Stool Testing – The Bottom Line

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Sneaky Peek

Poo is at the centre of a whole new form of medical diagnostics and treatment. And a handful of organisations now allow us, as citizen scientists, to find out, via a sample of our poo, some more information about the microscopic life inside our guts. Read on for more information.


INTRODUCTION

Bacteria, in all their microscopic majesty are fundamental to human existence. I am beginning to think that maybe, we humans live in a microbial world, not the other way round. One well-known scientist in this field has gone so far as to ask “are humans just elaborate vessels for the propagation of microbes?”

We need to know about them to understand ourselves. Our ability to do this has increased exponentially since we have been able to identify them via their 16S rRNA gene. This gene is found in all bacteria but parts of it differ in each species, making it a very good means of identification. We no longer have to propagate bacteria in petri dishes in order to find out who they are.

This technological advance has facilitated a giant leap in terms of learning about our resident gut bacteria, because a great many of these bacteria are so well adapted to life on the inside, they refuse to grow on petri dishes. Before 16S, we didn’t even know they were there. 16S has allowed us to begin to identify the full gamut bacteria that inhabit our gut and other body niches (1).

For a fee, companies such as Ubiome, Mapmygut and Genova Diagnostics now test poo, so that individuals can have their gut microbiome profiled and their results explained to them.

Although we have no specific health concerns, I really wanted to support the British Gut Project so earlier this year, I took a poo sample from each of us (6 in total) and sent them off. This is not a diagnostic test but a crowd-funded project to gather some baseline data on what a “normal” microbiome looks like in humans. The results they have gathered so far, illustrate the huge variation between individuals in terms of their bacterial inhabitants. Whilst we are over 99% identical to other humans in terms of our human genes, our bacterial overlap is only around 30%.

So far, I have only looked in detail at my own results. These consist of a long list of bacteria with their percentage relative abundance and some very basic information on how my sample compares to those on a similar diet to me, those of the same gender, similar BMI and similar age. It also tells me which bacteria are most abundant within me and which ones are more abundant in me compared to the “average population”.

I have researched every genus of bacteria listed as inhabiting my poo. Not only that, I have created a bacterial spreadsheet of the information I gathered, so now I have a reference document that I will add to as I work my way through the rest of the family’s poo data.

 

MY BACTERIAL PROFILE - SOME BACKGROUND

You can look at the contents of my poo – bacterially speaking, I mean – don’t worry, there are no photos, although there is this infographic.

To try to give an overview, I calculated the percentage of each major phylum of bacteria within me (plus Archea, which aren’t strictly bacteria) and then the percentage of each class of bacteria within those phyla.

It may help to hold in mind the taxonomic ranking system used to identify things in biology – Kingdom (bacteria) Phylum, Class, Order, Family, Genus, Species, Strain. Kingdom is the most general ranking and strain is the most specific. The major bacterial phyla are Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Verrucomicrobia, Tenericutes, Cyanobacteria & Fusobacteria. Got that?!

As another way of looking at the information, I colour-coded the different genera into my own classification system based on WHAT THEY DO rather than WHO THEY ARE.

 

THE RESULTS

I contain more Firmicutes than Bacteroidetes. We used to think this was an important distinction but it appears not to be so decisive now. I don’t have much Actinobacteria and I would like more of these. This phylum includes Bifidobacteria, which we know to be beneficial. I would prefer less Proteobacteria – to me this phylum is a bit like Slytherin in Harry Potter – I appear to have an eclectic mix of these lot but in relatively small amounts. We don’t know all that much about Verrucomicrobia but my Mollicutes from the phylum Tenericutes may be parasitic. I have a small amount of Cyanobacteria which is interesting to me because they photosynthesise. Maybe the sun really does shine out of my backside? And I have a small amount of Archea which are also single celled micro-organisms but they are distinct from bacteria.

From my attempt to colour-code my bacteria based loosely on function, I can see that the “green” ones (which break down complex carbohydrates – plant material) dominate. This is not a surprise given the vegetables we get through.

I have a some ”BLUE” (special) bacteria. Take Akkermansia for example, so far as I know, the only known species of Akkermansia is Akkermansia muciniphila – this is a helpful bacteria which feeds on our mucus lining, produces helpful by-products, is linked with healthy metabolic responses, healthy gut barrier function & leanness. (2, 3, 4). I have a small amount (about which I am glad) but I am not surprised that I don’t have much. I also have a small amount of bacteria from the Christensenellaceae family, hopefully this is Christensenella genus as this is linked to people of a healthy weight. Roseburia is a whole grain/complex carbohydrate digester and produces the important by-product butyrate (which helps keep my gut lining healthy) and Dorea is also a butyrate producer. Finally – Bifidobacteria – this is a highly beneficial genus to possess. it is not unusual for these to be present in small numbers but ideally, I’d like some more!

I have a little of the “YELLOW”  bacteria – Rikenellaceae – fat LOVERs. And I am a fat LOVER. I try to be sensible about my intake of fat and stick to “good fats” so I am glad I don’t have lots and lots of fat-loving microbes. I think if I’d done this test before we started on our Wondergut journey, there may have been more of these.

There are quite a few “ORANGE” bacteria (o.k. if not too many of them), mostly in relatively low numbers and I hope it stays that way. This is where I think diet and lifestyle choices come in. I imagine my bacteria as an orchestra. The main composition stays reasonably fixed – a wind (!) section, a strings section, keyboards, percussion and voice but the proportions and their relative influence alters depending on the environment with which I provide them. What I eat, the level of stress I am under, how much I am sleeping, the medication I am taking (none), my interactions with nature – all affect who gets the upper hand and therefore, who is maintaining my gut lining, managing my immune system, influencing my gut nervous system & talking to my brain via my vagus nerve. (Find out how to CARE FOR YOUR MICROBIAL SELF - ideas on how we can maximise our chances of keeping our orchestra balanced).

I am not surprised I have some “RED” bacteria (not wanted so far as we currently know) – my digestive system does border on the sensitive (although this has been much improved since we started our Wondergut approach) and I have no doubt these blighters are sitting waiting for their 15 minutes of fame – I intend to minimise their chance of getting any sort of a foothold by doing my best to follow the main tenets of the Wondergut Wisdom Brain Prompt.

The “PURPLE” ones (some unhelpful, some helpful) sum up for me just how complicated our relationship with our bacteria is. In just one genus, there are many species and even strains of species. Different species and strains do different things and have differing effects – both on us and on the other bacteria around them. Even the same species can have a beneficial effect in some ways and a detrimental effect in others – the classic example is Helicobacter pylori – maligned for causing stomach ulcers but now shown to protect against asthma and gastro-oesophageal reflux disease (5).

Finally, we come to the “BLACKS” which drove me nuts because as a non-scientist, I could find very little information about them at all.

I have 4 types of rare bacteria – Rhizobiaceae – a bacteria that is symbiotic to plant roots – helping to fix nitrogen from the soil. Anaeroplasmataceae – a family apparently found in the rumens of cattle and sheep. We did own some goats once…….. Delftia – the only species I could find is Delftia acidovorans which makes gold nuggets with the assistance of another microbe, Cupriavidus metallidurans. I am disappointed that I don’t have Cupriavidus, so I won’t be sifting my excretia for golden nuggets any time soon. And finally, Cerasicoccaceae, about which I can find no information at all.

 
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SO WHAT?

I have found the entire process very enlightening and I am glad to have been able to support The British Gut Project. I intend to do another sample in due course through Ubiome or MapMyGut in the hope that the results come with a bit more information to make it easier for me to interpret. I did these tests early on in our adoption of the Wondergut approach to eating and living, so I will be interested to see what, if anything, has changed on the next test. I am also looking forward to going through the results of the rest of my family to see what we have in common and where we differ from a bacterial perspective.

Perhaps most enlightening though, has been what I have learnt about the shortcomings of these tests. I have learnt that there are a number of reasons why we should exercise caution before relying on the results to take strong measures, such as taking antibiotics or cutting out food groups in order to shift our microbial population. This especially applies if we are trying to do this without professional guidance.

 

Reason 1: Detail To Genus Level Is Not Enough Detail

The British Gut Project classifies bacteria down to genus level but there is enormous diversity of function within the same genus – at species level and even at strain level beyond that. I contain Bacillus genus – this could be bacillus anthrax which can cause the potentially fatal bacterial illness, anthrax or it could be Bacillus subtilis, a normal inhabitant of human guts. I have Pseudomonas genus but there are 191 species of Pseudomonas, many of which are pathogenic but many are not. I have Bifidobacterium genus but which species – longum, bifidum, breve, infantis, dentium, ruminantium?

In order to really understand which bacteria are journeying with me, I need information down to species or even strain level. If you intend spend the cash on a stool test, ensure they will identify your inhabitants down to species level and, where relevant, strain level.

 

Reason 2: Poo Is Not The Perfect Substrate

Our poo is around 30-40% bacteria. Getting a bit of poo on a sample stick and posting it to a laboratory is a non-invasive means of sampling the bacteria that were living in our colon, have died and have come out in our poo. However, some scientists question how accurate a sample this can be. Does it fully represent those bacteria living in the ascending colon (the bit straight after the small intestine) to the same extent that it does the descending colon – the bit before our anus? If we had sampled our poo the day after, would the results be very different? What about the difference between an early morning poo and a night time poo?

Some bacteria live deeply embedded in the mucus lining of our gut. To what extent will they be represented in that piece of poo into which we dabbed the sample stick? Taking a biopsy of the gut lining and mucus layer would probably give us a more accurate idea of what is going on in there but that is an invasive procedure which cannot be carried out in our bathrooms.

 

Reason 3: How Accurate Are The Results?

It doesn’t take much googling online to find the person who tested the same piece of poo with two different stool test providers and got a list of different bacteria. Since the genetic information obtained from sequencing the 16S rRNA gene is then put through an algorithm to identify to which bacteria it belongs and the methods of interpreting this information differ – there can be disparity in bacteria identity in the final result. In short, which bacteria the test says you have depends on the algorithm and reference database used. Whilst matches greatly outnumber mismatches, we need to be aware of the potential for mismatches. Treatment needs to be based on sound information – if we carry out one stool test we think we know which microbes inhabit us, if we carry out two, we are no longer totally sure (6).

 

Reason 4: Does Function Matter As Much As Identity?

Bacteria adjust quickly to survive in changing environments and one way in which they do this is by sharing genetic information with one another. This is what makes them so adaptable. So even if we think we have pinned down WHO they are, do they stay who they are if they take on board some useful genetic information from another species? How relevant is knowing who they are if this can change? Distinguishing bacteria in terms of their identity without also identifying them in terms of function may be preventing us from seeing true patterns and connections.

A number of research projects (1) have contributed to trying to characterise a baseline of the diversity of microbes and their functions in humans. Rob Knight has recently taken it one step further with a sample of 265 people but instead of sampling once at a number of body sites, they did so repeatedly over a period of time. Instead of one microbial snapshot, they took several of them.

Amongst other things, they found that in more than 75% of participants 258 bacterial metabolic functions were found to take place in more than one body site, 176 were taking place in multiple body sites and 28 were taking place in all 6 targeted body sites (nostril, inside cheek, gum, back of tongue, poo and inside the vagina) and with those 28 metabolic pathways, at least 70% of bacterial genera carried them out. This means that there are some jobs nearly all bacteria carry out, no matter which body site they inhabit. These results suggest that the community of bacteria we carry may be primarily driven by function rather than a requirement for specific species of bacteria.

Not only that, but even in Knight’s small and uniform sample of humans, with extensive sampling over time, they found bacterial strains that have not yet been fully genetically identified. Knight concludes that many key properties of the human microbiome – both in terms of identity and function remain to be characterised even in healthy people. To be able to identify and repair an “abnormal” microbiome, we need to first understand the personalised microbiome in human health and that is going to be no mean feat to achieve (7).

Imagine the largest and most complicated jigsaw puzzle of interaction you can possibly imagine, that is what is going on inside you and what scientists are trying to piece together.

 

Reason 5: Devil In The Detail

Whilst we may be starting to be able to identify, with reasonable accuracy, which bacteria we contain, we also have to work out the influence each individual strain has on us as well as the extent to which they act in consort with one another, which they certainly do. Bacteria exhibit an astonishing level of organisation amongst themselves (google quorum sensing or try bacterial biofilms for just a couple of examples) and that is before we even begin to delve into their interactions with the fungi, viruses and parasites (such as worms) that also inhabit us. Are some more influential in their particular community than others (keystone species)?  There are hundreds of thousands of potential studies to be done to try to start to piece the jigsaw together. It really is a mind-boggling endeavour. Decoding the human genome was child’s play compared to this.

 

Reason 6: What Is Normal Anyway?

The aim of various major research projects (1) has been to get some idea of what is “normal” in terms of the population of our gut microbiome but Jeff Leach and others believe that we can’t really get a good baseline whilst looking at Western populations because our Western lifestyle has already affected our microbiome. This has prompted a drive to look at ancestral populations that have not yet been exposed to the Western lifestyle. There are not many of them left but Jeff Leach, Maria Dominguez-Bello, Martin Blaser and of course Jeff Gordon are doing this – looking at rural populations in Malawi, Burkina Faso, Namibia, Botswana & the Hadza tribe in Tanzania. We need to know about the inhabitants of the guts of this baseline population – we need the “before” of the “before” and “after” to see the impact of Western lifestyle on our gut microbes.

Mainly, research is showing that these ancestral populations have a much more diverse gut microbiome compared to us Westerners, along with almost non-existent levels of non-communicable diseases – diabetes, autoimmune illnesses, cancer and cardiovascular disease. They are also finding that there are no clear patterns in terms of humans and their microbe populations – certainly not based on the questions and assumptions that have been the starting point of our analysis so far.

Until we really know what healthy looks like, it is going to be difficult to start to make sense of what diseased or dysbiotic looks like, let alone to know how to intervene to change this.


Wondergut Wisdom

Despite these caveats, I think these tests are remarkable. They are starting to show what is going on inside us in terms of microbial life. This has got to be a good thing. We have to start somewhere. We must remember though, that this area of science is still very much in its infancy, so we need to be circumspect about interpreting the results of our stool tests too categorically, whilst at the same time acknowledging that the state of our poo is a very useful general indicator of the state of our gut health.

Even if we feel we have no need for stool testing, we can start looking down the loo after we poo and comparing it to the Bristol Stool Scale Chart to start to be more in tune with what our guts are doing because we now know this to be inextricably linked to our health.


References

  1. Human Microbiome Project – 5 year programme by U.S National Institute of Health launched in 2007 involving 250 healthy young adults.

    MetaHit Project – a survey which began in 2008 by scientists in Europe involving the study of the gut bacteria of almost 300 Europeans to establish associations between gut microbes, health and disease.

    American Gut Project – Rob Knight’s lab at University of California runs a crowd-funded project founded by him and Jeff Leach. It is aimed at gathering as large a data set as possible – basically looking at the poo of as many people as possible to see which microbes inhabit them to start to give us some idea of what “normal” is if, indeed, there is a “normal” in terms of our gut microbes. As of early 2017, there have been over 10,000 participants.

    British Gut Project – the British Equivalent of American Gut Project but led by Tim Spector and run by the Department of Twin Research at Kings College London.
     
  2. Pill-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function by Noora Ottman et al 1 March 2017 journal PLoS ONE 12
  3. Microbe Profile: Akkermansia muciniphila: a conserved intestinal symbiont that acts as the gatekeeper of our mucosa by Willem M.de Vos May 2017 Microbiology.
  4. Strategies to promote abundance of Akkermansia muciniphila, an emerging probiotic in the gut, evidence from dietary intervention studies by Kequan Shou March 2017 Journal of Functional Foods 33 194-201.
  5. Missing Microbes by Martin Blaser
  6. Segal’s Law 16S rRNA gene sequencing and the perils of foodborne pathogen detection within the American Gut Project by James B Pettengill and Hugh Rand
  7. Strains, functions and dynamics in the expanded Human Microbiome Project by Rob Knight et al 10.1038 nature 23889