Prof Philip Hugenholtz on Metagenomic Stool Analysis for Optimal Gut Health

This episode is brought to you by Nourish Me Organics. We've partnered with a company called Microbar who are leaders in the metagenomic stool testing space. They have an amazing service where they can test for bacteria, yeasts, fungi, parasites, gut metabolites like GABA and a whole suite of others. And this service also includes personalized nutritional consulting based on your unique microbiome.

So.

So if you're interested in the test, head to www.nourishmeorganics.com and use the code guthealthgurus for 10% off. Hey, guys. Criben gavender from the Gut Health Gurus podcast. I've got a background in food science, and for the context of today's discussion, I've also got two years of background training in computer science, with also a major in microbiology and food microbiology. So just to give you a context on the conversation, and today we've got a fascinating guest and I'm a huge fan of the man. I'm going to totally butcher his name for sure. Professor Phil Hugenholtz. Did I get that right?

Yeah, that's very good.

Is that okay? Didn't butcher it till you pronounce it okay. It must be good. Must be good. Firstly, Phil, huge gratitude, huge fan of your work. I'm so nervous to have you on and have this conversation with you because I'm a bit of a fanboy, but huge welcome to the show.

Thank you.

And Phil, what we like to do at the start of every podcast is to get it from the horse's mouth. Who is Professor Phil Hugenholz?

Well, I'm a microbiologist. I've been doing this for about 30 years now, so getting on a bit. And I have a particular interest in microbial evolution and ecology. And I've been very fortunate to live through a period in the life sciences where we have a whole new toolkit that we can ask evolutionary and ecological questions, and that's been driven to a large part by sequencing technology. So I think most people would be aware of the human genome being sequenced, and that's a major milestone for us as a species, let's say, and that was driving the technology to improve sequencing. So sequencing is determining the order of bases or letters in your genome in order to sequence, in order to get access to the blueprints. And the blueprints tell you then what an organism is capable of. So microorganisms have been a very large benefactor of this improved technology. And in fact, it's much easier to sequence the blueprints of a microorganism than, say, us, because their genomes are on average, a thousand times smaller than ours.

So I'll just pause you there for a sec, Phil. I'm really interested before we dig in deep. And this whole conversation is going to be centered around metagenomics, Phil. He's a founder of an amazing company called Microbar here in Australia. And the whole context is going to be around that metagenomics. But what I like to start off is just to get a context of your origin story. Now, going back, just get into the, I guess, the memory banks and think back. Where did it all start for you in terms of your passion for microbiology?

My backstory. So I've always been fascinated by this concept of scale. So early on I was amazed to learn the size of the universe being so much larger. And thinking about, you know, you keep going through these orders of magnitude scale differences, and I knew that that went in both directions. So I knew that you could go down in scale and you could see down to atoms. And so that concept fascinated me. When I was about 10, I did a. In the bio in my science class, I got a microscope and looked at all the creepy crawlies that were in a lake. And that was just fascinating to see this whole unseen world of microorganisms. And I guess that's where I really got interested in the first instance. And so knowing that, I went on, when I went to university, I did a science degree and I did microbiology. And I really liked using the microscope and seeing these organisms, and I was trying to classify them. And back in those days, because this was the late 80s, we tried to characterize them by growing them on an agar plate on a petri dish and, and then looking at properties of them. How big were they? What pigment did they produce? Could they use oxygen or not use oxygen? And the frustrating part of that is that you couldn't really classify microorganisms very well at that point with those characteristics because they're just too simple in order to get a good classification and understand how they are related to each other. And so I was very fortunate that I was in the department of Microbiology at the University of Queensland at that time. And a big German professor came and became the head of the department, Professor Erko Stackerbrandt. And he brought with him this great new sequencing technology where you could actually now relate organisms to each other not by seeing how they look, but looking at their blueprints, at their genes. And that's when I and I suddenly realized that this was something was pretty good at because I like doing puzzles. For instance, I've always had a real knack for jigsaw puzzles or Sudoku or things like that. And looking at DNA sequences was like one giant jigsaw puzzle Sudoku. And so I really gravitated towards that and I grabbed that, that happened during my PhD. He arrived during my PhD and I just immediately cottoned onto that. And ever since then I've been a real fan of sequence data and what it can tell us about living things.

Absolutely. And so I'm going to throw a couple of names out here and let's explore when I say the names Carl Woes, Norm Pace and Craig Venter, what does that mean to you?

So Carl Woese is. Well, he's. He died in 2012, but he was an American microbiologist and he's famous for doing the first sequencing of a market, what they call a marker gene. So you can think of that like a barcode when you go to the supermarket. So where you sequence an individual gene and then relate that to other genes to see how organisms are related to each other. So in the 70s he had read a paper written by ZuckerKant in the 1960s saying, you know what, we may be able to relate organisms to each other by looking at their DNA or their proteins. And he took that and he ran with it and he focused his research attention on a particular gene in the ribosome. So the ribosome is the protein manufacturing powerhouse in our cells and it's highly conserved. So he reasoned that if he sequenced part of that ribosome and he'd be able to use that as the way of relating organisms to each other. And the part of the ribosome that he ended up focusing on was the 16s or small subunit ribosomal RNA gene. And he all through the 70s he was looking at those and he was kind of considered a bit of an eccentric in his home department. And he's kind of a grumpy guy. Anyways, my take on it, so he was a bit ostracized, but he certainly proved all the naysayers wrong because he then Produced the first 16S ribosomal RNA based tree of life. And his big finding was that all these microorganisms which people had just lumped together actually formed two very distinct groups, the bacteria, all the ones we used to like E. Coli and Bacillus, and then these things which he originally called archaebacteria or old bacteria for archaea. And now 30 years later, it actually looks like we may be, have arisen from this archaeal microorganism group, from these archaea, they may be our direct descendants. So he discovered the archaea, and that was a huge finding and really shook up our understanding of how life evolved. Now, Norm Pace is a colleague, or was a colleague of Carl Woes, and he was fully aware of all of the business going on with the 16 srna gene, and he was interested to apply it to various ecosystems that he was interested in, ecosystems he looked at in Yellowstone National Park. He was interested in the organisms that could grow in boiling water there, for instance. He was trying to grow these organisms so that he could get hold of this gene, the 16 srna gene. And he wasn't having much luck with that. And then one day just realized that maybe he didn't need to grow the organisms in order to get hold of the gene. If he just took. If he just took a chunk of the, of the hot spring, say biofilm, and then threw it in a bucket of phenol, he could get access to that gene. And that was, that was the beginning of this culture independent approach. Now, why is that important? And the reason that's important is because it's actually a bit of a trick for a microorganism to grow on an agar plate. It turns out that a relatively small percentage of microorganisms in the environment can be grown in pure culture like that. So particularly in hot springs, maybe less than one in a thousand organisms can be grown in culture. So this opened up the door to looking at all of this diversity that hadn't been explored previously, that we were unaware of because we hadn't been able to grow it on an agar plate. And he was characterizing that using the 16 srna gene. So that was Norm Pace's pivotal contribution right there, which opened up the doors to all this diversity. Now, Craig Venter, he is well known because he was the guy that got shotgun sequencing to sequence the human genome. So he's well known for that. And he also applied that to microorganisms and also to communities. So you can think of it this way. You can think. Carl Woes, first of all did the evolutionary thing to get the idea of the structure of the tree of life. Norm Pace then applied that one gene, the 16S gene, in order to characterize all the uncultured diversity. And then Venta then took it up to the next level. So rather than looking at one gene, he's looking at all of the genes within the full blueprints of microorganisms by shotgun sequencing. And that's looking at the full genome. And so essentially he applied that shotgun sequencing or genomics to environmental samples that Norm Pace had shown by 16s and that became what's now known as metagenomics. Right? Meta meaning above genomics. So that's been an amazing few decades in the life sciences and microbiology in particular, where we now are able to go into any given environment, whether it be a hot spring, whether it be a handful of soil, whether it be a poo sample, and then extract the DNA from that environment and then sequence the genomes of the organisms that are there and put them back together. So now we have the blueprints for all the organisms without having this bottleneck of culturing through an agar plate. So for the first time and only recently have we had this very spectacular high resolution view of these environments. And that's been driven. So those guys make pivotal contributions to that. But it's also been driven by huge improvements in sequencing technology. So all through the time that WOES and Pace and Venta were active, we were using the first generation sequencing technology, which was Sanger sequencing, named after Fred Sanger, who put the. Who came up with it and got a Nobel Prize for that. And that then gave way only in the mid-2000s, so 2004, 2005, that the next generation, so called next generation sequencing came online. And, and what this did was you could sequence a lot more, you could sequence it much cheaper. And it's just phenomenal now how much you can sequence for how cheaply. So it's really opened the doors. And that coupled with increases in how computers, the processing speed of computers really has opened up the doors. So to give you an example, the first metagenomic studies, which were only dating back to 2004, one of them was sponsored in Jill Banfield's group, was sponsored by the Department of energy. And about 70 million base pairs of sequence data were done, and it cost about half a million dollars in those days. Using today's sequencing machines, you can sequence the same amount of DNA for less for a couple bucks.

I'll just press pause there for a second so we can just reflect on the conversation. Because some people listening to the podcast is like, oh my God, this is some pretty deep science. We're going to. We do go deep in the science in this podcast, so no issues there. But if we could just summarize very quickly. We started off very early discovering microbes and we didn't actually cover that. Van Leeuwenhoek, I believe that discovered?

Oh no, I didn't go back that far.

Was it who actually first saw microbes under a microscope?

Yeah, it was Van Layer Hoek. So I mean, it was Ronald Hook before he saw. He saw fungi and larger microorganisms and protozoa and then Leeuwenhoek. He was the first one, I guess, to see bacteria. And it was because he worked out a way to make lenses for microscopes that up until that point people had been grinding down the lenses. He worked out that you could just heat up glass and extrude them and make really perfect lenses, which allowed you to get be able to see under the microscope much easier. And he wouldn't tell anybody that for decades. And he kept that a secret. So everybody's like, how does he manage to do this? And the first organisms, microorganisms he saw amongst the first were bacteria in his own mouth.

Okay.

So we knew about these very small, you know, these bacteria for now, for a couple hundred years. Few hundred years. But the first characterization beyond that then relied on growing them on an agar plate koc k o c h 50 years later that came up with the first solid medium which allowed these microorganisms to be grown and essentially bring them up to our level so that we can interact and do stuff with them. Cool.

So we went from seeing them or discovering that they exist, then we went to actually trying to classify these things using morphology and whatnot. And then it's evolved into using RNA or DNA extracted to classify so basically.

Using their genetic blueprints in order to characterize them.

Okay. So that's the basic evolution of discovery all the way to where we're at currently. So I'm really fascinated now to talk about, for instance, your company Microbar. Fantastic progressive company. And there's other companies in the world that are also pretty dominant. There's Eubiome and fiome and certainly Microba is definitely in that kind of space as well. So how did you conceptualize. Firstly, I'll ask you this question. What is GreenGenes?

Okay, well, GreenGenes is a database of 16s RNA genes. So we talked about 16s and Karl woes non pace. And so that is using that marker gene, that 16s to classify organisms. And that was an initiative that was born out of the LBNL in California and when I was working over in the States and I was curator of that database. And so it's now unfortunately demised and there's a lot of hate on the Twitterverse, I noticed the fact that it's not maintained anymore. But that's using the 16 srna gene and so going. And then where does that fit in? So the idea then is that you mentioned ubiome. So ubiome has been offering these community profiling service of your gut microbiome, basically your fecal microbiome, by using that 16s RNA gene. Right. So, and that's what I was doing earlier in my career too, in the 1990s, using that same idea in Norm Pacer's group, that you use that 16 srna gene to tell you, give you, give you an indication of what's present in a given sample. And so that's where green genes came in too, because you need to have a reference database that when you pull out a new 16s sequence, what is it? You do it against this reference database. But what we saw when it came to Microba, and this is myself from my co founder, Gene Tyson, is that we could improve upon, rather than just using that single gene, the 16s, we use Craig Venter's shotgun sequencing and metagenomics to characterize the community. So once you go beyond the single 16s marker gene, which just tells you who's there, and it's got some limitations around it as well, which I won't go into, and you go to metagenomics, you can not only tell who's there, but you can tell what they're capable of. And that was really. And the metagenomics, as I mentioned before, really kicked off in about 2004. So it's only 15 years old. It's pretty new technology and it's really taken off just in the last five years. Right. So it's only been very recently that you could offer metagenomics as a service, say to a customer. To a customer. In the last few years, when the sequencing cost came down to a point where you could do it at a reasonable price. So that's where, that's where microbial was born out of. So, you know, Gene and I were looking at, we were seeing, you know, people were getting into the, into the commercial space and we see the, the potential of sequencing the gut microbiome because it's such an important sentinel for health and disease. And we said to each other we could really use metagenomics and scale it up and do a lot of. We were very excited about the idea of being able to do the gut microbiomes of thousands of individuals because we're interested in analyzing big data sets. And if you've got data from thousands of individuals, you can start to see patterns, you can Start to see, well, which organisms are important under which conditions. You can see, well, what's the function that's important that associated with that population, for instance. So that's where we go. And that's where microbial came from.

Fantastic. And in terms of. So you were heavily involved, I guess, with the curation of the green genes database at some point and correct me if I'm wrong. So just to summarize, so you're using the green genes as a reference database to then use that to identify genus level types of bacteria?

Exactly, yeah.

Is that. So that's, Is that the limitation of 16s?

Yeah, that's the. It's because, I mean it was. Carl Woes chose that gene in the first instance because it's so conserved, you need it. If you wanted to work out, if you wanted to be able to relate all organisms like including E. Coli and ourselves in a single tree, evolutionary tree, you needed to pick something that's really conserved and that's why you picked it. But that, that hyper conservation kind of works against you when you want to work out closely related things. So that's why you get genus level resolution. Like my 16s. We're not discernible from other primates, for instance.

In terms of conservation. What does it actually mean?

When the gene is conserving, it's changing at such a slow rate that there are not enough differences in the sequence to be able to distinguish closely related things. So many instances you can't distinguish between species. So as a rule of thumb, we talk about 16s having reliable identification down to genus. Whereas if you use the whole genome via metagenomics, you now have really great resolution. You can not only resolve down to species level, you can resolve below species, you can resolve at subspecies or end strain level. So that's, that's one of the benefits of using metagenomics.

And how about the ITS gene? How's that working to the conversation?

The ITS is in between the 16s gene and other structural RNA genes in the ribosomal RNA operon. So that's less conserved than the 16s gene. So people have been using the ITS because it's got higher resolution. Again, that's true. And they use it a lot in fungal classification. But if you can go beyond a single marker to the whole genome, you're really cooking with gas. And so really that's where we are now in 2019, where we've used the entire genome to get this high level of classification resolution and also be able to tell what the organism is capable of doing.

Right. That Makes perfect sense. So you're going from a pretty, I guess, a low resolution photograph of the microbiome to get insights to now a higher resolution shot, which I'm sure also encompasses other things like viruses and parasites as well.

Absolutely. So you can think of it like, remember if you look at the TV quality in the 1960s, opposed to the high def pictures you get now, that's kind of, you know, that's an analogy for going from 16s to genomes.

Cool.

So that's one way of thinking of it. What was the other part of your question then?

I was gonna, I was just gonna say is, what's the workflow?

Oh yeah, sorry, you said about viruses and other things.

Viruses and parasites.

That's a really important point because the way that you fish out the 16s gene is you use PCR, the polymerase chain reaction, which is dependent on being able to target that particular gene. So if your organism doesn't contain the gene, then obviously you can't target it like this. Like viruses don't have ribosomes, so there's no 16s to target. So 16s profiles can't tell you what viruses are present, whereas metagenomics can. But also you're also dependent on the, on, on the specificity of those primers. And so often those primers will miss all of the protozoa and all these other things. And so that again is an advantage of the metagenomics because it gives you a view of all these other life forms that may be missed by 16s.

Yeah. So just, just for my own understanding, it's a bit of a selfish question. Just for me, try to understand this as best I can with my limited amount of knowledge compared to Phil, who's an absolute guru when it comes to this kind of stuff.

Well, I reckon you're doing pretty well. You're asking a whole bunch of quite detailed questions.

So in terms of, so you've got 16s or its using a primer, you're finding the gene, you're using PCR to amplify the quantity and then you're using that as, then using a database to actually identify it. So that's the 16s process. Now, what does the metagenomic process look like?

So the metagenomic process is where you go, you extract the DNA from the sample and then you just sequence all of it. And that's called shotgun sequence, you randomly sequence it. So that was first demonstrated on isolates. I think the first bacterium sequence was Haemophilus influenzae and Craig Vendor did that. So Basically, you shear up that genome and you sequence it. Because the sequencing technology, we haven't got a sequencing technology that can just sequence a genome all in one piece yet. Maybe it will come. But at the moment, you sequence it in short pieces called reads, and then you can think of that like pieces of a jigsaw puzzle. So you've got all of these pieces and you put them back together to get the picture on the box, and that's your genome. So if you've got an isolate like Haemophilus, you've only got one jigsaw puzzle. With metagenomics, where you're looking at a community of organisms or a microbiome, it's this like if somebody got, you know, 500 jigsaw puzzles, grabbed bunches of pieces from different boxes and stuck it all into one thing and then said, okay, put that back together. And so you're actually making 500 jigsaw puzzles in parallel. And that's metagenomics. And that sounds tricky and complex and it is, but the technology is improving so much, including the way we process the data and put these jigsaw puzzles back together, that we can actually pull out fairly complete genomes for organisms within a metagenome. So it's an amazing advance, in my opinion, in the last few years only. And so now suddenly we're pulling out all of these so called metagenome assembled genomes or mags for short, at an astonishing rate out of environmental samples. So actually human poo is like the poster child of this process. And so in the last two years alone, there have been three major studies. And in the last one, they pulled out 160,000 mags out of the data set. 160,000 genomes in one paper. It's insane. And so we're just getting. So once you. So then essentially those genomes are your reference database. So with the 16s with green genes, you now have databases that are based on genomes rather than 16s. And so we have one that we developed here at the University of Queensland called gtdb, which stands for Genome Taxonomy Database. And anybody in the world can then bring their genome and put it up against this reference database to tell them what's the organism.

Wow.

And so that's what microbial. Microbial takes. So let's say we take a sample from you, we sequence maybe 7 million reads or 5 gigabases of data, we take all of those reads and now we match them up against the reference database, and then that gives us that really nice high resolution picture or profile of the organisms that are present in your gut.

Wow, that's astonishing. It's amazing. I mean, it's so exciting to see where this technology is headed and what hope it can offer for people suffering with all sorts of things. So where do we go next? I did have a question brewing in my mind. So did you want to say something?

I can give you a couple of anecdotes. So we've sequenced over 5,000 gut microbiomes from the Australian public, and we've got an opt in option. So we say to people when they're getting their profile, would you like to opt in? So that these profiles can be compared to a database in order to give us. So we can analyze it and look for patterns. And the majority of people actually opt in because I think most people appreciate, you know, that this really helps in scientific endeavor. And so about 75% of people opt in. So we've been able to analyze those data and they make an amazing. You get amazing correlations. So, for instance, we're able to predict inflammatory bowel disease with a 97% accuracy. Based on these profiles, we're able to what the major type of IBD is, whether it's Crohn's disease or ulcerative colitis, also with 97% accuracy. And I guess the most exciting part is that when the gastroenterologist is trying to treat these conditions, they have a number of treatments at their disposal, but generally the efficiency depends on the patient for unknown reasons. So you might say, okay, try this treatment and maybe one in five of your patients will have a positive response to it. Well, we can also predict what your response is going to be to a particular treatment by just through your gut microbiome. I think this has huge potential. We see these very strong statistical signals for pretty much any condition you look at. You can see particular microbial populations that are elevated or reduced in people with depression, people that have predisposition for particular types of cancer. Pretty much every condition, you'll see some signal in the gut microbiome. And the reason you do that is because the gut microbiome is essentially part of us. It's evolved with us, and it plays an important role in our health. But also when things go off out of whack, you get changes in the gut microbiome that can exacerbate the problem, make it worse. And, and often the guts are one of the first things that reacts to changes in what's going on in you. So you can use it as almost an early warning of things coming up and so the potential is huge. And that's why there's so much research focused on this at the moment. We're seeing hundreds and hundreds of papers published every month. It's really quite difficult to stay on top of everything that you've got this huge potential to be able to use this information to guide treatments of different diseases, to let you know ahead of time that you may have a disease coming. And this is very new technology. So the regulatory authorities are just coming to terms with this. How do we deal with this? So we have to. In a startup company like this, we have to be careful about what we say in terms of diagnostics, because it has to be all scientifically and rigorously shown. But I can tell you that at the research part of the startup company, we are definitely seeing these very strong statistically significant correlations that we know will be very good as diagnostics. And then the next thing you can do after you. So where the future is going is that when you diagnose it, then you also know, well, this organism is absent or reduced and needs to be there, or this organism shouldn't be there. You can start to design some therapeutics around that. So one of the interesting things is that we looked at the healthy cohort, and so we had a number of characteristics that would define the healthy cohort, which works out to be about 8% of the total number of profiles. I actually don't belong to the healthy cohort because I don't meet all the metrics. But the healthy cohort were characterized by having a number of microbial populations that were enriched in the healthy cohort relative to everybody else. So then we can start to think about, well, if we can get those populations in culture, they essentially would be probiotics that you find in healthy humans. That could help. Right. And the interesting thing, remember I mentioned before that the majority of organisms have not been cultured. So in the gut, actually, it's a little better than, say, in the hot spring. So about 30% of the species in the average human gut have been cultured, but 70% haven't. 70% of species have not been cultured. And what we're finding is that through the big data analysis, that a lot of these populations that are in healthy people haven't been cultured before. So we're spending time trying to isolate those. And because we have their full genetic blueprints, we can use that genome information to direct their isolation efforts because we know what they're potentially capable of by reading the genes. And then we can design growth media and selective conditions around that in order to culture them. And so that's a hugely exciting area as well. So potential enormous, Enormous.

I've got a couple of follow up questions and I have to ask this is a million dollar question. What does a healthy gut microbiome look like?

Well, we think we do know what a healthy gut microbiome looks like, at least for Australians because there may be variants as you go to different cultures and different ethnicities that will have an effect. So we base this healthy, we first of all base it on survey looking for healthy lifestyle people that haven't are not on medications, people that have no reported disease states. And we use that as our centering where the healthy microbiome is. And then we, as I mentioned we had, we found particular populations that are enriched in people with healthy states. So I would call this as the healthy microbiome. Right. And the other day we found one lady that was right smack in the bang of this healthy. She was at 100% because we ranked people according to this healthy profile. And there was a lady that was smack bang in the middle of it and she was, she's 70 years old, on no medication, healthy, has been on a high fiber diet since she was quite young. And she would be what I call the apex of the healthy gut microbiome. So if you ever need to have a fecal transplant, you know about those things, this would be the lady I'd want to go to and ask about as a donor can define it. Yes.

What's the specific? So when we look at, are you looking at a phylum level? You're looking at class, genus.

This is all species level.

A species level. So what?

This is all species level definition of a healthy microbiome. In fact, you know, we're in the process of going to subspecies and I suspect that some of it signal is going to be at the subspecies rather than just the species. So yeah, when you phylum in class, they're way too coarse, they just don't have the right resolution. You really need to be at the species and subspecies level.

Could you share a couple of these species that we want to have in that gut for a healthy person?

Well, they don't have names because they haven't been cultured. So they've got identifiers which don't mean a lot.

Okay.

I mean they mean a lot to us at the moment.

So part of it, yeah. What type of, what species are they? They're Lactobacilli or they're Bifidobacteria. Are they really speak of.

So some of them are cultured. So some of them people will be familiar with like Phecali bacterium Pranatski is one of them. But actually when you, when you go to the genome level, that species actually is actually seven or eight species, it's not just one species. And then one of those, those species carry genes which again this is only a relatively recent discovery, carry genes which encode a protein that has an anti inflammatory effect. So I think a lot of this centers around inflammation actually. So when things are going bad in the body it's often associated with inflammation. So organisms that have natural anti inflammatory properties I think are associated with a healthy microbiome and organisms which tip the balance and cause things to go off rails sometimes have the opposite, have an inflammatory response. Right, so that's part of the story. Yeah. And there are many other parts of it. And so as we're identifying the species, we're also trying to find out what is it about this organism that makes it part of a healthy microbiome or part of a, what they call a dysbiotic microbiome or an unhealthy microbiome I hear.

So we're looking for some keystone species, subspecies that can produce health giving benefits and we're trying to.

First of all it's an interesting thing because everybody's guts are unique and individual. In fact, if we could use it forensic science, if I could sample, if everybody had had a poo profile, you could work out who's been there because their profile is unique. It does change over time, but it is unique. So you find the samples taken from one individual, it's quite diagnostic of that individual. However, when you look at thousands of individuals you see those signals and that boils down to often the case maybe a handful of species. We found 20 species in IBD that are changes in about 20 species that are characteristic of IBD.

Anything specific that you could share?

I can't remember off the top of my head. I'm probably not supposed to say anyway, but yeah, definitely that you can pin it down. So even though there's a lot of individualism amongst the gut profile, there are these strong signals. So it's a very complex ecosystem as opposed to say that hot spring ecosystem because you've got the added component of our own immune system which adds a lot of complexity. But I do believe that despite the complexity of all those, you know, Maybe there's about 500 to a thousand species in your average human gut. So you've got a lot of species complexity You've got a lot of additional complexity because of the immune system and the signaling and communication going on between the gut and ourselves. But despite that, I think we have quite strong signals that we can use to affect change in a positive way.

How do we do that?

And I guess the proof will be in the pudding in the future because the research is very young in this area. And I mean there's a lot of. The problem is when you get a new area, you get this. Things can be overhyped and a lot of people can come in and make lots of claims. But really what you want to look at is the good solid science behind it. And I believe that we will see in the next five to ten years very specific therapies for conditions that would either help to eliminate a disease or manage a disease state based on the microbiome.

Cool. Phil, we're pretty much coming up to time. I've got a couple of burning questions if you wouldn't mind. The first question is. So we mentioned some keystone species that are beneficial. What about some that could be negative, have negative connotations? Something like Bilophila deosulfovibrio Helicobacter pylori. How do these things fit into the puzzle?

Well, I mean the poster trial for that would be Clostridium, or it's Clostridioideum now, I guess. Difficile C. Diff. Yeah. So they're well known pathogens. If they get a foothold in the gut, they can really throw things out. Bilofila zworthii is one that is a normal. You find it in most people's guts, but under certain conditions it can get out of control and start having deleterious effects. So what people typically refer to as opportunistic pathogens. So under a certain set of conditions, an organism can step up and then create a problem. And you've named a couple of them there, but most of those are in the normal commensal gut environment. So it's the conditions that. Under which they can then become a problem. And also I think again, this, some of this comes down to subspecies resolution because they are like, let's go back to Faecali bacterium, for instance. There's one subspecies of Faecali bacterium that has a mucin degrading genes in it which then under certain conditions can cause problems in the gut. So yeah, I think we're going to increasingly know more and more about this. And almost every week there's a new study which has pinpointed a particular organism under a particular condition which creates a good or bad thing in the gut.

Great. And I've actually had my stool tested by you guys, and it was really insightful and interesting to have a read and see what's in the gut. And I was. Your percentage, I can't remember from the top of my head. I think I was very high in definitely protobacteria and I had very little bifidobacteria to speak of. But I did notice as part of the profile, there was a whole section on metabolites. So you're able to speak to that a little bit about your metabolite part of your test?

Sure. So this is again courtesy of the fact that we've got the metagenomics. So if we've got the metagenomics, we've got the full blueprints, so we can predict what functions the organism is capable of. And part of that is we can predict which metabolites they're producing. So metabolites, essentially chemicals that get either used up or produced by organisms. Right. So one example is gaba, which is an acronym for something I can't remember, which is used. Actually, it's a modified amino acid and it's used just as a. By organisms, for example, just as a carbon and nitrogen source, so food source. But it also happens to be one of our major neurotransmitters that we use in our bodies. So that means that organisms in the gut can produce gaba, which is beneficial to us, or consume the gaba. There are studies that show the level of GABA production or consumption in the gut is related to tendency for depression, for instance. So there is that connection between the gut and the brain. Gut, brain axis. So that's just one of the metabolites. There are many in the report. You'll see several that are highlighted. So the idea is that from the metagenomics, you predict your ability to produce those metabolites. What I think will happen in the future is once we have a really good handle on all the pathways and they're fully articulated, we may actually just be able to sequence the metabolites directly. And there's a whole branch of, in the life sciences, metabolomics, which allow you to do that. And that's also come along in leaps and bounds. The issue is that we're still learning what those genomic blueprints are coding for. So we don't have a complete picture of it yet. But yeah, that's one thing that why we might expect in the future that we just measure the metabolite profile rather than reading the DNA. And that might happen in 10, 15 years.

So you guys are pretty much, I'm guessing, using something like kegs.

So keg is the metabolic pathway. That's the Kyoto encyclopedia that's developed in Japan, which basically tells you metabolic pathways. So that's telling you about a chunk of the genes in the genome, not all of the genes. And that's an important resource, reference resource for interpreting the metabolic potential of the organism. Yes.

And what you're saying is in future, maybe they're going to use something like HPLC or gas chromatography to directly test the metabolites.

Yes, I think that's what will happen. Yes. And the moment is that you can produce those profiles, but a lot of the peaks, you don't know what the metabolites are.

Right.

So as our understanding of the metabolism goes up, and a lot of that's based on being able to do this comparative genomics, that we'll be able to identify more and more of those peaks. And so we'll get a higher and higher resolution picture. And if we can read the metabolites out directly, that has the advantage that you're reading. You're getting the output of the metabolites directly rather than inferring them from the DNA. But we're not quite at that point yet. But I would predict that you will see that coming up in the next decade or so.

Totally. I'm 100% with you there, Phil. I think it's fascinating also with the drug interaction side and where that pathway is headed as well. That's fascinating. Drug efficacy based on a microbiome.

Exactly.

Fingerprint, which is absolutely fascinating where it's headed. So, a couple more questions and then we'll wrap. So, in terms of where the technology is headed, is there anything particular particularly exciting that stands out in mind and the future of this technology?

Well, we already touched on the idea of doing the metabolomics directly, so I think that's definitely going to go. And that's exciting because that will bring the price down again by an order of magnitude, which means that now we might be able to profile for 10 bucks or 15 bucks, which means that that opens it up to a much wider audience. So at the moment, people might look at the price and go, 350 bucks we're offering, which sounds. Oh, that's. You give that a bit of serious thought about whether you're going to get that done or not. But if you consider that the only other commercial offering just five years ago cost €6,000 to do the Same thing. So the prices are continuing to come down, so sequencing technology continues to improve. And so the cost of doing these profiles is going to get cheaper and cheaper and then if we go to metabolomics, even cheaper. I'm very excited by the prospect of looking at hundreds of thousands or millions of profiles because every time you increase the size of the database that you're looking at, your resolution goes up, your statistical significance goes up. And so I think it's going to be a very exciting time over the next few years as we increase that size of the database and make it more accessible to people to use that. We fully expect that in years to come. That the same way that you go now to get your cholesterol measured through a blood test, your GP can say, get your gut microbiome profile. And we'll have that as another set of information to assess how you're going, what your health is like.

Do you think they could maybe put a sensor in the toilet?

Yeah, people have talked about that. People have definitely talked about that. I think there's even a prototype for that. I mean, that's actually another whole area looking at the volatiles produced by the microorganisms, so measuring just the volatiles that are coming out of the microbiome. And so that's a branch essentially of the metabolite mix.

And how about like. Because you guys must be dealing with a huge amount of data. So is there machine learning algorithms that are being developed to actually data. Mine is.

That's how we're getting our predictability. So I mentioned earlier about being able to predict IBD 97% precision. So we are using machine learning to help us with that because it's complex data set and so it can look at patterns that we may not be able to see by eye. And so this is the standard ways of applying. There's lots of different ways you can do machine learning. There may be 20 different algorithms, so we applying a whole bunch of different ones and then you can train it and then you have a test set and that's how you assess the efficiency of the machine learning. So, yeah, everybody's into machine learning these days and it's for good reason because it's quite a, it's a powerful technique and machine learning loves data. So the more information you can give it, the better it can predict things.

Absolutely fascinating to see where this technology is headed and what a wealth of information. And for someone that's, that's looked at all this data and this developed this founder of this amazing company, you've Got all this data mining happening. In terms of insights, what's your top recommendations? I want to give the audience something actionable to take away from this conversation. Coming from someone who's one of the world leaders in this space, what's some actionable steps that people can take to improve their gut health at home?

Well, I mean, at the moment, because it's such a new area and we're going through all the diagnostic thing, you can't just tell people, you have to go through the road. All has to all be tested. The best that we can do at the moment is in the most immediate way you can affect your microbiome is by diet. Diet has a huge driver and that's why these tests give you specifics about things that you can alter your diet if you're deficient in something or you get too much or something. But the interesting thing is that varies from person to person. So there was a very interesting study that came out of Day two in Israel and they just showed depending on you on your host genetics, it's in your setup that you're giving people bananas and chocolate. So the majority of people, they get a better response to bananas than chocolate. But in a subset of people, chocolate's actually better than bananas. So you always hear the generic things eat more high fiber and more vegetables, which is true by and large. But I think the really interesting thing is when you get your personalized profile that what's good for you may not be exactly what's good for somebody else. So I can't give you, I can tell you eat more vegetables because generically that's a good idea. But you know, getting your personal profile done, you can tell. And I think the precision is just going to go up and up and up as we get, as we get improved knowledge that you can really get a personalized diet or hopefully supplemented with prebiotics or probiotics that actually are effective and help you. This is the whole precision medicine thing, the personalized medicine. I do believe that's going to become much more to the fore in years to come.

How about the general recommendations? You hear about people or dietitians or people in that profession, health profession, saying eat more fiber because of butyrate. What's your thoughts on that?

Well, butyrate is an important short chain fatty acid in the gut and it contributes to the health of your epithelium. But with everything there's a happy medium. So too little is no good, but too much is also no good. So that again can be predicted from looking at your Gut microbiome and the pathways for the organisms that produce butyrate, whether you produce too much or too little. So again, it comes back to the personalized business that if you take a test and then you from that you can get recommendations about what you should eat or not eat in order to try and optimize that. You should probably talk to a nutritionist for this part to get recommendations.

That's okay. You've been absolutely brilliant in terms of sharing the nuts and bolts of the technologies. And I think my takeaway from this conversation is that it's probably a good idea to consider having a test done and then working with a practitioner, like a dietitian, to actually look at improving the diet based on the results of the test.

Yes, I think so. I mean, I'm a bit biased agreeing to that. Yes. As I say, I think that the test is going to become more and more useful over time. And so as the same thing, when you get your human genome tested and they tell you come back and check out your profile because we'll have more information when you come back. It's going to be exactly the same with the microbiome profile. So we know that there's a lot more information in the data that can be presented and actioned on by individuals. That's what we're aiming to do.

Cool. I always ask the question at the end of the show. We'll wrap now. You've been absolutely brilliant, Professor Phil. So what are you doing for your gut health?

What am I doing? Well, I definitely changed my diet, so I've cut down on my meat consumption and particularly processed meat consumption because of the trimethylamine. So I try to eat more vegetarian meals now. Personally, exercise is definitely an important part of it. You see changes, so changes in diet and exercise. Almost the first thing that changes or improves is mental health. That's like one of the early indicators and you see changes, corresponding changes in the gut microbiome as well. So I'm trying to do those things. I mean, in terms of personalized things. There have been some in my profile, I talk. There were recommendations for particular food groups which I adopted. And I feel pretty well for somebody my age. You look great.

You're looking really healthy and vibrant. Cool.

Terrific.

Well, we'll wrap there. Huge amount of gratitude for you coming onto the podcast and sharing your absolute wealth of knowledge. Hopefully I didn't ask too any questions that were too difficult and I appreciate there's some information.

No, that was fun and, and you've clearly done a lot of reading, so it's great.

All good. Thank you so much, Phil.

All right, no worries.

Hey guys, we hope you enjoyed this episode of the Gut Health Gurus podcast with Professor Phil Hugenholz from Microbar. Now, if you enjoyed the episode, head to Apple Podcasts and please leave us a five star review and a kind comment. So guys, if you're interested in this particular service provided by microbar, head to www.nourishmeorganics.com and search for stool test in the search bar. We've bundled together the Microba service with a 40 minute nutritional consultation to optimize your GUT microbiome based on your unique profile using dietary interventions. So guys, thank you so much. Until next time.