Tag Archives: microbiome

Nur Hasan, CosmosID
Food Genomics

Metagenomes and Their Utility

By Gregory Siragusa, Douglas Marshall, Ph.D., Nur A. Hasan
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Nur Hasan, CosmosID

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.

Go to page 2 of the interview below.

David Chambliss, IBM Research
In the Food Lab

Scientific Breakthrough May Change Food Safety Forever

By David Chambliss
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David Chambliss, IBM Research

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.

Metagenomics, Food Safety

Preventing Outbreaks a Matter of How, Not When

By Maria Fontanazza
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Metagenomics, Food Safety

When it comes to preventing foodborne illness, staying ahead of the game can be an elusive task. In light of the recent outbreaks affecting Chipotle (norovirus, Salmonella, E. coli) and Dole’s packaged salad (Listeria), having the ability to identify potentially deadly outbreaks before they begin (every time) would certainly be the holy grail of food safety.

One year ago IBM Research and Mars, Inc. embarked on a partnership with that very goal in mind. They established the Consortium for Sequencing the Food Supply Chain, which they’ve touted as “the largest-ever metagenomics study…sequencing the DNA and RNA of major food ingredients in various environments, at all stages in the supply chain, to unlock food safety insights hidden in big data”.  The idea is to sequence metagenomics on different parts of the food supply chain and build reference databases as to what is a healthy/unhealthy microbiome, what bacteria lives there on a regular basis, and how are they interacting. From there, the information would be used to identify potential hazards, according to Jeff Welser, vice president and lab director at IBM Research–Almaden.

“Obviously a major concern is to always make sure there’s a safe food supply chain. That becomes increasingly difficult as our food supply chain becomes more global and distributed [in such a way] that no individual company owns a portion of it,” says Welser. “That’s really the reason for attacking the metagenomics problem. Right now we test for E. coli, Listeria, or all the known pathogens. But if there’s something that’s unknown and has never been there before, if you’re not testing for it, you’re not going to find it. Testing for the unknown is an impossible task.” With the recent addition of the diagnostics company Bio-Rad to the collaborative effort, the consortium is preparing to publish information about its progress over the past year.  In an interview with Food Safety Tech, Welser discusses the consortium’s efforts since it was established and how it is starting to see evidence that using microbiomes could provide insights on food safety issues in advance.

Food Safety Tech: What progress has the Consortium made over the past year?

Jeff Welser: For the first project with Mars, we decided to focus around pet food. Although they might be known for their chocolates, at least half of Mars’ revenue comes from the pet care industry. It’s a good area to start because it uses the same food ingredients as human food, but it’s processed very differently. There’s a large conglomeration of parts in pet food that might not be part of human food, but the tests for doing the work is directly applicable to human food. We started at a factory of theirs and sampled the raw ingredients coming in. Over the past year, we’ve been establishing whether we can measure a stable microbiome (if we measure day to day, the same ingredient and the same supplier) and [be able to identify] when something has changed.

At a high level, we believe the thesis is playing out. We’re going to publish work that is much more rigorous than that statement. We see good evidence that the overall thesis of monitoring the microbiome appears to be viable, at least for raw food ingredients. We would like to make it more quantitative, figure out how you would actually use this on a regular basis, and think about other places we could test, such as other parts of the factory or machines.

Sequencing the Food Supply Chain
Sequencing the food supply chain. Click to enlarge infographic (Courtesy of IBM Research)

FST: What are the steps to sequencing a microbiome?

Welser: A sample of food is taken into a lab where a process breaks down the cell walls to release the DNA and RNA into a slurry. A next-generation sequencing machine identifies every snippet of DNA and RNA it can from that sample, resulting in huge amounts of data. That data is transferred to IBM and other partners for analysis of the presence of organisms. It’s not a straightforward calculation, because different organisms often share genes or have similar snippets of genes. Also, because you’ve broken everything up, you don’t have a full gene necessarily; you might have a snippet of a gene. You want to look at different types of genes and different areas to identify bad organisms, etc.  When looking at DNA and RNA, you want to try to determine if an organism is currently active.

The process is all about the analysis of the data sequence. That’s where we think it has a huge amount of possibility, but it will take more time to understand it. Once you have the data, you can combine it in different ways to figure out what it means.

FST: Discuss the significance of the sequencing project in the context of recent foodborne illness outbreaks. How could the information gleaned help prevent future outbreaks?

Welser: In general, this is exactly what we’re hoping to achieve. Since you test the microbiome at any point in the supply chain, the hope is that it gives you much better headlights to a potential contamination issue wherever it occurs. Currently raw food ingredients come into a factory before they’re processed. If you see the problem with the microbiome right there, you can stop it before it gets into the machinery. Of course, you don’t know whether it came in the shipment, from the farm itself, etc. But if you’re testing in those places, hopefully you’ll figure that out as early as possible. On the other end, when a company processes food and it’s shipped to the store, it goes onto the [store] shelves. It’s not like anyone is testing on a regular basis, but in theory you could do testing to see if the ingredient is showing a different microbiome than what is normally seen.

The real challenge in the retail space is that today you can test anything sitting on the shelves for E. coli, Listeria, etc.— the [pathogens] we know about. It’s not regularly done when [product] is sitting on the shelves, because it’s not clear how effectively you can do it. It still doesn’t get over the challenge of how best to approach testing—how often it needs to be done, what’s the methodology, etc. These are all still challenges ahead. In theory, this can be used anywhere, and the advantage is that it would tell you if anything has changed [versus] testing for [the presence of] one thing.

FST: How will Bio-Rad contribute to this partnership?

Welser: We’re excited about Bio-Rad joining, because right now we’re taking samples and doing next-generation sequencing to identify the microbiome. It’s much less expensive than it used to be, but it’s still a fairly expensive test. We don’t envision that everyone will be doing this every day in their factory. However, we want to build up our understanding to determine what kinds of tests you would conduct on a regular basis without doing the full next-gen sequencing. Whenever we do sequencing, we want to make sure we’re doing the other necessary battery of tests for that food ingredient. Bio-Rad has expertise in all these areas, and they’re looking at other ways to advance their testing technology into the genomic space. That is the goal: To come up with a scientific understanding that allows us to have tests, analysis and algorithms, etc. that would allow the food industry to monitor on a regular basis.