Where do we stand in unravelling the gut microbiome?

Delft, 1681. Antoni van Leeuwenhoek, the father of microscopy, places a smear of faeces under his microscope and sees more than a thousand living 'animalcules' crawling around. It is the first evidence of the microbiome. Yet, it is not until the start of this century that research into the microbes in our gut -collectively known as the gut microbiome - truly takes off. Modern techniques such as DNA sequencing make it possible to map entire communities of bacteria, fungi and viruses in faecal samples. Scientists soon discover that the gut is teeming with microbes and, contrary to what was previously thought, most of them are not harmful. In fact, they help keep us healthy.

Scientists collected vast amounts of data from large groups of people and identified links between the composition of the gut microbiome, eating habits, environmental factors and disease. They observed that certain microbial patterns, known as enterotypes, occur more often in people with conditions such as obesity, Crohn’s disease or Parkinson's disease. That raised high expectations. If a particular bacterial composition goes hand in hand with disease, could we change that composition to treat patients?

In some cases, the answer is yes. One example is infection with Clostridioides difficile, a bacterium that produces toxins that damage the intestinal wall. Symptoms include severe diarrhoea, fever and abdominal pain or cramps. The bacterium is also notoriously difficult to treat with antibiotics, and frequently returns after treatment. Wageningen microbiologist Willem de Vos demonstrated, together with doctors at Amsterdam UMC, that 'renewing' the microbiome can help these patients. In a stool transplant (or faecal microbiota transplantation), patients receive the microbiome of a healthy donor, whose bacteria outcompete the harmful pathogen.

More than ten years after that groundbreaking study, the Netherlands now has a dedicated faecal transplant bank, which stores stool samples from carefully selected healthy donors. Risks remain, however. ''The faecal transplant bank tests stool for pathogens, but researchers cannot screen for every disease-causing organism, so there is always a chance that a transplant also passes on unfavourable gut residents,'' explains Gwen Falony, associate professor of Host-Microbe Interactomics at Wageningen University & Research (WUR). Scientists are therefore working on a safer and more patient-friendly alternative: a pill containing a cocktail of microbes designed to achieve the same effect.

Beyond this particular infection stool transplants have made little headway. For other conditions, such as Crohn’s disease and ulcerative colitis, the results are far less clear-cut. A faecal transplant can change the microbial composition of the gut, but this does not necessarily lead to clinical improvement. This is despite the established link between the microbiome and disease. ''Previous studies have convincingly demonstrated that gut bacteria play a role in Crohn’s disease. We have also found that eighty per cent of people with the condition have a characteristic microbiome profile, which we find in only ten to fifteen per cent of healthy people,'' says Falony.

Even so, that correlation does not prove that one causes the other. An altered microbiome could simply be a consequence of the disease. The relationship between host and microbiome runs in both directions: the microbiome influences how the gut wall functions, but an inflamed gut also changes the environment in which microbes live, potentially altering their composition. ''In the case of inflammatory diseases, researchers can ‘restore’ the microbial side, but that does not necessarily cure the patient,'' Falony says.

In faecal microbiota transplantation, doctors use the microbiome of healthy donors. But defining exactly what constitutes a healthy microbiome remains a challenge. Researchers are, however, gaining a clearer understanding of which microbial patterns are less desirable: low numbers of bacteria, limited diversity and few butyrate-producing microorganisms. The latter have attracted increasing attention in recent years. As their name suggests, these microorganisms produce butyrate, an important energy source for the cells lining our gut. ''When the intestinal epithelium uses butyrate as an energy source, the cells consume oxygen from the blood,'' Falony explains. This is beneficial because it keeps the gut free of oxygen. When butyrate production declines, gut cells switch to anaerobic glucose metabolism. Oxygen supplied by the blood then seeps through the intestinal wall into the gut, which disrupts the microbiome’s composition.

The number of butyrate producers in the gut may be related to intestinal transit time: the time it takes for food to travel through the digestive tract. This determines, in part, how much time bacteria have to process nutrients and multiply. According to a hypothesis currently being investigated by researchers, this process in the large intestine works like a microbial relay race. First, fast-growing bacteria such as Bacteroides and Bifidobacteria pounce on the relatively simple carbohydrates. When those run out, other microbes, including butyrate producers, take over. Some of them feed on acetic acid and lactic acid, waste products left behind by the previous group. Others break down more complex dietary fibres.

When food passes through the gut very rapidly, the relay race stalls before the finish. Bacteria further down the chain, including many butyrate producers, then have too little time to feed and grow. Too slow a passage is not ideal either, says Falony, because bacteria then switch to fermenting protein. ''That can produce toxins that damage the gut wall and burden the liver.'' With an optimal transit time, protein fermentation remains limited. At the same time, butyrate producers get the time and space they need to thrive.

Alongside transit time, diet, and above all dietary diversity, plays a major role in shaping the gut microbiome. During a study, Falony and his colleagues asked participants to eat a monotonous diet of oatmeal, milk and water for six days. ''We observed a shift after just one day,'' he says. ''The gut microbiome contained fewer microbes, particularly fewer butyrate producers.'' After three days, some participants showed dysbiosis, an imbalance in the gut microbiome. Different types of bacteria need different food sources, which explains the effect. A varied diet with fibre from vegetables, fruit, pulses, nuts and wholegrain products therefore nourishes a broader range of bacteria and maintains both butyrate producers and microbial diversity. But more fibre is not necessarily better, Falony warns. In people with irritable bowel syndrome, extra fibre can increase gas production and worsen other symptoms. The key is to consume sufficient amounts of a variety of fibres, tailored to the individual's gut and needs.

Despite all the knowledge gained from large datasets, researchers are now reaching the limits of what modern DNA analysis can reveal. ''DNA tells us which bacteria live in the microbiome, but not necessarily what they are doing there,'' explains Laura Valk, a microbiome scientist and project leader at WUR. Although scientists can use the genes present to predict the functions of microorganisms, this approach has its limitations. Valk cites genes involved in butyrate production as an example. ''The fact that a bacterium carries a gene encoding butyrate production does not prove that it actually produces that compound in the gut. The gene has to be active, and that depends heavily on the surrounding conditions.''

This has prompted a shift in research priorities in recent years. Rather than focusing primarily on which microorganisms live in our gut, scientists now want to understand what they actually do there. They study which genes are active, how bacteria convert nutrients and which substances they produce as a result. That means returning to basics: culturing bacteria in the laboratory. That is no easy task. The gut contains no oxygen, and many gut microbes therefore cannot tolerate oxygen: they are anaerobic. For some, even the smallest amount of oxygen will kill them. WUR has developed a distinctive position in this field. With specialised culture rooms, growth media and years of experience, our researchers manage to isolate and study even these challenging gut residents. Clara Belzer, Professor of Microbiology at WUR, sums up the approach with a wink: ''Everyone says these anaerobic bacteria are so difficult to culture. We say: not for us!''

This cultivation work has already led to practical applications. A well-known example is Akkermansia muciniphila, a gut bacterium isolated and characterised by Wageningen researchers. The bacterium influences metabolism and may improve insulin regulation. It is notably scarcer in people with overweight or chronic gut inflammation. Akkermansia has since been developed into a postbiotic: an inactivated bacterium whose outer molecules can influence human metabolism and insulin regulation. Another example comes from research into breast milk. Research showed that breast milk contains human milk oligosaccharides (HMOs): complex sugars that babies cannot digest themselves, but that certain gut bacteria, such as bifidobacteria, can use as food source. That insight led manufacturers to add similar fibre mixes to infant formula to support babies' gut microbiome development. WUR contributed to this research and is now investigating how new insights into the gut microbiome can improve infant formula further. In this way, Wageningen's fundamental microbiome research translates into a tangible food product.

Akkermansia is a remarkable success story. In most cases, however, isolating and studying a single bacterial species does not work, according to Belzer. Gut bacteria live together, exchanging nutrients and waste products: the by-product of one bacterium may be a food source for another. They also stimulate or inhibit one another. As a result, a bacterium may behave very differently in the laboratory than it does among its natural neighbours in the gut. Wageningen researchers are therefore building so-called synthetic communities: artificially assembled, simplified microbial communities that resemble those found in the gut. Scientists compose these communities themselves, sometimes using just five bacterial species, other times several hundred. These communities allow them to investigate precisely which species depend on one another, what functions they perform and how bacteria respond as a community to different foods or dietary fibre.

This approach offers opportunities not only for fundamental research, but also for the food industry. Companies want to know, for example, whether a new type of fibre, an ingredient or a food product influences the microbiome. Instead of launching a large, costly study with volunteers straight away, they first test different products in a simulated microbial community at WUR. This helps identify which ingredients are promising enough to warrant further investigation. ''This fits into the broader research pipeline offered by WUR,'' says Valk. ''First, we use computer models to predict which interactions or effects to expect (in silico). Then we test them in the laboratory with cultured bacteria or synthetic communities (in vitro). Finally, we study the results in volunteers. Each step helps us approach the next one in a more targeted way and with a stronger evidence base.''

Where does gut microbiome research stand today? The much-anticipated prospect of a simple stool or DNA test that can diagnose diseases and then tell people exactly which bacteria they need has yet to become a reality. Researchers can identify groups of people with certain conditions based on their microbial composition, but they sometimes find the same patterns in healthy individuals. ''Moreover, different diseases sometimes show similar microbiome profiles,'' Falony says. He stresses that this currently limits the diagnostic value of the gut microbiome.

Belzer nevertheless sees potential for the future. ''A disease changes the environment in the gut,'' she explains. ''Think of changes in temperature, acidity, hormones and nutrients. The microbial community adapts to these conditions. As a result, the gut bacteria of people with the same condition may begin to resemble one another.'' Belzer expects that researchers may eventually be able to use such patterns to recognise certain diseases more accurately. But we are not there yet. First we need more insight into what bacteria actually do and how they interact and cooperate with their host.

Still, almost 350 years after Van Leeuwenhoek discovered his 'animalcules', we now know a great deal about the microbiome. We know many of its inhabitants by name, we understand how they process our food together and in what order, and we can culture them and even rebuild small communities. We also know that the microbiome is not simply a list of good and bad bacteria, but a dynamic ecosystem. Through laboratory cultivation, gut models and studies involving human participants, we are gaining an increasingly detailed understanding of how nutrients and our overall dietary patterns shape the microbiome.