The gut microbiome in health and disease

We live in a microbial world. Microorganisms are everywhere, from the depths of the ocean to hot, acidic volcanic lakes. They are on and inside us. Our own ancient ancestor was a single free living cell and the tiny power-plants inside our cells (the mitochondria) were once free-living bacteria that invaded our single celled ancestor.

Our bodies are inhabited by a large number of micro-organisms that live in many distinct habitats: the skin, mouth, airways, gut, oesophagus, genitals, external auditory channel, hair on head, and nostrils (Spor et al., 2011; Marsland et al., 2013). While it was often cited that the number of bacterial cells outweighs the human ones by a ratio of 10:1 new estimates place the ratio rather on a 1:1 (Sender et al., 2016) - still a huge number of 38 trillion bacterial cells.

The composition, density, and species-richness of the microbial communities varies greatly between these different habitats. In the intestine the density of microorganisms increases from the start point to anus (Schroeder and Bäckhed, 2016). There’s a large diversity in the classes of microorganisms that make up our gut microbiome. It consists of bacteria, viruses, archaea, fungi, and other maybe even other Eukaryota. In the gut the bacteria and viruses dominate in numbers and both groups may be about equally abundant (Ogilvie and Jones, 2015). Yet of all classes of microorganisms present in the gut we by know by far the most about bacteria and hence most of the discussion in this article will be focused on bacteria.

A substantial part of the viruses present in the gut are bacteriophages (or phages for short) (Abeles and Pride, 2014). Phages are viruses that prey on bacteria. Phages have been found in the bloodstream of healthy humans and it is believed that phages can cross mucosal surfaces (such as the lining of the gut) (Abeles and Pride, 2014). ((Even more surprising is the fact that bacteria, probably in dormant form, have been observed in the human bloodstream (Damgaard et al., 2015; Païsse et al., 2016; Potgieter et al., 2015))

Every individual harbors a combination of approximately 160 bacterial species in their gut. In total around 1,200 bacterial species have been identified in the human gut, hence each human harbors only a small set of the potential bacterial diversity (Schroeder and Bäckhed, 2016).
While everyone has this different composition of bacteria in their gut we can identify three large groups, the so called enterotypes. The three enterotypes are characterized by the relative abundance of bacterial species belonging to one of the following bacterial ‘groups’ (correct term is genera): Bacteroides (type 1), Prevotella (type 2), Ruminococcus (type 3) (Arumugam et al., 2011). This classification has been questioned (Knights et al., 2014). These three groups are likely not as discrete as the original work suggested but this classification remains useful to help us thinking about this complex bacterial community. This enterotype system also points out that certain species of bacteria co-occur in the human gut. The reason for that is that certain species of bacteria rely on each others metabolic activity while being in competition with other species that share the same niche (Schroeder and Bäckhed, 2016).

The composition of our gut microbiome is determined by various factors including host genetics, age, diet, lifestyle, smoking status, hormonal cycles, delivery mode (birth), use of specific drugs (e.g. antibiotics), hygiene, cold exposure, pro- and prebiotics (D’Argenio and Salvatore, 2015; Falony et al., 2016; Sommer and Bäckhed, 2013; Schroeder and Bäckhed, 2016; Zhernakova et al., 2016).

Is was conventionally thought that fetuses are sterile and that colonization with bacteria starts during birth. However, recent studies suggest that bacteria may be present in the womb (Schroeder and Bäckhed, 2016). Babies born in the natural vaginal delivery mode are coated with a microbiome from the mother that is absent in those born through cesarean section (Neu and Rushing, 2011). Breast milk feeding also has a big impact on the gut microbiome of babies because human breast milk contains specific oligosaccharides, different from those present in cow milk, that influence the microbiome composition (Barile and Rastall, 2013).

Family members tend to have similar microbiomes, which could reflect either similar environment or the effect of host genetics on microbiome composition (Spor et al., 2011). People who consume a mostly animal protein-based diet tend to have a Bacteroides enterotype (type 1) while people who consume a lot of carbohydrates tend to have a Prevotella enterotype (type 2) (Wu et al., 2011).

Interestingly, the effect of diet on gut microbiome can be age-dependent. In a recent study published in BMC Microbiology researchers fed chicken protein to young and middle aged rats and observed that in young rats chicken protein caused an increase in the probiotic bacteria belonging to the Lactobacillus group while in middle aged rats the relative abundance of Lactobacillus species was decreased by the chicken protein (Zhu et al., 2016a).

Normal physiological role of our microbiome

Our gut microbiome has various roles in health and disease. Some of these have been known for a long time (for example the role in digestion), while others are more recent discoveries (such as the role in various diseases of other organ systems). The gut microbiome has been described as the “forgotten organ” owing to the large role it plays in normal physiology (O’Hara and Shanahan, 2006).

The roles of the normal gut microbiome

• Aiding digestion (e.g. complex carbohydrates)
• Vitamin biosynthesis (e.g. folate, riboflavin, B12, vitamin K,...)
• Production of numerous natural products (short-chain fatty acids, secondary bile acids, conjugated linoleic acids, neurotransmitters, hydrogen sulfide,...)
• Suppress the growth of pathogenic bacteria
• Metabolizing drugs and environmental toxins
• Modulating immunity
• Influence the development and homeostasis of host tissues (e.g. bone and gut wall)

Collectively, the bacteria in our guts harbor more than 150-fold the number of genes present in the human genome (Lepage et al., 2013). This ‘extra genome’ greatly increases the metabolic capacity of the human body. Bacteria inside our guts help us to digest our foods, detoxify drugs and environmental toxins, produce vitamins, and a whole host of other beneficial metabolites. One surprising example of detoxification by our gut microbiome is that of dietary advanced glycation products (AGEs) (Bui et al., 2015). Studies performed in the last two decades illustrate that dietary AGEs may promote the development of a range of diseases including diabetes, dementia, and heart disease (Vlassara et al., 2016).

The gut microbiome may be very important in the development of our immune system. In a recent study the researchers observed that pet store mice had a much higher survival rate than laboratory mice after bacterial infection. Interestingly, laboratory mice were protected from the infection by co-housing them with pet store bought mice indicating that the immunity was (Beura et al., 2016). The "hygiene hypothesis" has long proposed that the lack of exposure to infections early in life leads to immune system abnormalities such as allergies and autoimmune diseases (Willis-Karp et al., 2001). More recently with the realization that the normal microbiome plays a larger role the “old friends hypothesis” and “disappearing microbiota hypothesis” suggest that our modern lifestyle leads to the loss of “old friend” microorganisms that are needed to properly educate our immune system making us more vulnerable to allergies and autoimmune disorders (Candela et al., 2012).

The bacteria in our gut can be classified in four groups based on their relation with their human host: mutualists, commensals, pathobionts, and pathogens. Mutualism is when both partners (the bacterium and its human host) benefit from the association. Commensalism is when one partner benefits from the association and the other is not harmed. Pathobionts are symbiotic bacteria that can turn pathogenic under certain genetic or environmental conditions. And finally, pathogenic bacteria cause disease . Researchers use the term “dysbiosis” to indicate that there’s an unbalance in the microbiome that is capable of initiating or propagating disease (Round and Mazmanian, 2009).

Gut microbiome in aging and disease

The father of immunology and gerontology Elie Metchnikoff conducted, among his extensive diversity of research, also studies on the gut microbiome in bats, horses, birds and humans (Cavaillon and Legout, 2016). Metchnikoff suggested that aging was caused by the toxic effects over a lifetime of putrefaction products produced in the colon (autotoxins). He also observed that people in some Southern European countries where long lifespans are common tend to consume a lot of sour milk and yogurt containing lactic acid bacteria. He suggested that lactic acid bacteria could prevent putrefaction and hence promote longevity (Korenchevsky, 1961). While Metchnikoff’s bacterial autointoxication theory is now considered dead, the idea that our microbiome is involved in human disease is very much alive (Bested et al., 2013).

A large number of chronic diseases have been linked to the gut microbiome including cardiovascular disease (Wang et al., 2011), rheumatoid arthritis (Scher and Abramson, 2011; Zhang et al., 2015), obesity (Turnbaugh et al., 2006), certain cancers (Schwabe and Jobin, 2013), asthma (Huang and Boushey, 2015), inflammatory bowel disease (Kostic et al., 2014), and psychological problems (such as anxiety and depression) (Cryan and Dinan, 2012). In many cases we are not certain if a change in the microbiome is causing the disease or if the disease is causing the change in the microbiome - but for certain diseases interventions that change the microbiome improve the disease (at least in animal models).

I still remember when I looked through the table of content of the April 7th, 2011 issue of Nature and saw a paper titled “Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease”. Before that day I had not really given any thought about a possible role for microorganisms in aging or non-communicable age-related diseases. I assumed that the 100-year old bacterial autointoxication theory of Metchnikoff had long been disproven and hence bacteria didn’t deserve my time. Then this Nature paper pointed out that dietary choline was transformed by the gut microbiome into the vascular toxin trimethylamine (TMA). When mice were fed choline they developed atherosclerosis but this was prevented by treating the mice with antibiotics (Wang et al., 2011). Trimethylamine is later by the liver oxidized to trimethylamine oxide (TMAO) and to my knowledge we still don’t know if the harmful effects come from TMA, TMAO or both. TMAO levels are higher in omnivores than in vegan/vegetarian people and challenge with a 250mg dose of carnitine (another dietary molecule that is converted into TMA by the gut microbiome) increases plasma TMAO levels in omnivores but not in vegan/vegetarians (Koeth et al., 2013). Possibly a vegan/vegetarian diet shifts the microbiome away from TMA-producers. People with higher plasma TMAO levels have more atherosclerosis (Randrianarisoa et al., 2016) are at greater risk for a cardiovascular event (Tang et al., 2013; Zhu et al., 2016b).

Mice that have been engineered to develop rheumatoid arthritis (joint inflammation) have a much milder presentation when they are raised in germ-free conditions compared to normal (SPF, see above) conditions. Furthermore, the introduction of a single bacterial species (segmented filamentous bacteria) is enough to induce arthritis in germ-free mice (Scher and Abramson, 2011; Wu et al., 2010). In a recent study researchers wondered why autoimmune diseases (allergies and type 1 diabetes) are more prevalent in Finland and Estonia compared to Russia (Vatanen et al., 2016). The researchers discovered that the microbiome of children born in Finland and Estonia differs from Russian babies. Bacteria species present in Finnish and Estonian children have lipopolysaccharide (LPS) with a chemical structure that is different from the LPS found in the bacterial species common in Russian children. LPS is part of the bacterial cell wall and is a strong pro-inflammatory molecule. However repeated exposure to LPS will induce tolerance, meaning that further exposure to LPS will no longer induce inflammation. The researchers discovered that the LPS produced by bacteria from the Finnish and Estonian children does not induce tolerance. The researchers injected LPS from either Finnish/Estonian or Russian children into animals that were genetically engineered to develop autoimmune diabetes. Treatment with LPS-derived from Russian but not from Finnish/Estonian bacteria caused a delay in disease onset.

Given the high bacterial load in the inside (lumen) of the gut it is of vital importance that the gut barrier prevents the translocation of these bacteria to the bloodstream. This barrier is based on a mucus layer that keeps the gut content away from the wall, an epithelial cell layer, and active antimicrobial peptides and antibodies that are secreted near the surface (Ostaff et al., 2013). However in certain conditions this barrier may be broken and bacteria and bacterial products are then able to invade the gut tissue leading to inflammation. Several factors such as alcohol consumption, energy-dense diets, major trauma (hypoperfusion of the gut), toxins, changes in the microbiome, and virus infections are known to increase the permeability of the gut (Bischoff et al., 2014). Increased gut permeability has been observed in people with or at risk for type 1 diabetes (Vaarala et al., 2008; Li and Atkinson, 2015). Increased LPS levels (Creely et al., 2007) and gut bacteria (Sato et al., 2014) have been found in the blood of patients suffering from type 2 diabetes. Increased LPS levels in plasma have also been observed in old compared to young mice and this might be (partially) responsible for the chronic inflammation (inflammaging) that is observed to occur with age (Kim et al., 2016).

Roundworms grown on UV-killed bacteria live longer than worms grown on living bacteria (Garigan et al., 2002; Gems and Riddle, 2000; Win et al., 2013). However, the roundworm were exposed to the geroprotective drug metformin then their lifespan was actually reduced in the absence of bacteria! In contrast when living bacteria are present metformin-fed worms live longer. In worms metformin increases lifespan by influencing bacterial metabolism (Cabreiro et al., 2013). Interestingly, recent research shows that intestinal bacteria may also be involved in the therapeutic effect of metformin in humans (Forslund et al., 2015). Roundworms fed the lactic acid bacterium L. gasseri SBT2055 (Nakagawa et al., 2016) or Bacillus subtilis (Sánchez-Blanco et al., 2016) live longer than those grown on the traditional E. coli bacteria. Another study investigated the effect of bifidobacteria on lifespan in C. elegans (Komura et al., 2013). These authors observed a dose-dependent lifespan extension of bifidobacteria mixed with E. coli. However the question remains if worms grown on probiotic bacteria will also live longer than bacteria grown on UV-killed bacteria or on bacteria-free medium.

Germ-free mice live longer than conventionally raised mice (Gordon et al., 1966; Snyder et al., 1990; Tazume et al., 1991). Interestingly, calorie restriction does not further extend the lifespan of germ-free mice (Snyder et al., 1990; Tazume et al., 1991). Germ-free mice have reduced IGF-1 serum levels compared to conventionally raised mice (Schwarzer et al., 2016) and transplantation of the gut microbiome from conventionally raised mice into germ-free mice results in increased IGF-1 serum levels (Yan et al., 2016). The IGF-1 pathway is one of the best established targets in modulating longevity of laboratory animals. Animals with decreased IGF-1 pathway activity live longer (Junnila et al., 2013).

The gut microbiome changes with age in humans (Claesson et al., 2011; Biagi et al., 2016; Kong et al., 2016; Odamaki et al., 2016) and in fruit flies (Clark et al., 2015). The microbiome composition differs between centenarians and 70-year olds. Centenarians have higher levels of pathobionts and lower levels of symbiotic bacteria this is associated with an increased inflammatory state. The researchers also identified that Eubacterium limosum and its relatives are more than 10-fold increased in centenarians providing a ‘microbiome signature’ of exceptional longevity (Biagi et al., 2010). The loss of microbiome diversity has been linked to increased frailty and reduced cognitive performance (Claesson et al., 2012; Jackson et al., 2016). A recent study found that specific patterns of bacterial species that live on the teeth are associated with all-cause and diabetes-related mortality in humans (Chiu et al., 2016).

How can we influence the composition of the gut microbiome to treat diseases?

The most radical way is by a so called “fecal transplant” in which fecal matter from a healthy person is introduced in a sick person. Fecal transplants are successfully used as a last resort treatment for drug-resistant Clostridium difficile infections.

In a “fecal transplant” one transplants all micro-organisms from a donor to a host but likely only a limited number of them are really necessary to achieve the desired effect. So rather than transplanting a cocktail of hundreds of species we could just make pills that contain a few types of microorganisms. These are the so called probiotics that are for sale in many health food shops and typically contain one or more bacteria (often lactic acid bacteria) and sometimes also some yeasts. More advanced probiotics are being studied for the treatment of specific diseases including obesity. Last month researchers reported in the journal Nature Medicine that living Akkermansia bacteria and more surprisingly dead ones improved obesity, blood lipid profile, and insulin sensitivity in mice on a high-fat diet. The reason why dead bacteria have a physiological effect is because a heat-stable bacterial protein binds to the human Toll-like receptor 2. The authors also report the preliminary results from a human trial with Akkermansia showing that the use of this bacteria is safe. The trial is still ongoing and we will have to wait longer to see if the use of Akkermansia pills reduces obesity and improves metabolic health in the human research subjects (Plovier et al., 2016). Researchers are also working on genetically engineered bacteria that can be used in the treatment of human disease. For example, bacteria producing the anti-inflammatory protein interleukin-10 have been developed for the treatment of Crohn’s disease (Braat et al., 2006).

A third approach is to eat products that stimulate the growth of specific microorganisms. One such product are dietary fibers. Human breast milk oligosaccharides are currently being commercialized for infant nutrition (Barile and Rastall, 2013). Just last month the biotech company Inbiose that developed the technology to produce human breast milk oligosaccharides partnered up with DuPont to commercialize this technology. They plan to submit for regulatory approval in 2017 to bring infant nutrition supplemented with human breast milk oligosaccharides to the market (http://www.danisco.c...ents-to-market/).

Fourthly, we can kill off bacteria using antibiotics. Indeed, antibiotic treatment seems to have some beneficial effects on rheumatoid arthritis (Ogrendik, 2014) and can prevent choline-induced cardiovascular disease (Wang et al., 2011).

Finally, we can think of specific drugs to steer bacteria - such as those influencing quorum sensing (Hentzer and Givskov, 2003; Thompson et al., 2015).

Conclusion

The role of the microbiome in health has become a hot topic in recent times. For example, when one types "microbiome AND health" in PubMed one sees an exponential increase in papers published in the last 10 years from as little as 187 in 2009 to 2,612 this year. Nevertheless, we still have a lot of holes in our understanding of how the microbiome influences health and aging. For some diseases such as rheumatoid arthritis we now have evidence that suggests that the microbiome plays a causal role in the disease as the introduction of a single bacterial species can induce the disease in genetically susceptible animals. For other diseases such as psychological ones the role of the microbiome is less well established. It is also important to understand that the effect of the microbiome on health may be complicated. In the roundworm C. elegans the presence of living bacteria shortens lifespan while the presence of living bacteria is essential for metformin's life extension effect in the same species. While we have methods to influence the gut microbiome, the lack of understanding of how these changes may impact health currently restricts the use of these interventions. What we do know is that a healthy diet that contains enough fibers (which promote the growth of specific bacteria) improves health. More research will undoubtedly lead to insights in how microbiome manipulation methods can be used to improve our health.