The human microbiome: our second genome

Until recently, information about the role of microorganisms in maintaining human health, although it was very massive, was not “packaged” into a coherent logical system; at the same time, at least the approximate number and systematics of symbionts inhabiting humans remained unclear. It was only known that microbial animals (gnotobionts) lag behind in physical development and die earlier than their relatives raised under normal conditions — too many functions were taken over by “passenger bacteria” in the process of evolution. To address this huge gap, in 2007, the National Institutes of Health (NIH), USA, launched the 5-year Human Microbiome Project. His goal was to determine the entire species and quantitative composition of the microbiota of a healthy person by analyzing the DNA sequence of these microorganisms. The obtained genetic information was planned to be used in the future, among other things, to find out how the absence and presence of certain microorganisms is related to human health and the occurrence of diseases. Realizing the special scientific value of the information studied, the foundation not only provided open access to the data obtained in real time, but also regularly published the results of the research2,3. The first stage of the work was completed by the summer of 2012, and on June 14, about 15 articles appeared in Nature and the Public Library of Science series of journals presenting the results. The project brought together about 200 scientists from 80 multidisciplinary research institutes and cost the NIH $173 million. This is a truly historic event: by mobilizing their capabilities and joining an international research group, the scientists managed to rise above the previously known and see a new horizon, the whole picture. In 2012-2013 alone, more than 250 interesting articles on the human microbiome were published. Isn’t this a manifestation of the highest interest of the scientific community in the problem? The project “Human Microbiome” and vaginal microecology got their piece of “glory”.

The microbiome, the total number of genes of all bacteria inhabiting the human body, significantly exceeds the amount of genetic information encoded in the human genome, but until a certain time this fact was not considered as a determinant of health or disease. For too long, the human microbiota has been studied solely for the purpose of suppressing and eliminating pathogens, and only today can we say that the “kill the enemy!” paradigm is being replaced by a much more thoughtful “restore the biocenosis!” It is important to realize that microsymbionts make up about 90% of the total number of body cells, and only the remaining 10% are actually human cells.4 This information radically changes modern ideas about the role of bacteria in our lives, and today the human body, together with the microorganisms that inhabit it (and those in close contact with it), is commonly referred to as a superorganism. The analogy with cartoon “superheroes” works completely here: the superorganism, a single whole of a person and his microbiome, reacts comprehensively to external and internal stimuli and evolves in a similar way. The demonization of microbes in the age of antibiotics and antimicrobials turned out to be futile and even harmful, and a significant part of the former “enemies” turned out to be not only necessary for survival, but also recognized as an important reserve for the evolution of the species. Not villains, but superpowers!

Let’s do it again: The number of cells in a healthy person’s body is about 10 times less than the number of microorganisms inhabiting it5. Perhaps the reason is that prokaryotic cells are much smaller than eukaryotic cells in the human body. But not ten times! Anyway, the bacterial share in the symbiosis of “macroorganism and its microflora” impresses the imagination — just like the vastness of the starry sky on an August night. In the human body, symbiotic microbes live literally everywhere: on the skin and mucous membranes — in the nose, in the oral cavity, in the digestive tract, in the urinary tract and, of course, in the vulvovaginal locus. Many of the microbes perform important functions that are irreplaceable by the enzymes of the human body itself, for example, they digest plant fiber, synthesize vitamins and essential amino acids, and participate in the work of local immunity factors.

To get an idea of the structure of the normal human microbiome, the researchers collected tissue samples from 15 locations on the bodies of 129 men and from 18 locations in 113 women. All the volunteers were previously examined and found to be healthy; these are adults aged 18 to 40 years old living in Houston and St. Louis. The researchers took three samples from each of them from the mucous membrane on the inside of the cheek, nose, from the skin behind the ear and from the elbow, as well as fecal samples and vaginal fluid samples from women. According to the results of the genetic analysis of the collected material, it was found that more than 10 thousand species of various microbes live in the human body. However, they provide human vital activity with most of the genetic, and therefore protein, information: the microbiome brings about 8 million unique coding genes into the human body. In other words, there are 360 times more bacterial genes in humans than there are actually human genes! In general, the Human Microbiome project was designed to solve the following tasks.

• To investigate microbial genomes and preliminary characteristics of the human microbiome.

To study interspecific interactions in the human microbiome, as well as the links between changes in the microbiome and the development of diseases.

• Develop new tools and technologies for computer analysis.

• Create a data analysis and coordination center.

• Organize data warehouses.

• Predict the ethical, legal and social consequences of the project.

• Evaluate data on the role of the individual in the microbiome.

The researchers divided the listed global goals into specific topics and distributed them among themselves. For example, Christian Jobin, an immunologist at the University of Florida in Gainesville, studies the relationship between inflammatory bowel diseases (Crohn’s disease, ulcerative colitis, etc.), intestinal cancer, and intestinal microflora. “… The microbiome is still so poorly understood,” he says, “we are at some loss; we don’t know what give preference first.” The scale of the picture of microbiome interaction revealed by scientists is so fascinating that for many it becomes a temptation as a “thing in itself”: some project participants were too interested in studying the diversity of microorganisms and their systematization, it became an end in itself for them. At the same time, much more important issues that make it difficult to continue working on the project have not yet been resolved. Rob Knight, an environmental microbiologist at the University of Colorado (Boulder City), names three of them.

The conditions for collecting microbial material are not strictly standardized enough. For example, how long after taking a shower can I take a skin sample? What kind of diet should the subject follow on the eve of taking a stool test? Although the scientists who collected samples from healthy individuals tried to comply with certain standards, some of these conditions were still not taken into account. For example, some of the microbiome samples were taken from people suffering from a particular disease and differ from the norm.

In order to sequence and analyze the DNA of thousands of microbial species living in and on humans, a universal interdisciplinary team of scientists with knowledge of medical ethics, bioinformatics and engineering is needed, and assembling such a team is not an easy task.

It is still not clear whether the change in the microbiome is the cause of the disease or its consequence. Rob Knight believes that this task could be solved by examining the microbiome of a certain number of individuals before, during and after the disease. This information would make it possible to assess the risk of developing pathological conditions, such as obesity.10 Along the way, an analysis of the role of the microbiome in the development of diseases can stimulate the development of appropriate therapeutic drugs and fundamental changes in pathogenetic treatment regimens.

«…If you want to get rid of a particular bacterium, pharmaceutical companies will provide you with thousands of drugs. Influencing disease—causing genes and radically ridding the patient of them is a much more difficult task,” concludes Rob Knight. At the same time, the understanding that a significant amount of genetic information regulating human life is “borrowed”, that is, belongs to microorganisms, and in theory can be corrected, opens up truly dizzying prospects for both research and therapeutic potential. For example, literally in November 2013, as a direct consequence of the Human Microbiome project, a sensational message spread around the scientific world that a certain type of intestinal bifidobacteria effectively eliminated the manifestations of autism (!) in children by restoring intestinal wall permeability.5

From method to result, the Human Microbiome project itself became possible thanks to the scientific development of methods for studying bacteria. The presence of microorganisms in the environment and their impact on health became known more than 300 years ago: in 1675, using a hand-held light (optical) microscope, Anthony van Leeuwenhoek (1632-1723) was able to see unicellular organisms for the first time and called them “small animals”. Later, the founders of modern microbiology — Louis Pasteur (1822-1895), Ferdinand Cohn (1828-1898) and Robert Koch (1843-1910) — confirmed the complexity of the microbial world with their research. And van Leeuwenhoek’s “little animals” were named microorganisms 150 years later.11 The newly discovered world of unicellular organisms (as before — plants and animals) had to be systematized, and for this purpose it was necessary to study in detail.

Since the late 1800s, the “gold standard” for the identification and characterization of microbes has been the isolation and cultivation of bacteria on nutrient media with further study of the properties of colonies. The library of discovered species was constantly replenished, however, the large number of compared phenotypic traits and their equal importance made it difficult to classify bacteria. The nuances of the structure of bacterial cells made it difficult to study them using methods that have been successfully used in botany or zoology for centuries. In particular, descriptive technologies (the cytomorphological method) turned out to be unsuitable: bacteria are quite simply arranged, they do not have a typical nucleus and other organelles, and there is no sexual process.

It was only in the 20th century that it became possible to study microorganisms based on a very important functional feature — their enzymatic activity. The enzymatic spectrum is a taxonomic feature characteristic of a family, genus, and, in some cases, a species. Therefore, even now, to establish the systematic position of bacteria, the spectrum of enzymes produced by them is determined using special test systems. However, the enzyme method could not cope with the identification of a huge number of species, information about which had accumulated by the end of the century.

The discovery of the polymerase chain reaction (PCR) by a group of scientists in the late 80s of the last century12,13 provided an opportunity to look at the bacterial cell “from the inside”. In the PCR process, they accumulate (amplify) DNA is a material isolated from a bacterial sample, which allows for genomic identification and analysis of microbial communities. Frederick Sanger received the Nobel Prize in Chemistry in 1980 for developing the amplification method.

Methods for determining the sequence of DNA nucleotides have evolved to reduce costs and increase accuracy. Currently, many researchers in this field rely on the sequencing platform developed in 2000 by Jonathan Rothberg (launched in 2005). Today, this is the most convenient, cheapest and fastest analysis of DNA and RNA samples.

Sequencing of the microbial genome using a new generation of techniques is fundamentally changing our understanding of microbial diversity; this technology has had a huge impact on the decoding of individual microbial genomes. In 1995, the hemophilic bacillus Haemophilus influenzae became the first in the study of the bacterial genome.[14] The genome size was 1.8 MB, which is 10 times larger than the previously studied sequences. Recreating this genome using the Sanger method would take more than 3 months and involve 14 machines, while Next Generation sequencing technology allows you to spend 1 week and use only one machine. During the outbreak caused by E. coli in Germany in May–June 2011, which covered almost all regions of the country, the use of the Ion Torrent Personal Genome Machine diagnostic platform for sequencing made it possible to identify the pathogen almost immediately (serovar O104:H4) and eliminate the outbreak within 3 days15.

However, sequencing is no longer at the top of genetic research methods. The fact is that there are certain difficulties in studying uncultivated microorganisms due to the small amount of their genomic material. In these cases, the “top” method of modern microbiological research is used — metagenomics. This is a branch of molecular genetics that studies genetic material obtained directly from environmental samples (a metagenome, rather than individual bacteria and other cells). Traditional genome sequencing relies on cultured clones of crops, while metagenomics works with a set of all the DNA found in the medium. The main difference when using the metagenomic approach is that uncultivated microorganisms are taken into account along with cultured ones.

An example of metagenomic analysis is the study of microbial biomass conducted in various water wells, mine waters, activated sludge, hot springs, as well as in the upper layers of the Sargasso Seas16-19. In addition to well—known bacteria and phages, the researchers found representatives of recently established phylum, and completely new bacteri20,21. By the way, Atopobium vaginae was discovered in a similar way at the time – it was impossible to distinguish it from vaginal lactobacilli without genetic methods.

One of the main goals of the 5-year Human Microbiome project was to determine the “map of microbial diversity” in several areas of the body of a healthy adult in a large cohort of subjects (n=242)22. The list of studied loci includes the oral and nasal cavities, gastrointestinal tract, skin, and vagina in women. Metagenomic data, including a description of 16s rRNA, were analyzed from clinical samples taken at these sites (see the section “Relatives? It will show 16s of RNA!”). The study confirmed that each area of the body contains a dominant type of bacteri23-26: for example, lipophilic Propionibacterium predominate in the sebaceous areas behind the ear behind the ear fold, Bacteroidetes and Firmicutes predominate in the intestine, and Lactobacillus predominate in the vagina.

As for the vaginal biotope, there was a lot of new stuff. As is known, lactobacilli form lactic acid from glycogen to create an acidic vaginal environment dynamically, and this protects a woman from the invasion of new and excessive activity of transient pathological microorganisms. A clear correlation was found between the pH of the vaginal environment and the number of lactobacilli in the local microbiome: the higher the acidity, the lower the proportion of acid-producing microorganisms, and, conversely, with an increase in pH, the number of lactobacilli increases compensationally.28 Thus, the composition of the vaginal microflora in women can dynamically change within wide normal limits.

Scientists from the Universities of Maryland and Idaho monitored the state of the vaginal microbiome of 32 healthy women of reproductive age for 16 weeks. Observations have shown that there are five main classes of bacterial communities. Lactobacilli (L. iners, L. crispatus, L. gasseri or L. jensenii) predominate in four of them, and obligate anaerobes predominate in another one. The qualitative and quantitative composition of each class can change over a short period of time or remain relatively stable for a long time. Sometimes it depends on a woman’s menstrual cycle, and sometimes it doesn’t. All the changes taking place in the microbiome are very individual. However, these combined data allowed us to draw the most important conclusion about the dynamism of the vaginal biotope.”..Now the woman’s condition is assessed by one sample, but in fact it is wrong, — says the head of the project, Jacques Ravel. “Repeated studies of the vaginal microbiome for some time will reduce the frequency of overdiagnosis and unnecessary prescribing of broad—spectrum antibiotics.”29 Earlier studies have shown that the state of the vaginal microflora depends, in particular, on ethnic characteristics. This is due to the different levels of acidity in the vaginal environment: it is lower in Latin American (pH 5.0) and African (pH 4.7) women compared with European (pH 4.2) and Asian (pH 4.4) women. Accordingly, Latin American and black women are less protected from the penetration of pathogenic microflora and are more likely to suffer from bacterial vaginosis 26,28 — from 25 to 30% of all women face this problem (the most common reason for visiting a gynecologist).

«…If we could identify women at high risk of bacterial vaginosis, we could offer them measures to prevent the disease,” explains study co—author Dr. Larry Forney. Scientists do not expect that gynecologists will immediately reconsider the principles of treatment of their patients, but they hope for an individual approach to the treatment of women in the future.

The first-ever study of the intestinal microbiome during pregnancy was conducted by a group of scientists from Cornell University (Ithaca, New York) led by Ruth Ley. It turned out that during gestation, especially in the third trimester, a woman’s intestinal microbiome resembles that of patients at risk of diabetes mellitus. These changes cannot be regarded as a violation of the health of the expectant mother, but correlate with an increase in blood glucose and the accumulation of fatty tissue for the nutrition of the fetus.

Previously, Ruth Ley studied the microbiome of patients with metabolic syndrome, a well—known precursor to diabetes mellitus, which is also characterized by increased levels of inflammatory markers, hyperglycemia, and obesity. After sequencing microbial DNA from stool samples taken in early and late pregnancy, she discovered that fixed changes were occurring in microbial communities. In general, the degree of diversity in the intestinal microbiome decreases by the third trimester, but the number of proteobacteria and actinobacteria increases. The same thing happens in obese people or those suffering from metabolic syndrome. Ruth Ley’s colleague, Kjersti Aagaard, believes that this similarity makes some sense, since at this time the fetus is actively gaining body weight, and the mother’s body has maximum energy reserves accumulated for successful lactation.30 However, the biological feasibility and medical prospects of the discovered analogy have yet to be understood.

Just as a healthy reproductive system is unthinkable without a normal vaginal microflora, full-fledged treatment is impossible without a detailed understanding of how the human microbiome works and functions. Microbiology and medicine are reaching a new level of understanding of what is happening in the “human body and its microorganisms” system, and the prospects of this new knowledge are breathtaking. In any case, the cutting edge of science nowadays runs very close to practical medicine: today’s theories are turning into tomorrow’s methods of diagnosing and treating diseases before our eyes.

Source: elibrary.ru (The authors: Igor Nikolaevich Kostin, Doctor of Medical Sciences, Professor of the Department of Obstetrics and Gynecology with a course in Perinatology at RUDN University; Lyudmila Yuryevna Kuvankina, StatusPraesens, Hilda Yuryevna Simonovskaya, StatusPraesens (Moscow)