analyze spices being imported into the country at receiving warehouses before the product reaches retail markets. Lead concentrations above 10 ppm can be determined instantaneously with a handheld XRF analyzer.

Adulteration with Sudan Dye Has Triggered Several Spice Recalls

Food Safety Tech
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3 Ways to Ensure Food Safety for Packaged Foods

By Erica Montes
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Food Safety Tech

Food safety and hygiene are very important aspects of food production, processing and consumption. In the absence of proper hygiene and safety protocols, the entire food chain right from the farmer who grows the food till the consumer who eats it is compromised. Food safety lapses like contamination and spoiling of food pose major health risks.

There are many ways in which a perfectly safe food product can turn hazardous. Cross contamination from animal matter, lack of hygiene among workers in processing plants, poor sanitation procedures, inadequate preservation techniques and low-quality packaging can all adversely affect the shelf life of a food product. Raw food spoils much faster than processed food, so fresh vegetables and fruits used in food processing must be washed properly and stored at optimal temperatures before they are processed.

The following are a few critical factors that affect the safety, shelf life and hygiene of food products.

1. Hygiene in Processing Plants

Personal hygiene and excellent sanitation policies are essential to maintaining food safety. Processing facilities potentially have several points of food contact equipment and food contact surfaces. There must be well developed and written standard cleaning practices or sanitation procedures for all such high-touch areas in a food processing plant. All equipment, vessels and surfaces must be monitored for bioburden or presence of microbial matter.

The workers must also be aware of good personal hygiene practices. This will help prevent cross contamination and possible spread of foodborne diseases from humans. Workers suffering from contagious diseases should refrain from coming to work and regular employee health checkups must be carried out by doctors. All staff must be trained in food and personal hygiene, and strictly follow recommended methods of hand washing and drying. Proper usage of hygiene gear including masks, caps, gloves, overalls and footwear must be ensured.

Floors, walls, drainage facilities, narrow cat-walks and all surfaces in the processing area must be cleaned thoroughly using high quality cleaning materials. The standard cleaning practices must be diligently met each time and the supervisors should ensure that the crew is doing their job properly. Quality and consistent employee training, and effective instant monitoring methods like ATP testing will help achieve these goals.

2. Good Packaging Is Crucial

The quality and suitability of packaging are also very important in determining the safety, longevity and hygiene of food products.

Evolving consumer habits, growth of online marketplaces, increased consumption of high-protein foods, popular demand for smaller portions due to shrinking family size and the rise in new global distribution channels have all impacted packaging requirements.

Sustainable and responsibly sourced packaging materials are the hallmark of advanced packaging technology. They are environmentally friendly and do not deplete natural resources. Clean label packaging focuses on using recycled materials, high-pressure packaging technology, digital packaging and 3-D printing techniques, and outsourcing of more activities to save money, time and resources.

The need for reducing food waste has been an important objective of all recent packaging innovations. According to a recent report by The Guardian, almost half of all U.S. food produce is thrown away. Global food waste can be reduced by extending the shelf life of packaged foods, thereby avoiding early disposal and excessive purchasing. Latest innovations include in-built freshness sensors in packaging that alert customers when food goes bad, vacuum skin innovations, barrier bags and modified-atmosphere packaging.

3. Consumer Awareness Is Key

The end user or the customer who buys the food product for consumption also needs to be aware of good food use, preparation and storage methods.

Fresh veggies and fruits should be washed thoroughly, chopped, diced, and sliced, and stored in clear, airtight containers in the fridge. Prepare and cook raw foods like fish, poultry and meat to extend their storage life. Cooked food can be safely frozen for a long time. In addition, many food items like casseroles, soups, sauces, stir-fries and baked foods stay safe for cooking and consumption even beyond their typically assumed use-by date.

As responsible consumers, we must be aware of the difference between use-by, sell-by, best-before and expiration dates. This will prevent us from throwing away a whole lot of perfectly edible food items from our pantries.

Conclusion

Food safety is a matter of global concern and affects the well being of billions of people all over the world. Ensuring safety, hygiene, freshness and long shelf life of food items will help reduce food waste, hunger and starvation in the world. It will also reduce the burden on limited natural resources and will help ensure a sustainable and efficient food chain.

Question mark

FSMA IQ Test Part I: Foreign Supplier Verification Program

By Food Safety Tech Staff
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The FSMA Foreign Supplier Verification Program (FSVP) has many elements that must be met. Do you know the correct response to these questions?

Kestrel ManagementWorking with Bill Bremer, principal of food safety compliance at Kestrel Management, LLC, Food Safety Tech is continuing its FSMA IQ test series. Results will be posted monthly in our Food Safety Consortium newsletter leading up to the 2017 event.

Confirm your company’s responsibility in meeting FSMA FSVP compliance by answering True or False.

Erin Mann, Food Protection and Defense Institute
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Targeting Agent Detection with Horizon Scanning of Food System Disruptions

By Erin Mann, MPH
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Erin Mann, Food Protection and Defense Institute

Agent detection to identify contamination of food products is required in food safety and defense programs. Detection typically involves laboratory methods or technologies, such as biosensors, that are used in close physical contact with food products. While the field of food protection has benefited from the development of novel agent detection methods in recent years, the challenge of determining which food products to test remains. The sheer volume of food produced within and traded across U.S. borders makes agent detection a daunting, time-consuming and expensive task. The decision of when to utilize detection methods depends on the risk of a particular product being contaminated. Contamination may be unintentional or intentional, including economically motivated adulteration (EMA).

The risk of contamination fluctuates over time and is a function of several factors. Risk depends on the biochemical makeup of the product, supply chain characteristics such as complexity and transport distance, and a wide range of natural or manmade events that may disrupt supply and potentially incentivize intentional adulteration. This is particularly true in the case of EMA. Events include but are not limited to natural disasters that destroy or reduce the usual supply of an ingredient, political instability that disrupts usual trade patterns, interruptions of routine food safety inspections, and market fluctuations that impact global prices. While data exists to monitor these risk factors of contamination, optimal use of this information by government and private industry is hindered by several challenges. For example, valuable data often exists across multiple data systems with data across systems appearing in inconsistent formats. In addition, the amount of data that must be reviewed to find a signal within the noise is frequently overwhelming.

Erin Mann, Food Protection and Defense Institute
Read our recent Q&A with Erin Mann, “As Food Fraud Grows, More Comprehensive Tools Emerge”

To address finding signals within vast quantities of data sources and systems, the Food Protection and Defense Institute (FPDI) developed technology to curate and help make sense of this data. With support from both the FDA and the Department of Homeland Security, FPDI developed FIDES or Focused Integration of Data for Early Signals to perform “horizon scanning” of food system disruptions in support of food protection efforts, including agent detection. FIDES was designed to help users forecast, monitor and identify food system risk factors and adverse food events. The FIDES web application fuses multiple streams of data from disparate sources and displays information in the form of an online dashboard where users browse, search and layer both dynamic and reference data sets related to food system disruption events. Examples of data currently included in FIDES are import refusals, global disasters, animal health alerts, food defense incidents, historical food safety incidents, import data, price alerts and reference data on food production worldwide.

Events in recent years illustrate the value of gathering intelligence and utilizing data related to food system risks to inform decisions regarding product targeting. Tsunamis, crop failures and disease outbreaks in humans and animals around the globe have threatened supply of products such as shrimp, spices, cocoa and eggs. When supply is disrupted, companies are often forced to quickly identify new and sometimes previously unvetted suppliers, including spot market purchasing. Likewise, supply disruptions often lead to price increases. As prices increase in the absence of adequate supply, concerns about EMA also increase. In both of these instances, the risk of product contamination—both unintentional and intentional—may rise and an increase in product screening or a change in agent detection methods may be appropriate.

For example, the 2014–2016 Ebola outbreak had a significant impact on West Africa, the primary production region for the world’s cocoa supply. Disruptions from the outbreak, including border closures and other trade interference, led to uncertainty about supply availability and prices. This raised concern for EMA, particularly given that many cocoa products are sold as powders, butters and liquors— forms that are more vulnerable to EMA than raw ingredients. As a test case, FPDI reviewed FIDES data streams during the peak of the outbreak. Real-time data on the outbreak was layered with data on global cocoa production and import patterns. Import refusal data from multiple global systems was assessed to identify any concerning patterns. Historical food defense and food safety incidents were also reviewed to determine which cocoa products had been previously contaminated. A similar approach could be used by the food and agriculture sector to guide decisions about targeted inspections—which product(s) and region(s) to monitor, which method(s) to use and which contaminant(s) to test. FIDES could support targeted screening and enhanced awareness of product risk profile that would allow the food industry to assure continued supply of authentic and quality products.

Michael Taylor FDA

Food Safety Over Past 25 Years: ‘Everything Has Changed’

By Maria Fontanazza
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Michael Taylor FDA

The effect that the 1993 E. coli O157:H7 outbreak had on the food industry was tremendous. Responsible for more than 600 illnesses and the deaths of four children, the outbreak led to significant changes in the industry’s approach to food safety. “[It] drove a shift in food safety that many had been working toward for years,” said Rima Khabbaz, M.D., acting deputy director for infectious diseases at CDC during the “We Were There” CDC lecture series, adding that the focus moved to food suppliers and how they could make their products safer. “The outbreak drove a paradigm shift that opened the door to food safety,” said Patricia Griffin, M.D., chief of the CDC’s enteric diseases epidemiology branch during the lecture.

Deirdre Schlunegger and Michael Taylor
Deirdre Schlunegger, CEO of Stop Foodborne Illness, and Michael Taylor at Stop event celebrating Food Safety Heroes during the 2015 Food Safety Consortium.

Within a few years, several actions and initiatives paved the way for notable progress. In 1994, Mike Taylor, who was administrator of USDA’s FSIS at the time, made a speech that “shocked and outraged the industry,” said Griffin, where he stated, “we consider raw ground beef that is contaminated with E. coli O157:H7 to be adulterated within the meaning of the Federal Meat Inspection Act.” From there, the USDA worked on the first major advance in meat regulation. In 1996 the agency established the Pathogen Reduction Rule to improve meat inspection. The same year CDC’s PulseNet was born, the nationwide lab network that uses DNA fingerprinting to help identify outbreaks early, along with the Foodborne Diseases Active Surveillance Network (FoodNet), an epidemiological system that tracks incidents and trends related to food.

In a Q&A with Food Safety Tech, Mike Taylor, most recently the former FDA commissioner for foods and veterinary medicine, discusses the dramatic change that industry has undergone during the past 25 years, from FSMA to technology advancements to food safety culture.

Food Safety Past, Present and Future at the 2017 Food Safety Consortium: Recognizing the 1993 Jack In the Box E. coli outbreak as the event that propelled the current food safety movement. Mike Taylor, Bill Marler, Esq. and Ann Marie McNamara (Target Corp.), who took the reins from the late David Theno at Jack In the Box, will discuss Theno’s impact on the industry. The session continues through a timeline of the evolution of food safety from 1993 to present, and then the future, where we will cover the IoT, social media, food safety culture and technology. It will be followed by the STOP Foodborne Illness Award Ceremony. Wednesday, November 29, 2017, 4:00–5:30 pm | LEARN MORE

Food Safety Tech: Reflecting on how far the industry has come since the E.coli O157:H7 outbreak involving Jack in the Box in 1993, what key areas of progress have been made since?

Michael Taylor: I think there are very major ones obviously. You have to remember where things were when the Jack-in-the-Box [outbreak] happened. We were in a place where USDA programs said it was not responsible for pathogens in raw meat and that consumers are supposed to cook the product; [and] industry was operating under traditional methods. Microbial methods were typically conducted for quality not for safety; you had the loss of public confidence and a terrible situation in which consumers were pointing at industry, and industry was pointing at consumers, and no one was taking clear responsibility for safety of the product.

Now we are in a completely different environment where not only is there clarity about industry’s responsibility for monitoring pathogens, there’s also been enormous progress by industry to put in place microbial testing, something David Theno pioneered and is now a central part of food safety management systems for meat safety.

Everything has changed.

These [institutional] arrangements exist not only in the meat industry, but now across the whole food industry. There’s the emergence of GFSI taking responsibility for managing the supply chain for food safety, food safety culture taking hold broadly across leading companies in the industry, and FSMA codifying for 80% of the food supply that FDA regulates the principles of risk-based prevention and continuous improvement on food safety.

I think it’s rather dramatic how far the industry’s food safety regulatory system has come since [the] Jack in the Box [outbreak].

FST: How has FSMA helped to align industry priorities?

Michael Taylor FDA
Mike Taylor was on the front lines of change in the meat industry.

Taylor: Let’s focus on the events first leading up to FSMA—for example, the outbreaks or illnesses associated with leafy greens [and] peanut butter, and problems with imported products—those events in the world aligned industry priorities around the need to modernize the food safety laws and to enact FSMA. It was the coming together of industry and consumer interests, and the expert community around the principles of comprehensive risk-based prevention that vaporized into FSMA. Now FSMA is the framework within which companies are organizing their food safety systems in accordance with these modern principles of prevention.

And clearly what’s been codified in FSMA and some of the key elements are becoming organizing principles where industry is aligning our priorities for food safety. Environmental monitoring where that’s an appropriate verification control for a company’s hygiene and pathogen control—that’s clearly a priority that folks are aligning on. The issue of supplier verification for domestic and foreign supply is a priority that has been elevated by FSMA, and so has the whole issue of training and employee capacity, whether it’s in processing facilities or on farms, as well as food safety culture. If you’re going to be effectively preventive you need to deal with the human dimension of your food safety system.

These are examples of ways in which FSMA is aligning industry priorities.

Read the rest of the interview on page 2 (link below).