Metagenomic Stool Analysis for Optimal Gut Health

 

 

Did you realise that having a stool analysis can predict more than just your current gut health? I had the pleasure of speaking with Professor Philip Hugenholtz. He has been a microbiologist for 30 years. He is the co founder of a company called “Microba.” Read on to learn about how this test can help predict future illnesses, why personalised health profiles are the wave of the future, and why everyone should get a stool analysis (Hint: Gut Health is not a one size fits all approach, your body may benefit more from chocolate than a banana!)

What Got You Interested in DNA Sequencing?

I have always had an interest in microbial evolution and ecology. I have always been fascinated by the concept of scale. I was always thinking about the size of the Universe and how it is much larger and I kept going through the scales in both directions. When I was 10, in my science class, I looked through a microscope and looked at the creepy crawlies that are in lake water. It was fascinating to see a whole unseen world of microorganisms. That is what brought upon my interest in microorganisms.

When I went to University I studied Microbiology and I really liked using the microscope and seeing the organisms. Back in those days (the late 80’s) we tried to characterise them by putting them on a petri dish and looking at the properties such as:

-How big they were

-What pigment did they produce

-Could they use oxygen or not have to use oxygen

The problem was you couldn't classify microorganisms very well at that point with those characteristics because they were just too simple. I was fortunate that I was in the department of microbiology at the University of Queensland at that time. A German professor came to lead the department and with him, he brought a different sequencing technology. You could now relate organisms to each other not by how they look but by looking at their genes and blueprint. I like puzzles and I started looking at DNA sequences like one giant jigsaw puzzle. Ever since then I have been a fan of sequencing and what it can tell us about living things.

What is the Evolution of the Discovery of Microbes?

Breaking it down simply with each scientist is the easiest way to understand the evolution of microbes: 

Antoni Van Leeuwenhoek: Discovered Microbes. He made lenses that could see the microbes. Before this, they were grinding down the lenses. He found a way to heat them and make perfect lenses that you could easily see out of the microscope. He didn't tell anyone about this for decades. The first microorganisms he saw were bacteria in his own mouth.

Robert Koch: He came up with the first solid medium that allowed these organisms to be grown. This allowed us to bring them up to our level and be able to interact with them.

Carl Woese: Got the structure of the tree of life. He was an American Microbiologist that was famous for completing the first sequences of a marker gene. Think of a marker gene like a barcode at the supermarket. You sequence an individual gene and relate that to other genes to see how organisms are related to each other. In the 1970s, he had read a paper written by another scientist in the 1960’s saying that we may be able to relate organisms to each other by looking at their DNA. He took that and ran with it and focused his research on a particular gene in the ribosome. The ribosome proteins are manufacturing powerhouses in our cells and are highly abundant. 

He reasoned that if you conserve part of that ribosome you could use that as a way to relate organisms to each other. Part of the ribosome that he focused on was the 16s rRNA gene. He then produced the first 16s rRNA tree of life and his big finding was that all of these microorganisms that people lumped together actually formed two very distinct groups:

Bacteria

Archaea

30 years later it looks like we may have risen from this Archaea, our direct descendants! 

Norm Pace: Applied the 16s gene to characterise the culture diversity. He was a colleague of Carl Woese. He was fully aware of the findings of the 16s rRNA gene and he was interested in applying it to various ecosystems such as ones in Yellowstone National Park. He was very interested in the organisms that could grow in boiling water. He was trying to grow them so he could get a hold of these genes. He wasn't having much luck then he realised that he needed to go straight to the source. This was the beginning of this culture-independent approach.

This is important because it is a trick for an organism to grow on a plate. It turns out that a small percentage of microorganisms in the environment can be grown like that. In hot springs, less than 1/1000 organisms can be grown in a culture! This opened up the door to look at all the diversity that had not been explored before because we had not been able to grow it on a plate. He was characterising those organisms using the 16s rRNA gene. 

Craig Venter: Took it up to the next level and instead of looking at 1 gene he looked at all of the genes. He applied that genomics to environmental samples that Norm Pace had shown him. This is what became what is known as Metagenomics. He was the guy that was known for leading the first sequence for the human genome. He also applied that to microorganisms and communities.

This gave us the ability to go into any environment whether that be:

-A hot spring

-The soil

-A stool sample 

And then extract that DNA from the environment, sequence the genomes of the organisms that are there, and then put them back together. Now we have the blueprint for all of the organisms. It has also been driven by huge improvements in sequencing technology. All through the time that these scientists were active, they were using the first-generation sequencing technology (Sanger sequencing). The next generation of sequencing came about in the mid 2000’s and this was beneficial because:

-You could sequence a lot more

-It is cheaper

-It opened the doors for more people to access it

That was also joined with the processing speed of computers developing as well. To give you an example:

The first genomic studies (only dating back to 2004) was sponsored by the Department of Energy. It cost about half a million dollars in their day, using today's machine you can process the same amount of DNA for a couple of bucks!

We are going through a period of time in life sciences where we have a new tool kit where we can ask ecological and evolutionary questions. That has been driven by sequencing technology. Most people are aware of the human genome being sequenced and that is a major milestone for us as a species. Sequencing is determining the order of bases or letters in your genome to get access to your blueprint. This tells you what an organism is capable of. Microorganisms are a benefactor of this improved technology because it is much easier to sequence the blueprints of a microorganism than us. After all, the genomes are a thousand times smaller!

What is Green Genes?

Green Genes is a database of 16s rRNA genes that uses that marker gene to classify organisms. BUT it is now demised because no one is maintaining it anymore.

“Ubiome” has been offering community profiling of your gut microbiome by using that 16s rRNA gene. This will give you an indication of what is present in the given sample and that is where green genes came in because you need a database to see what is present when you pull out a 16s rRNA sequence.

When it comes to our company, “Microba”, instead of just using that 16s gene we can use shotgun sequencing to characterise the community. Once you go beyond the single 16s marker gene, it tells you who is there but ALSO what they are capable of! Metagenomics kicked off in 2004 and it has really taken off in the last 5 years. Metagenomics has only been offered recently to customers when the sequencing cost came down. After the cost came down, our company Microba was developed.

We saw the potential of sequencing the gut microbiome because it is SUCH an important factor in health and disease. We could use metagenomics, scale it up, and use it for thousands of individuals to create a database. We are creating this database to start to see patterns, see which organisms are important for which circumstances, and what is the function that is important with that population.

What is the Limitation of the 16s rRNA Gene?

The reason why Carl Woese chose this gene is because it is so conserved. You need something that is really conserved if you want to relate all organisms in a single tree. This hyper conservation works against you when you want to workout closely related things. With 16s, we are not discernable from other primates. The gene is changing at such a slow rate that there are not enough differences to distinguish between closely related things. In many instances, you can differentiate against different types of species. 

In comparison, If you use the whole genome like in metagenomics, you now have great resolution. Past the species level to subspecies and strain level. In the middle of the 16s and other ribosomal genes you have the ITS gene (internal transcribed spacer.) That is less conserved than the 16s gene and some people have been using it because it has more resolution than the 16s gene. It was used in fungal classification but if you can go beyond to the whole genome then you are cooking with gas. This is where we are now, using the entire genome to get a high level of classification resolution and be able to tell what the organism is capable of doing.

You are going from a low-resolution photograph of the microbiome to get insights to now a higher resolution shot which encompasses other things like viruses and parasites as well. -Kriben Govender

The way that you fish out the 16s gene is you use PCR (Polymerase Chain Reaction) which is dependent on being able to target that gene. If the organism does not contain the gene then you can’t target it. For Example: Viruses don’t have ribosomes so there are no 16s genes to target. The 16s profiles cannot tell you which viruses are present BUT metagenomics can. Also, you are dependent on the specificity of the primers. These primers may miss all of the protozoa and other things. That is another advantage of metagenomics because it gives you a view of all of these other life forms that may be missed by the 16s gene.

You can think of it like this: Think of the TV quality in the 1960s as opposed to the high definition picture you get now. This is like going from 16s to Whole Genomes.

What Does The Metagenomic Process Look Like?

You first extract the DNA from the sample and then you sequence all of it. That is called shotgun sequencing. This was first demonstrated in Haemophilus influenzae. You sheer up the genome and you sequence it in short pieces. We don't have a sequencing technology that can sequence a whole genome just yet. Think of this as pieces of a jigsaw puzzle. You have all of these pieces and put them back together to get the picture on the box and that's your genome. If you have an isolate like Haemophilus influenzae, you only have 1 jigsaw puzzle. With Metagenomics, you are looking at a community of organisms or a microbiome. It is like if someone got 500 jigsaw puzzles, grabbed every piece from the different boxes, and stuck it all into one box and said put this back together, Metagenomics is like making 500 jigsaw puzzles in parallel. This sounds complex but the technology is improving so much, including the way we process the data, that we can pull out fairly complete genomes. 

We are now pulling out these genomes at an astonishing rate. Human stool samples are the poster child of this process. In the last 2 years, there have been 3 major studies and in the last one they pulled out 160,000 MAGs (metagenome-assembled genomes) out of the dataset! Those genomes are our reference database and we now have databases that are based on genomes instead of 16s like Green Genes. 

We have a database that we developed at the University of Queensland called GTDB which stands for Genome Taxonomy Database. Anyone in the world can bring their genome and put it up against this reference database to then tell them what the organism is. That is what our company Microba uses. For Example: We take a sample from you, we sequence 7 million reads (a sequence of base pairs corresponding to all or part of a single DNA fragment), we then take all of those and match them up against the reference database and that gives us a nice high-resolution profile of the organisms that are present in your gut. 

It is so exciting to see where this technology is headed and what we can offer for people suffering from all sorts of things. -Kriben Govender

What Is The Future of Gut Health Analysis?

We have sequenced over 5,000 gut microbiomes from the Australian public. We ask them if they would like to opt-in so these profiles can be compared to a database to analyse them and look for patterns. 75% of people have opted in because most people appreciate that this helps a scientific endeavor. We have been able to analyse this data and we have found amazing correlations:

-We can predict Inflammatory Bowel Disease with a 97% accuracy based on these profiles

-We can identify what type of IBD it is, whether it’s Crohn’s or Ulcerative Colitis, with 97% accuracy. 

When your gastroenterologist is trying to treat these conditions they have several treatments at their disposal. BUT their efficiency usually depends on the patient. You might say “try this treatment” and only 1 in 5 people may see improvements. Not only can we predict developing the IBD, but we can also predict what your response is going to be to a particular treatment just through your gut microbiome stool test! We see strong statistical signals for any condition we look at…..

-We can see particular microbial populations that are elevated when dealing with people with depression

-People that have dispositions for different types of cancer

Every condition you will see some signal in the gut microbiome. The gut microbiome is part of us and it has evolved with us. It plays an important role in our health. And when things go out of whack, you get changes in the gut microbiome that make the problem worse. Your gut health is one of the first things that reacts to changes in what's going on with your health. It can be used as an early warning of things coming up. 

That is why there is so much research focused on it right now. You are seeing 100’s of papers published every month regarding gut health. We have a huge potential to be able to use this information to guide treatments of different diseases. It will let you know ahead of time that you may have a disease coming. It is very new technology so the regulatory authorities are just coming to terms with this. We have to be careful about what we say about diagnostics BUT I can tell you that the research part of our company is seeing very strong, statistically significant correlations which we know will be very useful for diagnostics. 

In the future, when you diagnose an issue, then you will also know which organisms are absent or reduced and need to be there. If a certain organism shouldn't be there, you can design some therapeutics around this. We look at the healthy cohort, which was 8% of the total profiles, they were characterised by several microbial populations that were enriched in a healthy cohort in comparison to everyone else. We can start to think about if we could get these populations in cultures, they would be probiotics that you find in healthy humans, and they could help others. 

The majority of organisms have not been cultured. It is easier to culture gut microorganisms than those in a hot spring but about 30% of the species in the average human gut have been cultured. That means 70% haven't! We are finding that a lot of those populations that are in healthy people haven't been cultured before. We are spending time trying to isolate those and because we have their genetic blueprints, we can use that to generate isolating efforts. The potential is enormous.

What Does A Healthy Gut Microbiome Look Like?

For Australians, we based this healthy gut on a survey looking at people with a healthy lifestyle. This included people that aren't on medications and that have not reported having a disease. We used this as the standard of what a healthy microbiome is. We found particular populations that are in people with healthy gut microbiomes. We rank people according to this healthy profile and we found a lady that was the epitome of health. She is 70 years old, no medication, high fibre diet for years, and she would be what I call the apex of the gut healthy microbiome. We are using these types of microbiomes to set that standard.

These bacteria are all species-level definitions of a healthy diet. We are in the process of going to subspecies because I believe more important information will be at that level. Most of these species don't have names yet because they have never been cultured! One of the best for a healthy gut microbiome is:

Faecalibacterium Prausnitzii

When you go to the genome level this species is 7-8 species, not just one! One of those species carry genes that contain an anti-inflammatory effect. A lot of health issues center around inflammation. When things are going bad in the body it is associated with inflammation. Organisms that have natural anti-inflammatory properties are associated with a healthy microbiome. As we are identifying the species, we also try to find out “what is it about this organism that makes it part of a healthy microbiome or part of a dysbiotic (unhealthy) microbiome.”

BUT every individual gut is unique. If you look at it from a forensics approach, if everyone had a fecal profile then you would know who has been where because it would be that specific. It may change over time but it is unique. When you look at thousands of individuals you see those signals. That boils down to a handful of species. Regarding IBD, we found 20 species that were characteristic of it!

There is a lot of individualism in each gut microbiome. So it is a complex ecosystem because: 

-You have the added immune system

-500-1000 species in your gut

-Signaling going on between our gut and ourselves. 

Despite that, we have strong signals that we can use to effect change in a positive way. When you get into a new area of research, a lot of things can be over-hyped and people can make a lot of claims. What you want to look at is good solid science. In the next 5-10 years, we will find different ways that we can help or eliminate the diseases or manage the disease state based on the microbiome.

What Species Have Negative Connotations?

The poster child would be Clostridium difficile. They are well-known pathogens and if they get a good hold on the gut they can throw things out of whack. Bilophila wadsworthia is one that we find in most people's gut microbiome and in certain conditions it can start having negative effects. People refer to these as opportunistic pathogens. Under a certain set of conditions, an organism can step up and then create a problem. Some of this comes down to sub-species. We are still learning more about this, every week there is a study about a particular organism under a particular circumstance which creates a bad effect in the gut.

I have had a stool test analysed by Microba and it was interesting to see. I had very little bifidobacteria and the whole section of metabolites was very interesting. -Kriben Govender

What is the Metabolite Part of Your Test?

This is possible because of Metagenomics. We have the full blueprints so we can predict what functions the organism are capable of. Part of this is what we can predict what metabolites they are producing. Metabolites are chemicals that either get used up or produced by organisms. One example is GABA (Gamma-Aminobutyric Acid): it is a modified amino acid. It is used by organisms as a food source. It happens to be one of our major neurotransmitters. That means that organisms in the gut can produce GABA which is beneficial to us. 

For instance, some studies show that the level of GABA production in the gut is related to depression. This showcases the gut-brain axis. There are many more metabolites in the report as well. The idea is that from metagenomics you predict your ability to produce those metabolites. In the future, we may be able to just read the metabolite profile instead of the DNA.

What is KEGG?

KEGG is the Kyoto Encyclopedia of Genes and Genomes. It was developed in Japan and it is telling you metabolic pathways and about a big chunk of the genes but not all of them. It is an important resource to interpret the metabolic potential of the organisms. As our understanding of the metabolism goes up we will be able to identify more and more of the peaks and we will get a higher resolution picture. 

Where is the Technology Headed?

Analysing the metabolites directly, this will also bring the price down to 15-20 AUD. This will bring it to a much broader audience. At the moment people might look at the price of 500 AUD and think that is a lot. But if you consider that 5 years ago it cost 10,000 AUD to do the same thing, that price is a lot more reasonable. The cost of analysing these profiles will become cheaper and cheaper (and if we go to metabolites it will get even cheaper!) I am excited to see millions of profiles. Every time you increase this database the statistical significance goes up. In the future, the same way you go to get your cholesterol tested at the doctor, your GP can also get your gut microbiome profile and have that as another set of information on how your health is.

Are There Machine Learning Algorithms Being Used?

This is how we are analyzing our predictability. We can predict IBD with 97% accuracy and we are using machine learning to help with this. It is a complex dataset and it can look at patterns that we may not be able to see by eye. We are applying algorithms, then training it, and then we have a test set. This is where you can assess the efficiency of machine learning. It loves data and the more information you can get, the better it can predict things. 

Should We Eat More Fibre Because of Butyrate?

Butyrate is a very important short-chain fatty acid because it contributes to health. Too little is not good and too much is not good either. If you take a stool test, you can get recommendations of what you should eat to increase or decrease it. Consider having a test done then work with a dietitian or nutritionist to look at improving your diet based on the results of the test! The test is also going to become more and more useful after time. Whenever you get your human genome test, they tell you to check out your profile often because they are always coming out with new information. This will be the same with gut microbiome profile testing.

What Are Your Top Recommendations for Improving Gut Health?

The best way I can tell someone to improve their gut health is by diet. Try a stool test and it will give you specifics on what to change in your diet. You may be deficient in something or you may have too much of something. This varies from person to person. This is an interesting study that came out of Israel: based on your profile they would give you bananas or chocolate. The majority of people get a better response from the bananas but in a small subset of people they benefited from chocolate! The generic thing to say is to eat more vegetables which is true but the interesting thing is when you get your personalised profile what is good for you may not be good for someone else. 

I can tell you to eat more vegetables but by getting your profile done you can get specifics. In the coming years, you will be able to get supplements and diet recommendations that are effective and for your specific profile. This is personalised medicine, I think this will be more popular in the upcoming years. 

I have been changing my diet. I cut down on my meat consumption and processed meat consumption. Exercise is important. One of the early benefits of changing your diet and exercising more is your mental health. And you see changes in your gut microbiome as well. In my profile, there have been some recommendations for different food groups that I adopted and I feel great! 

Stool analysis is the wave of the present and the future. These tests are predicting IBD, depression, and even some cancers at an affordable rate. With each test, you can also opt in to contribute to the scientific research! Remember that each individual is different and having a personalised test is the best way to determine what you should do to improve your gut health. Share this with a friend that would benefit from this information!