July 11, 2019 at 14:01Microbiota. History of study and research methods
We received a lot of comments on the last article and decided with Atlas to supplement the series with material about what other methods of studying microbiota are. At the end of the article, they added that you need to remember about studies of intestinal bacteria today so that they do not harm your health.
Illustration by Rentonorama
The intestinal microbiota is called a new organ in the human body. We have more or less known about other organs for a long time, but it became known only at the end of the 20th century that bacteria perform important functions for humans. The study of microbiota began in the XVII century. The creator of the microscope and the “father of microbiology,” Anthony Van Levenguk, first examined and described bacteria in the oral cavity and feces.
In 1828, Christian Ehrenberg introduced the new term Bacterium. At that moment, he was studying Escherichia coli (Escherichia coli), a species of bacteria without spores. For spore-forming bacteria, Christian coined the term Bacillus. This type of bacteria was actively studied by Robert Koch. He also revealed the relationship between pathogenic representatives of this genus and diseases such as anthrax and tuberculosis.
Already in the 19th century, researchers understood that human health is closely linked to bacteria. However, the full study of microbiota became possible after the discovery of gene sequencing technology by Frederick Senger. Not all species are able to live and grow in Petri dishes, so it was difficult to classify and determine the functions of bacteria in detail.
Along with the development of technology in the 70s, the microbiologist Karl Woese proposed classifying microorganisms based on sequencing of the 16s rRNA molecule, which makes it convenient to compare nucleotide sequences and determine the degree of affinity. According to the analysis, Karl divided all microorganisms into archaea, bacteria and eukaryotes. This classification is used now.
Eukaryotes are distinguished by the presence of a nucleus, but bacteria and archaea do not. Archaea are simple unicellular microorganisms that live in extreme conditions (in geysers, at the bottom of the seas and oceans). And they are the most ancient: archaea exist on Earth for about 4 billion years. Also, among them there are no parasitic and pathogenic microorganisms, and among bacteria there are, although not as many as we think - about 1%.
In the human intestines, archaea produce methane. Also, the more there are, the lower the risk of obesity, but the causal relationship remains unclear. Not all have archaea and rarely go beyond 1-2%.
Bacteria live in a wide variety of environments and we contact with them much more than with archaea. They differ in a number of functions. For example, bacteria can break down carbohydrate molecules and produce fatty acids, while archaea cannot.
Before diving into research, let's first refresh our knowledge of DNA, RNA, and protein synthesis.
The body needs proteins to function properly. Skin tissues, in order to maintain a protective function, require one; the cells of the eyes, so that the organ works correctly, are different. Sometimes different cells and tissues require the same protein. To create proteins, cells use DNA as an instruction.
First, the area in which the necessary information is contained is determined. Double-stranded DNA is unwound and only one side is copied. Then the DNA is wound back, and a copy of one of its sides (RNA) picks up the ribosome. She reads the sequence and builds on it a chain of amino acids, which then takes shape and becomes a protein.
This can be compared to cooking according to an old recipe book. First, we write out a recipe so as not to use the fragile, but valuable book once again. Further on the recipe, we combine different products, like amino acid ribosome, to get the finished dish. For a cell, a dish is a protein that it uses further for its needs. In addition to protein, microorganisms produce other compounds, such as fatty acids, which are considered metabolites.
To study microorganisms, four approaches are mainly used: metagenomic — DNA research, metatranscriptomic — RNA research, metaproteomics — protein research, metabolomics — metabolite research.
Metagenome - a set of genes of all organisms in the studied environment. The essence of this analysis is the sequencing of the 16s rRNA gene, which is responsible for the operation of ribosomal RNA, or the sequencing of all DNA. Such a study answers the questions “what organisms are in the sample and what functions do they potentially perform?” We talked about this technology in detail in the previous article , since the test “Microbiota Genetics” is based on it.
Usually, microbiota research refers to this particular type of analysis, because it was the first to be used on a large scale to study bacteria. After the advent of DNA sequencing technology, scientists launched a global project to study soils, seas, hot springs. Thanks to metagenomic analysis, the database of microorganisms has grown exponentially. Sequencing allows you to study bacteria in a natural environment, while in laboratory conditions, many of them die.
In 2007, US researchers began a project to study the human body microbiome Human Microbiome Project . It became the impetus for a large-scale study of the composition of intestinal bacteria based on metagenomic data. Following the HMP in Europe in 2008 launched a similar project to study human microbiota -MetaHit .
The essence of metagenomic studies is to understand which microorganisms live in the sample, how many there are and what functions they perform. The analysis does not directly evaluate which compounds the bacterial community produces. However, thanks to many metagenomic studies, we can predict this indirectly. For example, if a person has more bacteria-producers of butyric acid - his microbiota probably produces it well.
Metagenomic studies are widespread because they are easier to carry out in comparison with other methods. Studying RNA, proteins, and metabolites requires sophisticated sample purification and more time-consuming analyzes.
According to metagenomic data, we have the most results. This is clearly seen on the basis of all scientific articles and clinical research PubMed. A search for metagenomic microbiome returns about 4,500 different articles, a metatranscriptomic microbiome query total 225, metaproteomic microbiome 100, metabomic microbiome 1,600.
A transcript is the totality of all molecules of messenger RNA (mRNA) that a single cell or group of microorganisms synthesizes. In metatranscriptome analysis, RNA is studied directly, and not the gene that encodes it.
It happens that there is a bacterium, but it does not participate in the life of the microbial community: it has inactive genes that are not copied by the RNA molecule. Metatranscriptome studies allow us to evaluate exactly the active part of the microbiota. However, the RNA molecule is not as stable as DNA, and decays quickly. Therefore, it is more difficult and more expensive to isolate and save it for analysis.
Often transcriptome studies are used to study certain gene functions. In this case, the results of an RNA test are verified against metagenomic data. So scientists get more complete information about the work of microorganisms. Microbiome metatranscriptome studies may be useful to more accurately determine the potential for the synthesis of various metabolites.
With this approach, all proteins that are in the sample are studied. Metaproteomics provides information on the structure, functions and dynamics of the microbial community. Scientists will learn more about how organisms interact with each other, compete for nutrition, and produce metabolites.
First, proteins are isolated from the sample. Often, liquid chromatography is used for this. Then an additional analysis is carried out to determine the molecular weight - mass spectrometry. So we get information about protein fragments (peptides), but not about the whole protein. To collect the fragments into a single whole, special programs are used, and scientists receive ready-made data.
Metoproteomics are currently less popular than DNA and RNA studies. This is due to the complexity of the research and the high probability of error. A sample may contain many human or food proteins. However, metaproteomics can help scientists shed light on the interactions between bacteria and the disruption of the microbiota in people with diseases.
With this type of analysis, metabolites are studied - substances that bacteria produce. These can be amino acids, lipids, sugars, fatty acids (including butyric acid) and other compounds. Now about 40,000 metabolites of the human body are described, and all of them are recorded in a large database .
As a sample for the study of metabolites, you can use any fluid from the human body: blood, saliva, urine, intestinal lavage (flushing) and even cerebrospinal fluid. On average, about 4200 metabolites are contained in blood plasma, 3000 in urine, 500 cerebrospinal fluid, and 400 in saliva. However, lavage is used as a biomaterial for microbiota studies.
The metabolite study procedure is similar to protein analysis. Using the same liquid or gas chromatography, the metabolites are first isolated and then their molecular weight is measured using a mass spectrometer.
The study of metabolites has its limitations. For example, based on this study, we can’t find out exactly which metabolites are excreted by the intestinal microbiota, and which we received with food.
Also, it is impossible to calculate how many bacteria are contained in the microbiota. Therefore, for a more complete picture, data on metabolites are accompanied by the results of metagenomic analyzes. This approach is sometimes used to study how microbiota and its metabolites are involved in the development of diseases.
So far, no microbiota research method has been used in regular clinical practice. Sometimes, for a complete picture, a doctor may recommend conducting a metagenomic study of microbiota to evaluate the composition of intestinal bacteria.
We warn users that the Microbiota Genetics test is only for educational purposes and is designed for healthy people who are interested in getting to know their bacteria. If a person is sick, then he will be able to find out the composition of the bacteria, but the recommendations in this case will not be relevant. The microbiota of people with diseases is very different, and for them the “normal” profile will be different.
We do not advise children to conduct the study, because their microbiota data is much less. And unnecessary intervention and limiting the diet of children according to the results of the study is potentially dangerous, since the child may not receive the necessary nutrients or may suffer from overdiagnosis.
Today, residents of Russia and the CIS countries are offered a study of microbiota metabolites for children and adults using a sample of blood or saliva by gas chromatography and mass spectrometry. According to its results, according to the developers of this method, it is possible to assess the presence and absence of inflammation in the body.
However, there are no such recommendations in international clinical guidelines. Diagnosis of inflammation and disease should be carried out by methods that have a high level of evidence, a certain degree of sensitivity, a low probability of false positive results and complications of overdiagnosis.
Other articles about the intestinal microbiota:
Illustration by Rentonorama
How did the study of microbiota begin?
The intestinal microbiota is called a new organ in the human body. We have more or less known about other organs for a long time, but it became known only at the end of the 20th century that bacteria perform important functions for humans. The study of microbiota began in the XVII century. The creator of the microscope and the “father of microbiology,” Anthony Van Levenguk, first examined and described bacteria in the oral cavity and feces.
In 1828, Christian Ehrenberg introduced the new term Bacterium. At that moment, he was studying Escherichia coli (Escherichia coli), a species of bacteria without spores. For spore-forming bacteria, Christian coined the term Bacillus. This type of bacteria was actively studied by Robert Koch. He also revealed the relationship between pathogenic representatives of this genus and diseases such as anthrax and tuberculosis.
Already in the 19th century, researchers understood that human health is closely linked to bacteria. However, the full study of microbiota became possible after the discovery of gene sequencing technology by Frederick Senger. Not all species are able to live and grow in Petri dishes, so it was difficult to classify and determine the functions of bacteria in detail.
Along with the development of technology in the 70s, the microbiologist Karl Woese proposed classifying microorganisms based on sequencing of the 16s rRNA molecule, which makes it convenient to compare nucleotide sequences and determine the degree of affinity. According to the analysis, Karl divided all microorganisms into archaea, bacteria and eukaryotes. This classification is used now.
Eukaryotes are distinguished by the presence of a nucleus, but bacteria and archaea do not. Archaea are simple unicellular microorganisms that live in extreme conditions (in geysers, at the bottom of the seas and oceans). And they are the most ancient: archaea exist on Earth for about 4 billion years. Also, among them there are no parasitic and pathogenic microorganisms, and among bacteria there are, although not as many as we think - about 1%.
In the human intestines, archaea produce methane. Also, the more there are, the lower the risk of obesity, but the causal relationship remains unclear. Not all have archaea and rarely go beyond 1-2%.
Bacteria live in a wide variety of environments and we contact with them much more than with archaea. They differ in a number of functions. For example, bacteria can break down carbohydrate molecules and produce fatty acids, while archaea cannot.
How to study microbiota
Before diving into research, let's first refresh our knowledge of DNA, RNA, and protein synthesis.
The body needs proteins to function properly. Skin tissues, in order to maintain a protective function, require one; the cells of the eyes, so that the organ works correctly, are different. Sometimes different cells and tissues require the same protein. To create proteins, cells use DNA as an instruction.
First, the area in which the necessary information is contained is determined. Double-stranded DNA is unwound and only one side is copied. Then the DNA is wound back, and a copy of one of its sides (RNA) picks up the ribosome. She reads the sequence and builds on it a chain of amino acids, which then takes shape and becomes a protein.
This can be compared to cooking according to an old recipe book. First, we write out a recipe so as not to use the fragile, but valuable book once again. Further on the recipe, we combine different products, like amino acid ribosome, to get the finished dish. For a cell, a dish is a protein that it uses further for its needs. In addition to protein, microorganisms produce other compounds, such as fatty acids, which are considered metabolites.
To study microorganisms, four approaches are mainly used: metagenomic — DNA research, metatranscriptomic — RNA research, metaproteomics — protein research, metabolomics — metabolite research.
Metagenomic Sequencing
Metagenome - a set of genes of all organisms in the studied environment. The essence of this analysis is the sequencing of the 16s rRNA gene, which is responsible for the operation of ribosomal RNA, or the sequencing of all DNA. Such a study answers the questions “what organisms are in the sample and what functions do they potentially perform?” We talked about this technology in detail in the previous article , since the test “Microbiota Genetics” is based on it.
Usually, microbiota research refers to this particular type of analysis, because it was the first to be used on a large scale to study bacteria. After the advent of DNA sequencing technology, scientists launched a global project to study soils, seas, hot springs. Thanks to metagenomic analysis, the database of microorganisms has grown exponentially. Sequencing allows you to study bacteria in a natural environment, while in laboratory conditions, many of them die.
In 2007, US researchers began a project to study the human body microbiome Human Microbiome Project . It became the impetus for a large-scale study of the composition of intestinal bacteria based on metagenomic data. Following the HMP in Europe in 2008 launched a similar project to study human microbiota -MetaHit .
The essence of metagenomic studies is to understand which microorganisms live in the sample, how many there are and what functions they perform. The analysis does not directly evaluate which compounds the bacterial community produces. However, thanks to many metagenomic studies, we can predict this indirectly. For example, if a person has more bacteria-producers of butyric acid - his microbiota probably produces it well.
Metagenomic studies are widespread because they are easier to carry out in comparison with other methods. Studying RNA, proteins, and metabolites requires sophisticated sample purification and more time-consuming analyzes.
According to metagenomic data, we have the most results. This is clearly seen on the basis of all scientific articles and clinical research PubMed. A search for metagenomic microbiome returns about 4,500 different articles, a metatranscriptomic microbiome query total 225, metaproteomic microbiome 100, metabomic microbiome 1,600.
Metatranscriptome sequencing
A transcript is the totality of all molecules of messenger RNA (mRNA) that a single cell or group of microorganisms synthesizes. In metatranscriptome analysis, RNA is studied directly, and not the gene that encodes it.
It happens that there is a bacterium, but it does not participate in the life of the microbial community: it has inactive genes that are not copied by the RNA molecule. Metatranscriptome studies allow us to evaluate exactly the active part of the microbiota. However, the RNA molecule is not as stable as DNA, and decays quickly. Therefore, it is more difficult and more expensive to isolate and save it for analysis.
Often transcriptome studies are used to study certain gene functions. In this case, the results of an RNA test are verified against metagenomic data. So scientists get more complete information about the work of microorganisms. Microbiome metatranscriptome studies may be useful to more accurately determine the potential for the synthesis of various metabolites.
Metaproteomics
With this approach, all proteins that are in the sample are studied. Metaproteomics provides information on the structure, functions and dynamics of the microbial community. Scientists will learn more about how organisms interact with each other, compete for nutrition, and produce metabolites.
First, proteins are isolated from the sample. Often, liquid chromatography is used for this. Then an additional analysis is carried out to determine the molecular weight - mass spectrometry. So we get information about protein fragments (peptides), but not about the whole protein. To collect the fragments into a single whole, special programs are used, and scientists receive ready-made data.
Metoproteomics are currently less popular than DNA and RNA studies. This is due to the complexity of the research and the high probability of error. A sample may contain many human or food proteins. However, metaproteomics can help scientists shed light on the interactions between bacteria and the disruption of the microbiota in people with diseases.
Metabolomics
With this type of analysis, metabolites are studied - substances that bacteria produce. These can be amino acids, lipids, sugars, fatty acids (including butyric acid) and other compounds. Now about 40,000 metabolites of the human body are described, and all of them are recorded in a large database .
As a sample for the study of metabolites, you can use any fluid from the human body: blood, saliva, urine, intestinal lavage (flushing) and even cerebrospinal fluid. On average, about 4200 metabolites are contained in blood plasma, 3000 in urine, 500 cerebrospinal fluid, and 400 in saliva. However, lavage is used as a biomaterial for microbiota studies.
The metabolite study procedure is similar to protein analysis. Using the same liquid or gas chromatography, the metabolites are first isolated and then their molecular weight is measured using a mass spectrometer.
The study of metabolites has its limitations. For example, based on this study, we can’t find out exactly which metabolites are excreted by the intestinal microbiota, and which we received with food.
Also, it is impossible to calculate how many bacteria are contained in the microbiota. Therefore, for a more complete picture, data on metabolites are accompanied by the results of metagenomic analyzes. This approach is sometimes used to study how microbiota and its metabolites are involved in the development of diseases.
What to remember
So far, no microbiota research method has been used in regular clinical practice. Sometimes, for a complete picture, a doctor may recommend conducting a metagenomic study of microbiota to evaluate the composition of intestinal bacteria.
We warn users that the Microbiota Genetics test is only for educational purposes and is designed for healthy people who are interested in getting to know their bacteria. If a person is sick, then he will be able to find out the composition of the bacteria, but the recommendations in this case will not be relevant. The microbiota of people with diseases is very different, and for them the “normal” profile will be different.
We do not advise children to conduct the study, because their microbiota data is much less. And unnecessary intervention and limiting the diet of children according to the results of the study is potentially dangerous, since the child may not receive the necessary nutrients or may suffer from overdiagnosis.
Today, residents of Russia and the CIS countries are offered a study of microbiota metabolites for children and adults using a sample of blood or saliva by gas chromatography and mass spectrometry. According to its results, according to the developers of this method, it is possible to assess the presence and absence of inflammation in the body.
However, there are no such recommendations in international clinical guidelines. Diagnosis of inflammation and disease should be carried out by methods that have a high level of evidence, a certain degree of sensitivity, a low probability of false positive results and complications of overdiagnosis.
Other articles about the intestinal microbiota: