Learn more about FSMA compliance at the 2017 Food Safety Consortium | November 28– December 1 | The 60-page draft guidance addresses the use of heat treatments as a process control, providing information on understanding potential hazards, design and validation of the heat treatment, establishing and implementing monitoring procedures (and how often), verification, and record keeping.
FDA states that it intends to publish at least 14 chapters of the guidance. In just two weeks, the compliance date for the preventive controls for human food rule falls for small businesses (fewer than 500 full-time employees).
Hygiena, LLC has launched an alkaline phosphatase (ALP) testing system that can verify pasteurization efficiency in short shelf life dairy products in five minutes. ZymoSnap ALP requires minimal equipment and can be used without special technical knowledge or testing facilities. It is also designed to provide repeat results at low levels (25–100 mU/L). Campden BRI independently validated the test.
“Manufacturers of dairy products are under constant pressure to demonstrate compliance with international safety and quality requirements. That’s why they need to regularly monitor and verify the efficiency of their pasteurisation process. With this in mind, we have developed ZymoSnap ALP to enable tests to be carried out rapidly allowing for an immediate pass/fail assessment and trend analysis. It has never been easier or quicker to ensure that pasteurisation processes are operating efficiently.” – Martin Easter, Ph.D., chief scientific officer, Hygiena
A 100% recyclable device, the ZymoSnap ALP Positive Control Kit provides a reference point for the regulatory limit of liquid milk and validation of other dairy products. It is compatible with the company’s EnSURE monitoring system, a luminometer that is used to detect indicators and bacteria, including coliform, E. coli and Enterobacteriaceae.
On Friday the USDA announced a large recall of 325,000 pounds of meat and poultry fat and lard products by Supreme Cuisine. The Class I recall is due to a processing deviation that could cause bacterial pathogens to grow and survive in the products. The duck, beef and pork fat and lard products, which have a one-year shelf life, were produced and packaged from June 1, 2016 through May 8, 2017.
The issue was uncovered after Supreme Cuisine received a consumer complaint of a loose lid. There have been no confirmed reports of adverse reactions due to consumption of the products, and consumers are being urged to discard any of these products.
Last year, nearly 550 food products were recalled in the United States. Nearly half of those recalls were a result of biological contamination, a whopping 65% of which was due to Listeria monocytogenes, according to Rentokil. The company recently released an infographic about the cost of a product recall, pulling out some of the key trends in food product recalls in the United States and the United Kingdom. Next to biological contamination, mislabeling continues to be a large issue.
By Gregory Siragusa, Douglas Marshall, Ph.D., Nur A. Hasan No Comments
Recall that in article one of this series we wrote that there are two main techniques to obtain a microbiome, a targeted (e.g., bacteria or fungi) or a metagenome (in which all DNA in a sample is sequenced, not just specific targets like bacteria or fungi). In this column we will now explore metagenomes and some applications to food safety and quality.
We have invited Dr. Nur Hasan of CosmosID, Inc., an expert in the field of microbial metagenomics, to share his deep knowledge of metagenomics. Our format will be an interview style.
Safe food production and preservation is a balancing act between food enzymes and microbes. We will start with some general questions about the microbial world, and then proceed deeper into why and how tools such as metagenomics are advancing our ability to explore this universe. Finally, we will ask Dr. Hasan how he sees all of this applying to food microbiology and safe food production.
Greg Siragusa/Doug Marshall: Thank you for joining us. Dr. Hasan, please give us a brief statement of your background and current position.
Nur Hasan: Thanks for having me. I am a molecular biologist by training. I did my bachelor and masters in microbiology, M.B.A in marketing, and Ph.D. in molecular biology. My current position is vice president and head of research and development at CosmosID, Inc., where I am leading the effort on developing the world’s largest curated genome databases and ultra rapid bioinformatics tools to build the most comprehensive, actionable and user-friendly metagenomic analysis platform for both pathogen detection and microbiome characterization.
Siragusa/Marshall: The slogan for CosmosID is “Exploring the Universe of Microbes”. What is your estimate of the numbers of bacterial genera and species that have not yet been cultured in the lab?
Hasan: Estimating the number of uncultured bacteria on earth is an ongoing challenge in biology. The widely accepted notion is more than 99% of bacteria from environmental samples remain ‘unculturable’ in the laboratory; however, with improvements in media design, adjustment of nutrient compositions and optimization of growth conditions based on the ecosystem these bacteria are naturally inhabiting, scientists are now able to grow more bacteria in the lab than we anticipated. Yet, our understanding is very scant on culturable species diversity across diverse ecosystems on earth. With more investigators using metagenomics tools, many ecosystems are being repeatedly sampled, with ever more microbial diversity revealed. Other ecosystems remain ignored, so we only have a skewed understanding of species diversity and what portion of such diversity is actually culturable. A report from Schloss & Handelsman highlighted the limitations of sampling and the fact that it is not possible to estimate the total number of bacterial species on Earth.1 Despite the limitation, they took a stab at the question and predicted minimum bacterial species richness to be 35,498. A more recent report by Hugenholtz estimated that there are currently 61 distinct bacterial phyla, of which 31 have no cultivable representatives.2 Currently NCBI has about 16,757 bacterial species listed, which represent less than 50% of minimum species richness as predicted by Schloss & Handelsman and only a fraction of all global species richness of about 107 to 109 estimated by Curtis and Dykhuizen.3,4
Siragusa/Marshall: In generic terms what exactly is a metagenome? Also, please explain the meaning of the terms “shotgun sequencing”, “shotgun metagenomes”, and “metagenomes”. How are they equivalent, similar or different?
Hasan: Metagenome is actually an umbrella term. It refers to the collection of genetic content of all organisms present in a given sample. It is studied by a method called metagenomics that involves direct sequencing of a heterogeneous population of DNA molecules from a biological sample all at once. Although in most applications, metagenome is often used to refer to microbial metagenome (the genes and genomes of microbial communities of given sample), in a broader sense, it actually represents total genetic makeup of a sample including genomes and gene sequences of other materials in the sample, such as nucleic acids contributed by other food ingredients of plant and animal origin. The metagenome provides an in-depth understanding of the composition, structure, functional and metabolic activities of food, agricultural and human communities.
Shotgun sequencing is a method where long strands of DNA (such as an entire genome of a bacterium) are randomly shredded (“shotgunning”) into smaller DNA fragments, so that they can be sequenced individually. Once sequenced, these small fragments are then assembled together into contigs by computer programs that find overlaps in the genetic code, and the complete sequence of the bacterial genome is generated. Now, instead of one genome, if you directly sequence entire assemblage of genomes from a metagenome using such shotgun approach, it’s called shotgun metagenomics and resulting output is termed a shotgun metagenome. By this method, you are literally sequencing thousands of genomes simultaneously from a given metagenome in one assay and get the opportunity to reconstruct individual genomes or genome fragments for investigation and comparison of the genetic consortia and taxonomic composition of complete communities and their predicted functions. Whereas targeted 16S rRNA or targeted 16S amplicon sequencing relies on amplification and sequencing of one target region, the 16S gene region, shotgun metagenomics is actually target free, it is aimed at sequencing entire genomes of every organism present in a sample and gives a more accurate, and unbiased biological representation of a sample. As an analogy of shotgun metagenomics, lets think about your library where you may have multiple books (like as different organisms present in a metagenome). You can imagine shotgun metagenomics as a process whereby all books from your library are shredded, mixed up, and then you will assemble the small shredded pieces to find text overlap and piecing the cover of all books together to reassemble each of your favorite books. Shotgun metagenomics approximates this analogy.
Metagenome and metagenomics are often used interchangeably. Where metagenome is the total collection of all genetic material from a given samples, metagenomics is the method to obtain a metagenome that utilizes a shotgun sequencing approach to sequence all these genetic material at once.
Shotgun sequencing and shotgun metagenomics are also used interchangeably. Shotgun sequencing is a technique where you fragment large DNA strands into small pieces and sequence all small fragments. Now, if you apply such techniques to sequence a metagenome, than we call it shotgun metagenomics.
Between 1996 and 2016, sprouts have been responsible to 46 outbreaks in the United States, which has led to nearly 2500 illnesses and three deaths, according to FDA. They have presented a consistent challenge to operators, because sprouts are most often produced in conditions that are ideal for bacteria growth.
Today FDA issued a draft guidance to assist sprout operators in complying with the FSMA Produce Rule, which requires “covered sprout operations take measures to prevent the introduction of dangerous microbes into seeds or beans used for sprouting, test spent sprout irrigation water (or, in some cases, in-process sprouts) for the presence of certain pathogens, test the growing, harvesting, packing and holding environment for the presence of the Listeria species or Listeria monocytogenes, and take corrective actions when needed.”
Large sprout operators must comply with the Produce Rule (applicable provisions) by January 26. Small business must comply by January 26, 2018 and very small businesses by January 28, 2019.
National Steak and Poultry has recalled about 1,976,089 pounds of ready-to-eat chicken products over concerns of bacterial pathogen survival in its products. According to FSIS, the product was adulterated due to “possible undercooking”. The expanded recall (the original recall included more than 17,000 pounds of product) was a result of a food service customer compliant to an establishment on November 28 that a product appeared to be undercooked. The products of concern were produced from August 20 through November 30, 2016.
FSIS has provided a complete list of the expanded recall products on its website. There have been no reports of adverse events due to consumption of the products, but consumers are being urged to discard or return the items.
Food production facilities are facing greater scrutiny from both the public and the government to provide safe foods. FSMA is being rolled out now, with new regulations in place for large corporations, and compliance deadlines for small businesses coming up quickly. Coverage of food recalls is growing in the era of social media. Large fines and legal prosecution for food safety issues is becoming more commonplace. Improved detection methods are finding more organisms than ever before. Technologies such as pulsed-field gel electrophoresis (PFGE) can be used to track organisms back to their source. PFGE essentially codes the DNA fingerprint of an organism. Using this technology, bacterial isolates can be recovered and compared between sick people, contaminated food, and the places where food is produced. Using the national laboratory network PulseNet, foodborne illness cases can be tracked back to the production facility or field where the contamination originated. With these newer technologies, it has been shown that some pathogens keep “coming back” to cause new outbreaks. In reality, it’s not that the same strain of microorganism came back, it’s that it was never fully eradicated from the facility in the first place. Advances in environmental monitoring and microbial sampling have brought to light the shortcomings of sanitation methods being used within the food industry. In order to keep up with the advances in environmental monitoring, sanitation programs must also evolve to mitigate the increased liability that FSMA is creating for food manufacturers.
Paul Lorcheim of ClorDiSys Solutions will be speaking on a panel of Listeria Detection & Control during the 2016 Food Safety Consortium, December 8 | LEARN MOREPersistent Bacteria
Bacteria and other microorganisms are able to survive long periods of time and become reintroduced to production facilities in a variety of ways. Sometimes construction or renovation within the facility causes contamination. In 2008, Malt-O-Meal recalled its unsweetened Puffed Rice and Puffed Wheat cereals after finding Salmonella Agona during routine testing of its production plant. Further testing confirmed that the Salmonella Agona found had the same PFGE pattern as an outbreak originating from the same facility 10 years earlier in 1998. This dormant period is one of the longest witnessed within the food industry. The Salmonella was found to be originating from the cement floor, which had been sealed over rather than fully eliminated. This strategy worked well until the contamination was forgotten and a renovation project required drilling into the floor. The construction agitated and released the pathogen back into the production area and eventually contaminated the cereal product. While accidental, the new food safety landscape looks to treat such recurring contaminations with harsher penalties.
One of the most discussed and documented cases of recurring contamination involves ConAgra’s Peter Pan peanut butter brand. In 2006 and 2007, batches of Peter Pan peanut butter produced in Sylvester, GA were contaminated with Salmonella and shipped out and sold to consumers nationwide. The resulting outbreak caused more than 700 reported cases of Salmonellosis with many more going unreported. Microbial sampling determined that the 2006 contamination resulted from the same strain of Salmonella Tennessee that was found in the plant and its finished product in 2004. While possible sources of the contamination were identified in 2004, the corrective actions were not all completed before the 2006–2007 outbreak occurred. Because of the circumstances surrounding the incomplete corrective actions, ConAgra was held liable for the contamination and outbreak. A settlement was reached in 2015, resulting in a guilty plea to charges of “the introduction into interstate commerce of adulterated food” and a $11.2 million penalty. The penalty included an $8 million criminal fine, which was the largest ever paid in a food safety case. While the problems at the Sylvester plant were more than just insufficient contamination control, the inability to fully eliminate Salmonella Tennessee from the facility after the 2004 outbreak directly led to the problems encountered in 2006 and beyond.
Many times, bacteria are able to survive simply because of limitations of the cleaning method utilized by the sanitation program. In order for any sanitation/decontamination method to work, every organism must be contacted by the chemical/agent, for the proper amount of time and at the correct concentration by an agent effective against that organism. Achieving those requirements is difficult for some sanitation methods and impossible for others. Common sanitation methods include steam, isopropyl alcohol, quaternary ammonium compounds, peracetic acids, bleach and ozone, all of which have a limited ability to reach all surfaces within a space, and some are incapable of killing all microorganisms.
Liquids, fogs and mists all have difficulty achieving an even distribution throughout the area, with surfaces closer or easier to reach (i.e., the top or front of an item), receiving a higher dosage than surfaces further away or in hard-to-reach areas. Such hard-to-reach areas for common sanitation methods include the bottom, back or insides of items and equipment that don’t receive a “direct hit” from the decontaminant. Liquids, fogs and mists land on and stick to surfaces, which makes it harder for them to reach locations outside the line of sight from where they are injected or sprayed. Hard-to-reach areas also include ceilings, the tops of overhead piping lines, HVAC vents, cooling coils and other surfaces that are located at greater heights than the liquids, fogs and mists can reach due to gravitational effects on the heavy liquid and vapor molecules.
Another common but extreme hard-to-reach area includes any cracks and crevices within a facility. Although crevices are to be avoided within production facilities (and should be repaired if found), it is impossible to guarantee that there are no cracks or crevices within the production area at all. Liquid disinfectants and sterilant methods deal with surface tension, which prevents them from reaching deep into cracks. Vapor, mist and fog particles tend to clump together due to strong hydrogen bonding between molecules, which often leave them too large to fit into crevices. Figure 1 shows bacteria found in a scratch in a stainless steel surface after it had been wiped down with a liquid sterilant. The liquid sterilant was unable to reach into the scratch and kill/remove the bacteria. The bacteria were protected by the crevice created by the scratch, giving them a safe harbor location where they could replicate and potentially exit in the future to contaminate product itself.
Processing equipment and machinery in general contain many hard-to-reach areas, which challenge the routine cleaning process. In sanitation, “hard to reach” is synonymous with “hard to clean”. Figure 2 shows processing equipment from an ice cream manufacturing facility. Processing equipment cannot be manufactured to eliminate all hard-to-clean areas. As such, even with all the sanitary design considerations possible, it is impossible to have equipment that does not contain any hard-to-clean areas. While sanitary design is essential, additional steps must be taken to further reduce the possibility of contamination and the risk that comes along with it. This means that in order to improve one’s contamination control and risk management programs, improvements must also be made to the sanitation program and the methods of cleaning and decontamination used.
Chlorine Dioxide Gas
Food safety attorney Shawn K. Stevens recently wrote that “given the risk created by the FDA’s war on pathogens, food companies should invest in technologies to better control pathogens in the food processing environments.”1 One method that is able to overcome the inherent difficulties of reaching all pathogens within a food processing environment is chlorine dioxide gas (ClO2 gas). ClO2 gas is a proven sterilant capable of eliminating all viruses, bacteria, fungi, and spores. As a true gas, ClO2 gas follows the natural gas laws, which state that it fills the space it is contained within evenly and completely. The chlorine dioxide molecule is smaller than the smallest viruses and bacteria. Combined, this means that ClO2 gas is able to contact all surfaces within a space and penetrate into cracks further than pathogens can, allowing for the complete decontamination of all microorganisms with the space. It also does not leave residues, making it safe for the treatment of food contact surfaces. It has been used to decontaminate a growing number of food facilities for both contamination response and contamination prevention in order to ensure sterility after renovations, equipment installations and routine plant shutdowns.
“If food companies do not take extraordinary measures to identify Lm in their facilities, perform a comprehensive investigation to find the root cause or source, and then destroy and eliminate it completely, the pathogen will likely persist and, over time, intermittently contaminate their finished products,” wrote Stevens.1 Environmental monitoring and sampling programs have been improved in terms of both technology and technique to better achieve the goal of identifying Lm or other pathogens within a food production environment. The FDA will be aggressive in its environmental monitoring and sampling under the food safety guidelines required by FSMA. Food production facilities will be closely monitored and tracked using PulseNet, with contaminated product being traced back to their source. Recurring contamination by a persistent pathogen will be viewed more severely. While there are many reasons that pathogens can persist within a food manufacturing environment, insufficient cleaning and decontamination is the most common. Traditional cleaning methods are incapable of reaching all surfaces and crevices within a space. In order to eliminate the risk of pathogens re-contaminating a facility, the pathogens need to be fully eliminated from their source and harbor locations. ClO2 gas is a method capable of delivering guaranteed elimination of all pathogens to maintain a pathogen-free environment. With the new era of food safety upon us, ensuring a clean food production environment is more important than ever, and ClO2 gas is uniquely situated to help reduce the risk and liability provided by both the government and the public.
In the summer of 2015, multiple ice cream manufacturers were affected by Listeria monocytogenes contamination. Part two of this article will detail one such company that utilized ClO2 gas to eliminate Listeria from its facility.
A new rapid assay may help growers make faster and more informed decisions right on the farm. Researchers from the University of Massachusetts and Cornell University are developing a test that addresses the challenge of sampling produce and assessing risk in a timely manner. The dipstick would enable rapid detection of Salmonella in agricultural samples in about three hours.
How It Works
“Users simply place a leaf sample in a small plastic bag that contains enzymes and incubate it for about 1.5 hours. Users would then squeeze a small liquid sample through a filter and place it in a tube with bacteriophages—viruses that are harmless to humans but infect specific bacterium, such as Salmonella or E. coli. Some phages are so specific they will only infect one bacterium serotype while others will infect a broader range of serotypes within an individual species. Phages also will only infect and replicate in viable bacteria, ensuring that non-viable organisms are not detected. This distinction is useful if prior mitigation steps, such as chlorination, have already been used. The phages used in the test were engineered to insert a particular gene into the bacteria.” – Center for Produce Safety
“We have been developing dipstick assays for ultra-low detection limits,” the technical abstract, Rapid bacterial testing for on-farm sampling, states. “Our preliminary data suggests that our fluorescent dipstick will have a detection limit of Salmonella spp. cells which makes the test ideal for on-farm use and appropriate federal requirements.”
How safe is a raw diet? Could sterilizing our food actually make us more prone to sickness? Are vegans healthier than carnivores? In the last few decades, global food poisoning scares from beef to peanut butter have kept food scientists and researchers around the world asking these questions and searching for improved methods of handling and testing what we eat.
It’s been more than 150 years since Louis Pasteur introduced the idea of germ theory—that bacteria cause sickness—fundamentally changing the way we think about what makes our food safe to eat. While we’ve advanced in so many other industrial practices, we’re still using pasteurization as the standard for the global food industry today.
Although pasteurization effectively controls most organisms and keeps the food supply largely safe, we continue to have foodborne outbreaks despite additional testing and more sophisticated techniques. The potential health promise of genomics, and the gut microbiome genetics and bacterial ecosystems, could be the key to the next frontier in food safety.
The scientific community is once again at the cusp of a new era with the advent of metagenomics and its application to food safety.
What is metagenomics? Metagenomics is the study of the bacterial community using genetics by examining the entire DNA content at once. Whole genome sequencing of a single bacterium tells us about the DNA of a specific organism, whereas metagenomic testing tells us about the interaction of all the DNA of all the organisms within a sample or an environment. Think of the vast quantity of genetic material in the soil of a rice patty, a lettuce leaf, your hand, a chicken ready for cooking, or milk directly from a cow. All of them have thousands of bacteria that live together in a complex community called the microbiome that may contain bacteria that are sometimes harmful to humans—and possibly also other bacteria that help to keep the potentially harmful bacteria in check.
Metagenomics uses laboratory methods to break up cells and extract many millions of DNA molecular fragment, and sequencing instruments to measure the sequences of A’s, C’s, G’s, and T’s that represent the genetic information in each of those fragments. Then scientists use computer programs to take the information from millions or billions of fragments to determine from what bacteria they came. The process is a little like mixing up many jigsaws, grabbing some pieces from the mix, and figuring out what was in the original pictures. The “pictures” are the genomes of bacteria, which in some cases carry enough unique information to associate a given bacterium with a previously seen colony of the same species.
Genomics of single bacterial cultures, each from a single species, is well established as a way to connect samples of contaminated foods with reported cases of foodborne illnesses. With metagenomics, which essentially looks for all known species simultaneously, one hopes to do a better job of early detection and prevention. For example, if a machine malfunction causes pasteurization or cleaning to be incomplete, the metagenomics measurement will likely show compositional shifts in which bacterial phyla are abundant. This can make it possible to take remedial action even before there are signs of pathogens or spoilage that would have led to a costly recall.
Up until now, keeping food safe has meant limiting the amount of harmful bacteria in the community. That means using standard methods such as pasteurization, irradiation, sterilization, salt and cooking. To determine whether food is actually safe to eat, we test for the presence of a handful of specific dangerous organisms, including Listeria, E. coli, and Salmonella, to name a few. But what about all the “good” bacteria that is killed along with the “bad” bacteria in the process of making our food safe?
Nutritionists, doctors and food scientists understand that the human gut is well equipped to thrive unless threatened by particularly dangerous contaminants. The ability to determine the entire genetic makeup within a food could mean being able to know with certainty whether it contains any unwanted or unknown microbial hazards. Metagenomic testing of the food supply would usher in an entirely new approach to food safety—one in which we could detect the presence of all microbes in food, including previously unknown dangers. It could even mean less food processing that leaves more of the healthful bacteria intact.
More than 150 years ago, Pasteur pointed us in the right direction. Now the world’s brightest scientific minds are primed to take the food industry the next leap toward a safer food supply.