By E.M. Bik, Science Editor at uBiome
Translated from Dutch. Read the original publication in the December 2016 issue of the Dutch Journal of Medical Microbiology (Nederlands Tijdschrift voor Medische Microbiologie).
The human body is home to large numbers of bacteria and other microbes, which are collectively known as the microbiome or microbiota, and which have important functions for our health, including for our digestive and immune systems. Recent innovations have resulted in better sequencing techniques and bioinformatics, resulting in a spectacular increase in studies of the human microbiome, particularly those of the intestines. This article gives a brief overview of the techniques used in microbiome research and provides new insights into this — until recently — invisible world. In addition, potential diagnostic and therapeutic applications will be discussed.
Microbiome, microbiota, intestinal bacteria, food
Antonie van Leeuwenhoek (1632-1723) was perhaps the first man who saw bacteria. With his self-built, simple but very effective microscopes, he studied all kinds of objects such as dander, hair, insects, blood, and swamp water. In letters to the Royal Society of London, he described his observations as tiny “Animalcules” in well and canal water. In 1683, Van Leeuwenhoek wrote another letter to the Royal Society. Although he always cleaned his mouth with salt and a piece of cloth, he had seen what plaque between his teeth and material under his self-built
laid microscope. There, too, he saw hundreds of little “critters.” His drawings and descriptions of these oral bacteria are probably the discovery of microbes associated with our body .
In the three centuries that followed, microbiology flourished — mainly in the field of pathogens. Most bacteria and viruses were considered pathogens, a logical assumption given the high prevalence of diseases such as cholera, tuberculosis, smallpox, and whooping cough. With the advent of vaccines and antibiotics, these infectious diseases were reduced in the twentieth century ink, and microbiology could also focus on the study of microbes in and on the human body.
We now know that our bodies accommodate complex communities of microbes that live on our skin, in the mouth, stomach, and intestines. These microbial consortia consist of bacteria, archaea, protozoa, and fungi species, which together are called the human microbiota (all microorganisms) or the human microbiome (all present microorganisms and their genomes) . An individual houses hundreds to thousands of different microbe species, mainly bacteria, and most microbes live in the large intestine. A widely used but now outdated statistic claims that our bodies contain ten times more microbial cells than human cells. In a recent publication, these calculations were re-done and it is now estimated that the number of microbes in and on the human body is approximately equal to the number of body cells, namely 4 x 1013 .
People are not the only habitat of complex microbial communities. Microbes are everywhere. Almost all living organisms — plant or animal life — are associated with microbes. In addition, microbial communities are found in a wide variety of environments such as in soil, seawater, glacial ice, and all kinds of surface plains indoors.
In the last twenty years, DNA amplification and sequencing techniques have improved spectacularly. This has led to a large number of studies of microbial communities . Many of these studies make use of the unique properties of the 16S rRNA gene, which encodes the RNA, which is part of the small ribosomal subunit . Ribosomes are part of all living organisms and are therefore rRNA genes in each bacterial genome. The 16S rRNA gene has a unique “mosaic structure,” with both conserved and variable regions. The conserved domains may be used for the design of universal primers that will fit on nearly every bacterium genome, while the intermediate variable domains (V1 to V9) are unique to each bacterial species and can therefore be used for identification and characterization.
The new molecular techniques made it possible to study the microbial diversity of a variety of sample types without having to be dependent on culture. This led to the discovery of many unknown bacterial phyla, and now there are millions of bacterial and archaeal sequences published.
In addition, it is now possible to sequence the complete genomic DNA in a sample, so that all the genes in a complex community, and their possible functions, may be studied (metagenomics). Finally, science is able to provide a variety of other innovations in the area of transcriptomics, proteomics, and metabolomics — new insights into the expression of all these genes under certain conditions . In addition to innovations in the field of DNA extraction and -sequencing, there is great progress in bioinformatics to analyze the vast amount of data generated by these techniques.
Composition of the human microbiome
The new techniques made it possible to analyze the complex microbiota of the human body. Human microbial consortia were found to vary by anatomic site but also by individual and time [7, 8]. In 2008 two large-scale microbiota studies were launched. The European MetaHit- consortium focused on the metagenomic analysis of feces , while the American Human Microbiome Project examined several body parts . Two recent, large Dutch-Belgian studies showed a major impact by food and medication to the composition of our gut microbiome [11, 12]. These projects have greatly expanded our knowledge about the identity and functions of the microbial inhabitants of the human body.
In the beginning, colonization begins at birth
The microbial colonization of our bodies begins at birth and is more or less complete around the third or fourth year of life . How this colonization process proceeds depends partly on the type of delivery. During natural birth, the baby first comes into contact with vaginal and rectal bacteria of the mother; whereas, during a Caesarean section, the baby first comes into contact with primarily skin bacteria. These new insights, meanwhile, inspire some parents to smear their newborns in the vaginal microbiota of mother. Also, how babies are fed in the first couple months determines the development of their gut microbiome. Breast milk contains bacteria, and artificial milk is sterile. Babies born by Caesarean section or fed with artificial milk thereby possibly follow a disturbed colonization pattern with a slightly higher risk for asthma, allergies and obesity at later age . Other factors that determine the development of the microbiome during childhood are the presence of siblings or pets and the composition of the microbiota of other family members . Children who grow up with a dog, or on a farm, have less chance of developing asthma . This seems to confirm the hygiene hypothesis, which asserts that exposure to bacteria is required in early childhood for the development of the immune system and a higher tolerance to environmental antigens.
Functions of the microbiome
The microbes in our body, particularly those in the intestine, are of great importance to our health. The joint genes of our gut bacteria are a well regarded functional extension of our own genome. The human gut microbiome contains 150 times more genes than the human genome, which encode a wide range of enzymes that we cannot synthesize ourselves . An important function of the gut microbiota is the digestion of nutrients that we can not break down by ourselves. The majority of plant carbohydrates and fibers can not be digested in the small intestine and arrive undigested in the large intestine, where they are fermented by our intestinal bacteria. The presence of intestinal microbiota thereby enables mammals to extract more energy from food. Germ-free mice, born and bred in sterile incubators, eat 30 percent more food but are thinner than mice with a conventional microbiome .
In addition to the digestion and fermentation of complex carbohydrates and fibers, the intestinal microbiota is involved in the production of short-chain fatty acids (e.g.
butyrate), fat metabolism, the correct development of anatomical structures in the large intestine, control of the immune system, vitamin synthesis, and the filling of voids in the intestine so that pathogens can not colonize [8-10, 19]. There is also increasing evidence being found for the existence of mutual communication routes between the gut microbiota and the central nervous system, called the Brain-Gut Axis, where there are even indications that intestinal bacteria have an effect on the behavior and mood of their host . So, germ-free mice take more risks than colonized mice, but they also have a lesser memory. It is unclear whether such links between microbiota and the brain are also important in human behavior.
Because our gut bacteria are actually little chemical factories with a vast arsenal of genes, they can also break down or modify all kinds of chemicals. Drugs such as cytostatics and heart medications can be activated or deactivated by the intestinal microbiota. Different people can react very differently to the same drugs and require higher or lower doses, depending on which gut bacteria they carry with them .
Microbiota and nutrition
The human gut microbiome is generally fairly stable. In a study in which volunteers gathered their feces for a long time, intestinal microbiota proved more or less constant, except during a change in diet, during a stomach flu, or on an international trip [22,23]. Recent studies on the composition of the feces of non- Western people in traditional communities in Africa and South America showed a great influence of lifestyle and diet on the human gut microbiome. Hunter-gatherers, traditional farmers, and urban industrial populations had vastly different microbiomes [13,24-26]. The intestinal microbiota of hunter-gatherers contained the highest bacterial diversity of these three groups. This very likely is caused by the huge amount of fiber that these traditionally-living groups consume — ten times more than average Americans and Europeans. This hypothesis is supported by animal experiments in which mice that eat little fiber showed a progressive loss of intestinal microbiota diversity over generations , thus providing new insights to develop new therapies to increase the microbial diversity in western intestines and possibly reduce metabolic diseases such as obesity and diabetes.
Unintended side effects of antibiotics
In severe infections, antibiotics can be lifesaving, but they also have unintended side effects on our microbiota. Most antibiotics are broad spectrum, and they unfortunately do not distinguish between pathogens and beneficial bacteria in our bodies. Many standard antibiotic regimens have a large effect on the number of types of bacteria in our intestines, often without the patient noticing it. Recovery is often incomplete, even months after stopping the course of treatment [28,29]. The mouth microbiota seems much less sensitive to these disturbances than gut microbiota .
In addition, it has been found from experiments on animals repeatedly actual antibiotic treatments mainly in young animals result is transferable in a permanent disturbed microbiota  and that obesity by transplanting human intestinal bacteria in germ-free mice . This has led to the hypothesis that repeated antibiotic treatments in childhood can lead to intestinal microbiota with fewer bacterial species . American children have been given an average of three to six antibiotics over the first three years of life, precisely at the time when their microbiome develops. Although this figure is lower in the Netherlands, antibiotics are being repeatedly prescribed to children, and the resulting reduction in the diversity of the intestinal microbiota is possibly related to the increased prevalence of obesity and diabetes in the western world. These recent findings could lead to new clinical practices, including reluctance to prescribe antibiotics, the further reduction of antibiotics in animal husbandry, and possibly supplementing the intestinal microbiota with probiotics .
Clostridium difficile infections, and fecal transplants
One of the great success stories of microbiome research is the application of fecal transplantations for the treatment of patients with Clostridium difficile infections (CDI). C. difficile is a spore-forming and toxin-producing bacteria that is present in low numbers in the intestine in about 10 percent of the healthy population but in a higher percentage for hospital and nursing home patients. An antibiotic treatment can lead to an imbalance in the intestinal microbiota with C. difficile, which is relatively resistant to most antibiotics, suddenly causing it to grow to large numbers. diarrhea, abdominal pain, and fever may result. In the United States, antibiotics cause nearly half a million cases of CDI and 30,000 deaths per year . Until recently, the limited treatment options included specific antibiotics such as vancomycin or, in extreme cases, a resection of a portion of the colon. Recurrent infections are common. Research by the Research by the Academic Medical Center in Amsterdam showed that fecal transplants with the stool from a healthy donor are very successful for CDI patients. After a first transplant, the cure rate was more than 80 percent, and after a second attempt, the percentage increased possibly as high as 94 percent [34, 35]. Fecal transplants have therefore become an attractive alternative for the treatment of CDI and possibly also for many other bowel diseases. Although the complication rate of fecal transplants is small, there is a risk of transmission of pathogenic viruses or bacteria, or the aspiration of fecal material .
Inflammatory bowel disease
Inflammatory bowel diseases (IBD), such as Crohn’s disease and ulcerative colitis, are difficult-to-treat inflammatory conditions of the intestine with an unclear cause and erratic symptoms. Besides a genetic component, there is also evidence for a role of the intestinal microbiota. The intestinal microbiota in IBD is somewhat different from that of healthy people, with a lower bacterial diversity and an altered ratio of specific bacterial groups . However, it remains unclear whether this dysbiosis is the cause of the clinical picture or just the result of prolonged inflammation, medication, or dietary changes. Despite many publications and studies, so far no obvious microbial pathogens have been found. Among other things, the success of fecal transplants for patients with CDI depend on the recovery of the microbial diversity in the intestine. It has become an interesting new therapy option, but treatment with probiotics or fecal transplant supplements have, up to now, not resulted in high percentages of success for IBD patients .
Autism is an umbrella term for various developmental constraints in the areas of social interaction and with few treatment options. Compared with healthy children, autistic children have gastrointestinal problems such as diarrhea or constipation. Therefore, there is much interest in a possible role of the microbiota with this disorder . Studies on the intestinal microbiota of individuals with autism unfortunately appear to contradict each other and have not yet shown any clear differences from healthy people . Possibly, some of the different microbiota in autism is caused by certain behavior in these patients, often including a strong aversion to vegetables and fruit and a preference for starchy foods. Autistic individuals therefore possibly refuse certain nutrients, such as fibers. This is possibly the cause of the differences in their intestinal bacteria that are found in some studies. As with IBD, it is therefore difficult to distinguish cause and effect. Although the relationship between autism and intestinal bacteria is still unclear and has not yet led to clinical treatment options. Prebiotics, probiotics, and recently fecal transplants are increasingly popular as ‘self-medication’ by (the parents of) individuals with autism .
In addition to the growing realization that gut bacteria are good for our health, we begin to realize that we live clean. Compared with the current traditional hunter-gatherers, whose living conditions probably very similar to those of our ancestors, our western lifestyle brings us much less in contact with microbes. Babies are often born by cesarean section or fed sterile milk, children grow up with fewer siblings and play a lot less in the sandbox or on the street. Our food and drinking water are almost sterile, we are hardly in contact with soil, plants or animals, we regularly receive antibiotics, many soaps and shampoos contain triclosan, and antibacterial wipes or UV-holders to disinfect toothbrushes or phones are becoming more popular. All these factors, coupled with the food of little fiber, there will likely help ensure that our gut microbiota from bacteria species is much less than that of traditional- living communities. Since our microbiome is involved in many processes in our body, including the construction and control of the immune system, the increased use of antibiotics, decreased intake of fibers, and the reduced exposure to bacteria at an early age are potentially connected with the increase of many metabolic, allergic and chronic gut disorders . Obviously we do not want to return to the Middle Ages, where infectious diseases could kill half a continent’s population. Vaccinations are important, life-threatening bacterial infections should be treated with antibiotics, and it is good to wash our hands before we eat or start treating patients. But maybe we should expose ourselves a bit to bacteria by consuming fermented foods and probiotics, and be more active outdoors in activities such as walking, gardening, or playing in a sandbox. We also need to take care of our internal microbes by eating more dietary fiber and complex carbohydrates and fewer simple sugars.
The new insights arising from microbiome investigation also fit in well with the development of ‘personalized medicine’, where patients get tailored therapy appropriate to the individual layout of their genome and now also their microbiome. Determining a microbiome profile is likely to become a part of standard care soon, and the absence or presence of certain bacterial groups can be used for diagnosis or as a starting point to serve as treatment plan for diseases which are currently difficult to treat. A worrying development associated with this, the rise of commercial companies offering their special blend of probiotics and feces transplant pills. Many of these supplements are sold without scientific basis or quality. Although some probiotic strains in specific situations have a proven positive impact, the proliferation of these types of products makes it difficult to separate the wheat from the chaff. However, the microbiome research field is very grateful and exciting profession to work in and I expect that we will discover many unexpected features and effects of our little friends on our health in the future.
In addition to the growing realization that gut bacteria are good for our health, we begin to realize that we live too cleanly. Compared with the current traditional hunter-gatherers, whose living conditions are probably very similar to those of our ancestors, our western lifestyle brings us much less contact with microbes. Babies are often born by Cesarean section or fed sterile milk, children grow up with fewer siblings and play a lot less in the sandbox or on the street. Our food and drinking water are almost sterile; we are hardly in contact with soil, plants, or animals; we regularly receive antibiotics; many soaps and shampoos contain triclosan; and antibacterial wipes or UV-holders to disinfect toothbrushes or phones are becoming more popular. All these factors, coupled with too little fiber in our food, will likely help ensure that our gut microbiota from bacteria species is much less diverse than that of traditional-living communities. Since our microbiome is involved in many processes in our body, including the construction and control of the immune system, the increased use of antibiotics, decreased intake of fibers, and the reduced exposure to bacteria at an early age are potentially connected with the increase of many metabolic, allergic, and chronic gut disorders . Obviously, we do not want to return to the Middle Ages, where infectious diseases could kill half a continent’s population. Vaccinations are important, life-threatening bacterial infections should be treated with antibiotics, and it is good to wash our hands before we eat or start treating patients. But maybe we should expose ourselves a bit to bacteria by consuming fermented foods and probiotics and be more active outdoors in activities such as walking, gardening, or playing in a sandbox. We also need to take care of our internal microbes by eating more dietary fiber and complex carbohydrates and fewer simple sugars.
The new insights arising from microbiome investigation also fit in well with the development of ‘personalized medicine,’ where patients get tailored therapy appropriate to the individual layout of their genome and now also their microbiome. Determining a microbiome profile is likely to become a part of standard care soon, and the absence or presence of certain bacterial groups can be used for diagnosis or as a starting point that can serve as a treatment plan for diseases that are currently difficult to treat. A worrying development associated with this is the rise of commercial companies offering their special blend of probiotics and fecal transplant pills. Many of these supplements are sold without a scientific basis or quality control. Although some probiotic strains in specific situations have a proven positive impact, the proliferation of these types of products makes it difficult to separate the wheat from the chaff. However, the microbiome research field is a very gratifying and exciting profession to work in, and I expect that we will discover many unexpected features and effects of our little friends on our health in the future.
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