Microbiomes: A Tool for Food Manufacturers

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Sarah Curran: All right. Welcome, everyone. Thank you so much for joining us for our presentation today. Sorry for the slight delay. We were just having some technical difficulties here at home, but without further ado, we will get started in our presentation, Microbiomes: A Solution for Food Manufacturers. My name is Sarah Curran. I am the marketing specialist with Eurofins US Food division. Our presenter today will be Dr. Greg Siragusa, who I will introduce in just a moment.

 

Before we get started with our presentation, I'd like to let everyone know that this webinar is being recorded and a copy of the slides and recording will be available to all of you either later today or within 48 hours. There will also be time for a question and answer session after the presentation. To ask a question, all you have to do is use the question panel in your GoToWebinar dashboard. As you can see, there's a section in the menu called questions. You simply type your question and hit enter.

 

So, a little bit about Eurofins before we get started. Eurofins is driven by our mission to contribute to global health by offering the highest quality testing, training, auditing, consulting services to our customers. We strive to listen to our customers and not only meet but exceed their expectations. Our footprint is global. We have over 35,000 staff at 400 laboratories across 41 countries and a portfolio of over 150,000 analytical methods. Eurofins provides a unique range of analytical testing services to the pharmaceutical, food, environment, and consumer products industries and to governments around the world.

 

Eurofins has a large genomics division with locations across the US, Europe, and India. Our labs are able to accept genomic samples from around the globe for analysis, including whole genome sequencing and microbiomes, just to name a few. Without further ado, I would like to introduce our main presenter today. Greg Siragusa is a Senior Principal Scientist with Eurofins Microbiology Laboratories, a division of the global life sciences company, Eurofins Scientific. He has held research director positions with Danisco, DuPont, and Agtech Products, was a lead scientist in the USDA Agricultural Research Service and [inaudible 00:02:42] Service in Clay Center, Nebraska and Athens, Georgia, and an adjunct professor at the Universities of Nebraska and Georgia. He holds a bachelor's and master's degrees in Microbiology from Louisiana State University and a Ph.D from the University of Arkansas. He has over 100 peer-reviewed articles, chapters, and presentations, presented abstracts and talks. He has conducted and led a team of research in the area of direct-fed microbials or probiotics for poultry. Currently, he is focused on applying genomics to food microbiology problems including cultures. With that, I will hand it over to Greg.

 

Gregory S.: Thank You, Sarah. Good morning or good afternoon everyone, wherever you are. Glad you can make it. I hope this is going to be informative for you. I'd like to start with a couple of questions. The first one is using the information that you obtain from doing or having done plate counts, how well do you know your product's microbial population?

 

Next, can you relate to any of these questions? You're conducting microbiology or you're directing a microbiology laboratory. You look at a plate. You ask yourself, "These colonies. They sure do look interesting. I wonder what they are?" Or, perhaps you're dealing with a gas defect in a packaged product. You get a plate count. Your question is, "Are these the culprits? Are these the ones causing the gassy defect?" Or, the question might be, "We spent a lot of money on antimicrobials. Why didn't the antimicrobial work? Was there something resistant?"

 

Then, most likely you or your laboratory staff and technician will throw away the Petri dish. They'll throw away the agar dish, the culture plate and autoclave it. We miss a lot of what the data is telling us. So, plate count data is indeed a count, but, as we'll see as we walk through this, information on microbiomes, there's a lot more information that can be obtained from a sample, and there's a lot more information that can be obtained from that plate. Next. Next, Sarah, if you could. Thank you.

 

What is microbiome? Let's start out with a definition. A microbiome is the population or the community of commensal, symbiotic, starter, or even pathogenic microorganisms that share a space. Those organisms can be bacteria. They can be a virus. They can be a fungi. They can be algae. They can be protozoa. Microorganisms that share a space. You'll see a lot of pie charts. One of the ways of conveying the data from a microbiome is to present it as a pie chart. You can also use bar graphs and you can use tables, which is done in the analysis. I like a pie chart because, in a way, the round pie chart represents to me a different type of Petri dish.

 

I'll go in more detail later, but the method on the right is pretty straightforward. The main steps, we extract DNA, we amplify a bacterial gene if we're interested in bacteria or a fungal gene if we're interested in yeast and mold. That information is sequenced, then that data goes to a bioinformacist. Then, from there after the data is analyzed and put in a form that we can interpret, we help you interpret it and make from that recommendations and conduct consulting. Next, please. Sarah, can you go to the next?

 

Sarah Curran: It's going. Sorry, guys. We're having some technical difficulty here. Let's see. There we go.

 

Gregory S.: Okay, so for many years, microbiologists have used the plate counts or culture methods. It tells us in many is not a quantitative method, but it does give proportions in a sample. You'll see what that means. One thing to keep in mind, it detects DNA that can also be DNA from non-viable cells, but I'll address that in just a moment. Well, Sarah, if we can go onto the next.

 

There's been a complete revolution in microbiology because of the advent of the microbiome. Typical in the food microbiology settings, we're very busy making and producing food. That's what we do, so it takes a little while for food microbiology to either catch up or, in many cases, lead new areas, but this is some data from December 2016 to the end of November 2017. You can see there's a big difference. These publication numbers I obtained from PubMed change daily. There's a reason for that. One is the method is being applied to more and more things, but there's some other more down-to-earth methods. The cost of sequencing is dropping. The cost per genome is dropping. If any of you have done 23andMe, if you've had your own genome sequencing done, you can get a pretty good idea that, yeah, things are becoming quicker. They're becoming better. They're being done better, cheaper, faster. Same way in microbiome research. It's all sequence-based. That technology is greatly improving. We'll talk about that a little later, too. Next, please.

 

Microbiomes are wisely applicable. If you look at the literature as a whole, you'll see a lot of areas of microbial ecology that have been studied. Today we're going to talk about just food. Next, please. Just a reminder. There are a lot of microorganisms on Earth. If we talk just about bacteria and archaea, or the extremophilic bacteria, if you will. There was an estimate published in 1978 by Bill Whitman and Company and the University of Georgia. They calculated that there are approximately five times 10 to the 30 bacteria on the planet. If you look in the upper right, you'll see he looked at and calculated this from data obtained from aquatic habitats, ocean, soil, and then everything on the Earth's surfaces including animals, plants, et cetera.

 

But, of that 10 to the 30, which is a very large number obviously, we have only cultured about 1% of the different bacterial types on Earth. That's just something to keep in mind. Next, please. Sarah, if you would, click through four times. Okay. Well, that's great. We have all these bacteria on Earth. How have we looked at them? How have we studied them and learned about them? One of the earliest breakthroughs in microbiology was the technology of the plate count. It started after Leeuwenhoek said there are these very small things in rainwater. Then, after that, fast forward almost 200 years. An observation was made that bacteria can grow on the surface, in this case, the surface of a communion wafer.

 

A little bit later on, Louis Pasteur and Robert Koch came on the scene. They showed that you can grow organisms on surfaces in isolated colonies, but they didn't really have a good way to do that. At the time, one of the dominant methods was to use a slice of potato. Well, not everything grows on the slice of potato. So, at the same time, someone in the Koch laboratory, Dr. Hesse was married, living in New York. His wife was not only a very accomplished artist, but Fanny was a very accomplished chef. She saw this problem that her husband was dealing with. She said, "You know what? I have a solution and it's called agar." The agar was used in cooking and later on, it was used in canning hams. She was the one responsible for first suggesting we use agar. It's a big breakthrough. It doesn't sound like it might be such a big deal now. It was a big breakthrough if you consider we're still using this technology.

 

Then, a little bit later on, two individuals said, "We need something to put this in." They invented what we know as the modern Petri dish or culture dish. So, Sarah, if you go to the next? That technology has led us to today for the plate counts. All right. Go to the next, please. But there is something to keep in mind. I said earlier there are all these organisms on Earth and we've only cultured about 1% of them. Okay. Plate counts, as good as they are, they suffer from something called the great plate count anomaly. That is that when we look at bacterial samples under a microscope, they count the viable ones by using stains, viability staining. We see that there are a lot more there than we can culture, about a hundred times more under the microscope then you'd get calculating a population by culture. In the upper left there, that was the original work where they coined the term great plate count anomaly. In there, they were looking at lake water. The one on the left is microscopic examination. It's very dark. That indicates a lot of bacteria on the right. It's culture. It's very light. That indicates fewer organisms. Next, please.

 

There are a lot of reasons for this great plate count anomaly. I'll just mention a couple of them. One is that when we autoclave our media, we have to do so with all the components together. Now, you can add something separately. You can add carbohydrates separately, for instance, but phosphate as an example. When phosphate is added to media and then it's autoclaved, we see on the top row there that by the time you get to a concentration of about 1.5 to 2 millimolar phosphate, you stop seeing colony growth. If you add phosphate after autoclaving, as in the middle row, you see growth all the way up to 10 millimolar phosphates. If you add zero phosphate, you get what you get at the lower left. So, there are reasons why organisms don't grow on agar. Some of them are as straightforward as this. Others are completely unknown. Next, please.

 

All right, but we still do plate counts. They're still very functional, very utilitarian. They're very good sources of data. They provide us information. The way they do it, probably are well aware. We homogenize food, we dilute it, we deliver it to a plate, whatever that agar is. We incubate it under certain conditions. Then, we count colonies. Do a calculation. Normally, we throw that plate away. In some cases, we might keep it to identify bacteria like phenotype or genotype. Next, please.

 

All right. Plate counts are great indicators, but what if your job was to identify bacteria on a plate? If we use this as an example, there are about 100 colonies on this plate. If you pick them and grew them in isolation and then identified each, roughly, very conservative estimate is that it would be about $7.5 thousand, $7,500. For that much money, what do you get? Well, you get identities, but that's identities from things that grew on just one growth condition on a single agar.

 

So, what if you wanted to grow things anaerobically, or what about incubation in a cold temperature, or what if you wanted to use lactic acid bacteria media? You get the idea. You can't detect all in a single Petri dish culture experiment. Next, please. Okay. Right away, I just want to say, will microbiome technology do away with cultural microbiology? No, it will not, but hopefully, by the end of this presentation, you're going to see how it's going to greatly enhance our power of the plate count. Next, please. Culture is still king.

 

All right. If you go through the next four, please. So, what's the next breakthrough in microbiology? We already saw historically how a plate count was developed over time, concepts that were put forth in the literature. They came together. Sarah, if you hit it one more time. Okay. All right. Let's talk about the microbiome. This is pretty recent history for many of us, anyway. In early 1950s, I think 1952, a group in the UK, Watson and Crick. We all know James Watson, Francis Crick. Morris Wilkins and Rosalind Franklin discovered and elucidated the structure of DNA. After that, fast-forward to 1977 at the University of Illinois in Urbana-Champaign, Carl Woese discovered that there was a gene in bacteria that was highly conserved. That is, it was very old. It changed or mutated very little throughout evolutionary history. That was called the 16S gene.

 

All right. Upper right, three individuals figured out using PCR how to amplify those genes and make many, many copies of them. Lower left, people figured out how to sequence those genes, and then how to automate sequencing. Lower right, once you sequence and you have all this data, what do you do with it? Stephen Altschul was one of the members of a group at the National Center for Biotechnology Information, who invented something called BLAST, which is a way of taking that data, and from it, elucidating the name of an organism. Now, that's the field of bioinformatics. It's greatly exploded since then, but that's some of the basis of it. Next, please.

 

There we are. We're at the stage of the microbiome. So, I'd like for you to think of the microbiome much in the way that you'd think about plate counts. It's a tool in our tool chest. It's just one of many things that we can do to understand our food systems. Next, please. All right. How do we do biomes? Not totally dissimilar to the way we do plate counts. We homogenize food. We separate in a stomacher bag with lyse cells that are associated with that food. We extract that DNA and do a purification. We amplify the 16S genes and then we label them. Then, we sequence them. Then, at the bottom right, we analyze those sequences.

 

Those names you see there, Qiime and Mothur, those are different softwares. A lot of bioinformatic software’s have these very kind of cutesy-sounding names. There are a lot of acronyms in that field, but what they do is take that data and make it into a readable and interpretable pie chart or table of information about the names and the proportions of bacteria or fungi or microorganisms in your sample. That's the basic process. Next, please.

 

Sarah Curran: We're actually going to jump in here for a second to do a quick polling question of our audience. So, I'll launch the poll and give everyone about a minute or so to respond, but our question is have you or your business used microbiome analysis services before? Greg, if you could predict the answer, what do you think? Have most people been using microbiome analysis?

 

Gregory S.: Will I get a prize if I'm correct?

 

Sarah Curran: Yes.

 

Gregory S.: I would say probably not.

 

Sarah Curran: Probably not? All right. Let's see. I'm going to display the results. Give everyone just a couple more seconds to respond. All right, and I'll let everybody see the results here. Greg, you were right. How did you know? 70% answered no, 22% yes, and 7% have an analysis in progress.

 

Gregory S.: I can say Eurofin's microbiology has been in the process of changing that for about the last two years.

 

Sarah Curran: All right. Back to you, Greg.

 

Gregory S.: Who would use and get the most out of a microbiome approach in your food factory? If you're involved in quality and safety and your responsibilities are to understand spoilage, to extend shelf life, to conduct safety and hazard analyses or to understand preservation efficacy. If you spend money on a preservative, is it working and doing its job? If you're involved in new product development. If you're trying to remove salt. If you're trying to remove whatever it is, what does it do to the shelf life in the microbial ecology of your product? You have an interest in this. If your job is to obtain raw materials, you want to know what are the differences in those raw materials, whether they come from East Coast or the West Coast or from another country.

 

Then, maybe the most important thing to take away from this slide is that regulatory agencies in the world are using microbiomes. That includes our FDA. It includes the European Food Safety Agency and it includes the USDA. I would say if we give a similar webinar in six months or a year, that list is going to grow. More regulatories for getting benefit from using it. It's not punitive in any way. It's meant to help people in the countries they're representing make better food that's going to be safer to export. Next, please.

 

Okay, so talked enough about it. Let's get right to the point. This is Larry the laboratory technician. His job was to understand fish spoilage. He works in a fish plant. There was an issue. So, what he did was a standard, or, I say, a general plate count. Could have been an anaerobic plate count. It could have been a psychotropic count. He did a plate count experiment. What he got was that the counts were pretty similar. Next, please.

 

But Larry is a clever guy. He knew that there was a way to tell what organisms were dominant in the spoiled fish. He had a microbiome analysis done. The lower left says, "There's a variety. There's blue, green, orange, red, and purple," but on the spoiled side, bottom right, there's a lot of blue and there's less green and there's less orange and there's much less blue. It's the blue organism that we would hypothesize is causing this issue. That's the basis of using a spoiled pair. You have a point of comparison. Next, please.

 

So, what can you do with that information? He can use the information to selectively culture that spoilage organism and look for it in his environment. If he can do that, then he can control it. He has a way of knowing. Now, we have these fish coming in. This patch has a high level of this organism. This one has a lower level of this organism. We have to do something about it. Do we put the onus on the provider or do we somehow try and clean those fish, process them in our hands so that we lower the level of that organism?

 

The other thing that can be done is let's pretend that that blue organism is difficult to culture or if it takes a long time and you have fish. They're ready to go. You can't wait. Well, you can take that organism. You can sequence its 16S gene and use that information to make a PCR assay and cut down the analysis time from 36 to 48 or even 72 hours of culture to two to three hours of acute PCR assay. Next, please. All right. So, you can use microbiome analysis on a variety of things. I'm going to spend most of the rest of the time talking about examples, examples that came from our laboratory, examples that came from the literature. Next, please.

 

All right. So, before we dig into these examples, I want to give everybody a chance to stand up, stretch, take a break, get a drink of water, have coffee. Whatever you want to do. Sarah, why don't we do this for about two minutes. Would that be okay?

 

Sarah Curran: Sounds good.

 

Gregory S.: Okay. Do you have any other polling questions right now?

 

Sarah Curran: Yeah. We can do one right now. We'll leave it open for the duration of our break. Our next polling question is — This question's a little confusing. — have you personally performed a microbiome analysis? So, are you in the field of conducting these yourself? Just trying to learn a little bit more about our audience.

 

Gregory S.: I just want to get an idea and I would assume that maybe there's some of you out there who are actually hands-on microbiomic scientists and have done this. Sarah, while people are voting and people are stretching, if anyone has any short questions, we can take them now. We'll have time at the end, though, for more discussion and questions, but please, go ahead and ask.

 

Sarah Curran: Yeah. We haven't received any questions from the audience yet, but this is a great time to type your questions into the question action box in your GoToWebinar dashboard, so drop that down, type in your question. Just hit enter and those will get submitted to me. I can pitch them to Greg or you can send them to me in the chat. We'll give everybody, well, 30 more seconds to answer the question. We'll wrap up our break here. Someone asked a clarifying question, Greg. "In the old days, what we're calling microbiome today used to be called normal flora. Is that correct?"

 

Gregory S.: It's yes and no. So, first of all, the term flora is … Actually, I still use the term. In my dreams, I will dream about microflora, but the term that people tend to use today is microbiota because flora implies plants, but that's a minor point. Yes, the microflora is what we knew we could count by plate counts. So, for instance, if it were the microbiota of meat, well, we pretty well knew what was going to grow. We were going to get a variety of organisms and you could predict them. If it was fermented milk, you could predict the microflora or the microbiota of that fermented product was going to be mainly lactic acid bacteria. That's correct.

 

What's different now is we can discern to a higher degree of resolution which organisms are actually there. Yeah, they're lactic acid bacteria, but they also are pediococci or they're also Lactobacillus. If they're Lactobacillus, are they Lactobacillus plantarum? Are they Lactobacillus curvatus? We get a higher level taxonomic resolution and we have that little number to the side that says, "About 50% of the organisms that are there are based on DNA sequencing are Lactobacillus curvatus," so that's a much higher resolution microbiota profile or a microbial profile.

 

Sarah Curran: All right. Then, one more quick one before I move on. Someone asked, "Is it always or mostly the case that in a spoiled pair, the spoiled products will be less diverse in its microbiome?"

 

Gregory S.: It's a great question. The answer seems to be yes, that if there is a dominant organism, it would show up in a microbiome. It's not always a dominant organism. I can give one example. In the case of beef cattle, the rumen is very complex, that there are hundreds of different genera of bacteria, hundreds. It might only be a very small percentage that's doing something very profound.

 

For instance, there are propionibacteria in the rumen that will detoxify nitrates that animals get from eating poorly. That's a very small portion of it, but it makes a big difference if it's there or not. So, in general, in the case of food, I think we do see that dominant case, but not all cases, do we have a dominant culprit.

 

Sarah Curran: All right. Thanks, Greg. So, we're getting a lot of good questions. I'll save them for the end. Just know that if we don't have time for your question today, you will get a personal message from someone on our team answering your question, so we will follow up with every question whether it's live or after the fact, but let's keep going.

 

Gregory S.: Okay. All right. So, now comes the good part. I have some case studies that we performed. All but one was performed in our laboratory here. Let's start with case number one. Next, please. This really for me was a proof of utility of microbiome analysis. We were asked to study a problem in a plant that made a deli salad. The problem was that every once in a while, one particular salad had an aerobic plate count that was out of range, out of range for the customer. The customer said, "We want that plate count, the aerobic plate count, the SPC, standard plate to be less than 10 to the four colony-forming units per gram."

 

All right. So, this was a very modern plant. It had very good sanitation. It had very good managements. However, this problem was sporadic. It was almost impossible for them to predict it. It was very long-term. We asked them, "How long has this been going on?" The answer was, "13 years." 13 years! They tried a lot of different things. They spent a lot of money on new machines. They tried alternate ingredients, which affected the flavor, but they tried those. They also tried alternate sanitation providers. They put in automatic hand washers, for instance. In general, they were just getting sick over it and a lot of ulcers and gnashing of teeth. All right. Next, please.

 

So, we obtain from them what I'll call a spoiled pair, but this product wasn't actually spoiled. This product just had a high APC, so we had one sample, the first one was within range. It was less than 10,000. The other one greater than 10,000. We did two things. We cultured it. Then, we did a microbiome analysis. Now, in order to protect the customer, I took this data. I just more or less made a summary of it, but what we found in the sample that was out of range had a relatively high proportion of what was this purple pie wedge.

 

If we go to the next, what we found is that that purple piece was identified as Aeromonas species. Now, it wasn't a pathogenic Aeromonas. It was not Aeromonas hydrophila, but it was Aeromonas species, the genus. Then, we asked the question, "All right. Where can that be entering the system and why isn't it consistent?" Turned out that one of the ingredients were actually pre-boiled eggs. Shelled, refrigerated, hard-boiled eggs. You go to the next, and indeed we found that, on occasion, we could find Aeromonas in the condensate from the hard-boiled egg package.

 

That was brought back to the provider of the eggs who not only processed the eggs. Aeromonas is very susceptible to heat. Boiling kills it, period, but there apparently was recontamination after the fact. After these eggs were boiled and cooled, there was Aeromonas in their environment. It actually makes sense because this same organization that produced and processed hard-boiled eggs produced the eggs. They own the flock of hens that made eggs. So, when that material was coming in, it was likely that Aeromonas was there because Aeromonas is a common inhabitant of the hen oviduct.

 

So, this really, for me, solidified the proof of the utility of microbiome analysis. We would not have discovered this, had we not done a biome. Next, please. Okay. Second case, this was a customer that had problems with gas formation in its small packs of cheese spread. This is a very multi-component product, but it had dominantly a fermented component, the cheese. It was refrigerated, but it was linked to a very large distribution area. It was also linked to one particular recipe. Next, please.

 

So, we did a couple of things. One was we did standard culture, which included air and plate count and aero bacterial yeast and mold and lactic acid bacteria. The plate counts really didn't provide any information between the spoiled pairs. There wasn't a significant difference between those count. However, when we looked at the non-spoiled microbiome versus the spoiled microbiome, we could see a difference. If you just glance at that, you see these red bars on the right.

 

All right. If we go to the next slide, those red bars turned out to be Leuconostoc gelidum. What we did here is the difference between this graph and the previous is that, electronically, we removed the main bacterial component in that product. Because it was a fermented cheese, it had a very large population of Lactococcus lactis. All right, but that interfered with our ability to see what the culprit was, so that's another beauty of microbiome analysis. You can electronically remove things that might be blocking your view. We did that.

 

All right, so we found out that there was a high level of Leuconostoc gelidum. Why is that significant? Well, Leuconostoc gelidum requires a lower temperature to grow on lactic acid bacterial media. We did a standard LAB plate count, which is incubated at 30 degrees centigrade. It turns out that this Leuconostoc doesn't grow very well at temperatures above 25.

 

So, had we just looked at it with the lens of the plate count, we probably still be doing these experiments. So, here's another example of how biome analysis elucidated a problem that culture really couldn't identify. Next, please. This was a research project. We were working with a customer that produced cold-smoked salmon. The salmon was produced actually in two different geographies. They were all aquaculture salmon. As you know, smoked salmon is something that's susceptible to Listeria contamination, to refrigerated, vacuum packaged product. It had a very large distribution area within the United States. The question was whether or not nitrite addition would change the shelf life in the microbiota or the microbial progression of the product as it sat refrigerated. Next, please. Sarah, can you see my pointer?

 

Sarah Curran: No.

 

Gregory S.: No? Okay. I'll just describe it. Top panel are samples that receive no nitrite. Coal smoked, they had a standard flavor spice rub, but no nitrate. Bottom contained nitrite. So what happened? If we had just done plate counts on these, we probably wouldn't understand that, by day 34, the dominant organisms in the samples without nitrite were Photobacterium. Photobacterium is a gram-negative normal inhabitant of marine fish. We would expect that.

 

The bottom panel, we see that with nitrate addition, that microbial progression was driven toward lactic acid bacteria, in this case, Lactobacillales. That's the order that contains Lactobacillus. That was important to this company because what they were interested in knowing was whether or not they could use those organisms to naturally preserve their smoked salmon. Could they add a specific organism back and get a preservative effect?

 

This would indicate that if they would go back in and isolate those lactic acid bacteria, that they could probably introduce them at the beginning of processing or pre-packaging, anyway, and have a longer shelf life without having to add nitrite. They were trying to remove nitrite from their label. This was the research they needed. Next, please.

 

Okay. I'm going to speed it up just a little bit here. This was a fermented vegetable extract. I took this picture off of the internet. It wasn't actually turmeric, but it was concentrated fermented vegetable extracts. These are refrigerated products. They're still active. They make gas. There's a warning label on the lid. They have a low pH. The warning label says, "Open with caution. There is gas in this bottle." The company was trying to make a probiotic claim. They wanted to be able to say on their label, "This product contains healthy bacteria," if you will. Next, please.

 

Okay. What we found was we had two different flavors, if you would. On the left, those are microbiomes that were done. They are reported there at the genus level. So, remember there's kingdom, family, class, order, family, genus, species. Example of a genus, Lactobacillus. Example of a species, plantarum. All right. Well, there, we stopped just at the genus level. It doesn't tell you a whole lot, but if we go to the right, the DNA sequence data allowed us to go, in some cases, to the species level. Not all the time, but in some cases. What it told us was that there was a big variety of bacteria. Indeed, some of them would be considered as probiotic-type organisms.

 

But something to note here. If you look at the list from the top down, it goes from order of a frequency, low frequency to high. So, in the bottom is high frequency. If you look down, you see Vibrio. Immediately, what does everyone think of when you see Vibrio? Well, there are pathogens in the genus Vibrio, parahaemolyticus, vulnificus, cholera. All right. Whole bunch of bad guys.

 

Well, it turns out that these weren't those, but there also a big family of Vibrio in the ocean that are perfectly harmless to humans. In fact, most Vibrio bacteria are not pathogenic. Where'd they come from? Turns out that in the seasoning for these fermented vegetable extracts, they were using sea salt. Sea salt is known to carry ocean-dwelling bacteria. So, at first, there was a little bit of alarm. When we studied the problem using microbiome analysis and studied those sequences, we understood it was very logical that there was a source for Vibrio. The company got the answer it was looking for. Are there probiotic types of bacteria in their product? Next, please.

 

Sarah Curran: I'm actually going to swoop in here with one last polling question, since we are reaching the end of our presentation. Now that we're halfway through our case study, what's your impression of microbiome analysis as a tool? We still have a few more examples to show you. We had a few questions come in during the break asking what the practical applications are. So, just polling the crowd. What do you think?

 

If you're answering other, there's about 7% in the other category right now. If you can give us an explanation in the chat or in the questions box, what are your other thoughts? All right. Just a couple more seconds and I will close that poll. Give everyone a few moments to answer. Okay. I'll let everyone peek at these results because they're good. I'm happy to report that a majority, almost everyone feels that they're useful, so hopefully you get a few more. Then, if you were in the 6% that … Apparently, we have 101% on this call. If you are in that percentage that feels differently, drop us a note in the question box to let us know why. Back to you, Greg.

 

Gregory S.: Okay. Next, please. All right. So we were approached by a maker of probiotic tablets. They asked, "For purposes of labeling, will a microbiome be able to tell them the approximate percentages in the species of organisms in their product?" Next, please. So, this information was used along with plate domes. What it did was tell them that, in their product, they had top to bottom, in this case, high to low, these organisms. These were actually the organisms or the groups of organisms that they had in their product. They needed data to back up what was on their label. So, now they not only had plate counts, but they had identity data. Next, please.

 

All right. This is the last of the internally-derived case studies. Sarah, after this slide and the next, I'd like for you to proceed to about slide number 43 in the interest of time to make up for the technical difficulties. All right. Case number six. I went to the grocery store and had been buying the same brand of baby carrots for some time. I still use the same brand. As we all know, baby carrots are not actually baby carrots. They're adult carrots that have been processed and cut down to a certain size and dimension. These bags were within the best buy date. There was no discoloration. There's virtually no odor, but they were slimy. They were very slimy. If you move to the next …

 

We performed microbiomes of those internally. Here on the left are the bacterial microbiome results. On the right are the fungal microbiome results. We see, on the bacterial side, there are some differences. There are a number of interim bacterial groups gram-negative bacteria, many of those make extracellular polysaccharides. They also make enzymes that can begin to degrade carrot tissue, making it slimy. If we go to the right, there was a dominant fungi there, Mrakia frigida. It turns out that it's a yeast that's commonly associated with produce.

 

Now, this was an experiment we did internally. A customer didn't come to us saying they had slimy carrots, but it's a very common defect. We understand that carrot processors often use chlorine rinses. One thing chlorine rinses do well is they can reduce bacterial load, but they also make food products susceptible to other microbiota. I think we show that quite clearly.

 

All right. So, Sarah, if we could move to slide 43, I am going to leave it to people, if they're interested in all these other examples to contact Sarah. These are from the literature so they're freely available, but what I wanted to show next is that one of the real utilitarian uses of microbiome analysis is in the analysis of hard physical surfaces. So, Sarah, go to the next.

 

Okay. This is an example of work that was done on restroom surfaces. Very briefly, if you look at the red, the blue, and the green bars, even the orange bars, the top four. Those are things that a number of researchers in the area of skin microbiology call people stuff. By people stuff, they mean these are things that are commonly found on built surfaces that come from human contact, generally the hand.

 

Now, in this case, it wasn't just the hand, but what they see is more or less what they expected. As people go in and out of a restroom, as people touch things like a faucet or a soap dispenser, they leave an imprint of what is on their hands. Now, there is a little bit of a difference when you go down, but if you look at the lower left there, there's a component slot. It looks a little bit like a shotgun blast, but if you look, you can see that there's a separation between the biomes that were found on the toilet seat and on the toilet floor. Then, in the upper right, that's largely the stuff I refer to as people stuff. You see there are three main groups. That can only be done by a microbiome analysis. To try and do this by culture, if it were even possible, would take years. That's probably a pretty generous guess. Next, please.

 

You can use this for kitchen surfaces or deli surfaces. What I'd like to draw your attention to in the graph is that on the right-hand side, we see those are bars representing the number of species. Look at the number of species that were found on the floor. Well, you can imagine how do things get on the floor? It come from people's shoes, or, if you're in front of a working area like, for instance, a refrigerator, they can come from people shoes and they can come from doggy feet, if you have dogs and they're looking in the refrigerator with you. The point is, you can use this to analyze your surfaces, as well. Next, please.

 

All right. I put this in here just to show you that there are things that our databases don't cover. This was a study. It was published in science progress reports 2015. They studied different coffee pod machines, coffee makers. If you look up there you'll see green. It says, "Other." What does that mean? What it means, that there is a large bunch of bacteria that aren't in databases. It means that they're undescribed.

 

So, this brings us to another point. One of the utilities of the microbiome is to discover the unknown and to understand what is right under our noses, but we haven't been able to culture it, so we really don't know what it is yet. It's absolutely a new frontier. Next, please. All right. So, I'll summarize. We know what a microbiome is. It's a population or community. It's a very new and powerful tool, but it's one of many tools in your toolbox. It enhances cultural data. You can use it for discovery. Our regulatory agencies are using this right now.

 

Always remember, if you go to the next slide, the most informative samples are spoiled pairs. So, I encourage you, if you have a spoilage issue, if you have issues of pathogen presence, if you have anything that you're trying to understand that's of a microbial origin and you have an affected and a non-affected sample, please keep those samples. Put them in your freezer. We don't need much to extract DNA. In fact, just a few grams. Remember, spoiled pairs are the best samples. Next, please.

 

All right. Now, we can revisit these questions whereas before, we really didn't have a way to answer them. What are those colonies? What's causing gas formation? Why didn't my anti-microbial work? The bottom is a representation of how we can use culture data with microbiome data to now answer those questions. Finally, if we go to the next, I want to make a quick plug for the near future of DNA sequencers. In 2014, a company called Oxford Biosciences introduced this little gadget on the upper left there. It's called the MinION or the MinION. It's a handheld DNA sequencer.

 

Imagine this. If you go through the same process that I showed you to do a microbiome and you load that DNA onto or into the MinION, then it plugs into your computer. They can also plug it into your smartphone. It generates sequence. That data is uploaded to the cloud, where it can be automatically analyzed by Oxford Biosciences informatics software and delivers to you a microbiome in the form of a pie chart. Who would have ever thought that you can hold a sequencer in one hand?

 

Right now, the size of sequencers are still desktop. They're coming down in size, obviously, but you can actually do this. I want you to think ahead to end of 2018, end of 2019, end of 2020. What I hope to be able to present to you is that we can do DNA sequencing and biome analysis with instruments that we can hold in our hands, do them quickly and more frequently. Next, please.

 

So, I'll stop there. I would like to say that there are a number of us working in the microbiome area, myself, Dr. Marshall, you might know our Chief Science Officer, Jodi Benson in New Berlin, Wisconsin is our lead microbiome scientist. She did many of these microbiomes. Mehgan Styke is a microbiologist and also business development manager for probiotic analysis and microbiome. Sarah Curran is our marketing specialist. Here are their emails. I encourage you, please contact Sarah, myself, or anybody else on the list. Thank you.

 

Sarah Curran: All right. If you're willing to stay on the line for a few more minutes, we'll take a few questions, but like I said, we'll be following up with everyone individually with a direct message. So, if you still have questions, go ahead and submit them now and I'll pitch a few to Greg. I see a few questions about costs, as well. Those questions we'll answer offline just because everything is project-based obviously. So, earlier-

 

Gregory S.: Sarah, can I give…

 

Sarah Curran: Oh, yeah. Go ahead.

 

Gregory S.: I just want to indicate we're now talking, in some cases, less than a hundred dollars depending on the volume.

 

Sarah Curran: Good to know. Someone has asked, "Is it an issue that microbiome analysis does not distinguish between live and dead bacteria? How problematic is that?"

 

Gregory S.: In some cases, it is. DNA itself can be a very recalcitrant molecule. It can stick around for a long time, but DNA in a living system, and I consider foods to be living systems, the chances are that it's going to be degraded pretty quickly and that you won't see it. So, that hasn't proven to be a major problem. The same question that people asked when PCR testing hit the food safety market in the mid 1990s, and, yes, it was possible that you could have residual DNA, but what turned out to be the case is that that DNA just did not stick around very long when there were other bacteria there eating it. So, good question.

 

Sarah Curran: Someone else has asked, "Some of the experiments you showed data from very few replicates and is this a problem?"

 

Gregory S.: It is a problem and as a journal reviewer, I know that, in some cases, it was very difficult to even generate one or two microbiomes when this first started. So, we couldn't fault the scientists for not doing many of them, but now we should start thinking in terms of doing this in a way that's statistically valid, which means we should have more replications. I think that will come when the process gets less expensive and takes less time, but yeah. Absolutely. It's like any other method in biology. You have to have replication.

 

Sarah Curran: Then, a few questions have come in about typical turnaround time for microbiome analysis.

 

Gregory S.: So, microbiome analysis can be done in as little as five days. Normally, we tell customers it takes between three to five. We have had results back in as quick as two weeks. That's pretty quick. When we make a commitment for two week turnaround time, we are more than competitive with most laboratories. Most, they're not that many, but there are other providers of genomic services.

 

The reason it can take so long is because the sequencing itself is done in batches. So, by a batch I mean a 96-well plate. So, if we have two samples in a 96-well plate, we can do it, but the cost of that would go up exponentially. So, people wait until those plates are filled. Sometimes that takes longer than other. Once the sequence is obtained, informatics analysis again depends on the volume, but it can be pretty quick. It can be a matter of just less than a day if it's a few samples or maybe two days if it's a big body of sample.

 

Sarah Curran: All right. Then, since we're already over time, I'll just pitch you one more. Then, like I said, we'll follow up with leftover questions personally to answer a few other questions on here. If you joined late, yes, we will send you both a recording and a copy of these slides to everyone who both registered and attended. So, the last question was, "Greg, do you have any insight on if and when food regulations might reflect the utility of this new technology?"

 

Gregory S.: Wow $64,000 question. I would say this, that right now, we know that the US FDA is using whole genome sequencing, it's another type of sequencing, to track pathogens. That's happening right now. That's helping Public Health. That's helping control foodborne disease. Biome analysis is different in that it's going to have other uses besides just maybe detecting conditions for pathogens. We don't have a big database on that.

 

Well, our US FDA has initiated a large research project to get and collect microbiomes on normal products. By normal, I mean they're not out of spec. It's doing that right now. So, I don't know if that's going to translate into regulations per se, but hopefully it's going to translate into information that you can use as a manufacturer to make a better product and keep it under microbial control.

 

I would say, in 10 years we're going to see changes in our regulatory makeup, what those laws say. I think they're going to be including a lot of sequence data. It's not necessarily is going to be all based upon culture data. So, I'll predict. I'll give it a decade before we start to see big changes in print

 

Sarah Curran: Great. All right. Well, thank you so much, Greg, for your time today. As you know, everyone on our call really appreciates the insight that you shared. Thank you everyone who tuned in. At this time, we will say, "Thank you," and conclude our presentation. Have a great day, everyone.

 

Gregory S.: Good-bye.