Microbiota Study history and research methods
We received many comments on the last article and decided with the Atlas to add a series of material on what other methods of studying microbiota are. At the end of the article they added that it is necessary to remember about the studies of intestinal bacteria for today, so that they do not harm your health.
Illustration by Rentonorama
The intestinal microbiota is called the new organ in the human body. We more or less knew about other organs for a long time, but the fact that bacteria perform important functions for a person became known only at the end of the 20th century. The study of microbiota began in the XVII century. The creator of the microscope and the "father of microbiology," Anthony Van Leeuwenhoek, first examined and described the bacteria of the oral cavity and feces.
In 1828, Christian Ehrenberg introduced the new term Bacterium. At that moment, he was studying E. coli (Escherichia coli) - a kind 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.
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Already in the 19th century, researchers understood that human health is closely related to bacteria. However, it became possible to fully study the microbiota after the discovery of gene sequencing technology by Frederick Sanger. 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.
Simultaneously with the development of technologies in the 70s, microbiologist Karl Wöse proposed classifying microorganisms based on sequencing of the 16s rRNA molecule, which makes it convenient to compare nucleotide sequences and determine the degree of relatedness. 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, while bacteria and archaea do not have it. Archaea are simple single-celled microorganisms that live in extreme conditions (in geysers, at the bottom of seas and oceans). And they are the most ancient: archaea has existed on Earth for about 4 billion years. Also, there are no parasitic and pathogenic microorganisms among them, and among bacteria there are, although not so much as it seems to us - about 1%.
In the human intestine, archaea produces methane. Also, the more of them, the lower the risk of obesity, but the causal relationship remains unclear. Archaea is not all and rarely go beyond 1–2%.
Bacteria live in a wide variety of environments and we are in 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, but archaeans do not.
Before diving into research, let's first refresh our knowledge of DNA, RNA and protein synthesis.
The body needs proteins to work properly. Skin tissues, in order to maintain a protective function, are required alone; eye cells, so that the organ worked properly, - others. Sometimes different cells and tissues require the same protein. To create proteins, cells use DNA as an instruction.
First, determine the area that contains the necessary information. The double-stranded DNA is unwound and only one side of it is copied. Then the DNA is rolled back, and a copy of its one side (RNA) picks up the ribosome. It reads the sequence and builds on it a chain of amino acids, which then takes the form and becomes a protein.
This can be compared to cooking using an old recipe book. First, we write a prescription not to use the once again fragile but valuable book. Further, according to the recipe, we combine different foods, like an 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.
Four approaches are generally used to study microorganisms: metagenomic - DNA research, metatranscriptome - RNA research, metaproteomics - protein research, metabolomics - metabolite research.
Metagen - a set of genes of all organisms in the studied environment. The essence of such an analysis is in the sequencing of the 16s rRNA gene, which is responsible for the operation of ribosomal RNA, or in 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 Genetics of Microbiota test is built on it.
Usually under the study of microbiota, this type of analysis is meant, because it was first 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 microorganism database has grown exponentially. Sequencing allows you to study bacteria in the natural environment, whereas in the laboratory, many of them die.
In 2007, US researchers began a project to study the human microbiome of the Human Microbiome Project . It was the impetus for a large-scale study of the composition of intestinal bacteria based on metagenomic data. Following HMP in Europe in 2008 launched a similar project to study the human microbiota - MetaHit .
The essence of metagenomic research is to understand which microorganisms live in the sample, how many are there and what functions they perform. The analysis does not allow to directly evaluate what compounds the bacterium community produces. However, thanks to many metagenomic studies, we can predict this indirectly. For example, if a person has more bacteria producing butyric acid, his microbiota probably produces it well.
Metagenomic studies are widespread because they are easier to conduct in comparison with other methods. The study of RNA, proteins and metabolites requires complex sample cleaning and more time-consuming analyzes.
According to metagenomic data, we have the most results. This is clearly seen in the database of all scientific articles and clinical studies PubMed. A search for a metagenomic microbiome results in about 4,500 different articles, a metatranscriptomic microbiome query - a total of 225, a metaproteomic microbiome - 100, a metabolomic microbiome - 1600.
A transcript is a collection of all molecules of messenger RNA (mRNA) that are synthesized by a single cell or group of microorganisms. In metatranscriptome analysis, the 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 an RNA molecule. Metatranscriptome studies allow us to estimate the active part of the microbiota. However, the RNA molecule is not as stable as DNA, and quickly disintegrates. Therefore, it is more difficult and expensive to isolate and save it for analysis.
Often, transcriptome studies are used to study certain functions of genes. In this case, the results of the study of RNA are compared with the 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 about the structure, functions and dynamics of the microbial community. Scientists will learn more about how organisms interact with each other, compete for food, produce metabolites.
First, proteins are isolated from the sample. Often, liquid chromatography is used for this. Then conduct an additional analysis to determine the molecular weight - mass spectrometry. This is how we get information about protein fragments (peptides), but not about the whole protein. To assemble the fragments into a coherent whole, special programs are used, and scientists obtain ready-made data.
Metoproteomics is currently less popular than DNA and RNA research. This is due to the complexity of the research and the high probability of error. There may be a lot of human protein or food in the sample. However, metaproteomics can help scientists shed light on the interactions between bacteria and microbiota disruption in people with diseases.
With this type of analysis, metabolites are studied - substances that bacteria produce. It can be amino acids, lipids, sugars, fatty acids (including butyric) and other compounds. About 40,000 metabolites of the human body are now described, and all of them are recorded in a large database .
As a sample for the study of metabolites, you can use any liquid from the human body: blood, saliva, urine, intestinal lavage (flush), and even cerebrospinal fluid. On average, plasma contains about 4,200 metabolites, 3000 in urine, 500 in cerebrospinal fluid, and 400 in saliva. However, lavage is used as a biomaterial to study the microbiota.
The study of metabolites 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 cannot find out exactly which metabolites the gut microbiota secretes, and which we received from food.
Also, it is impossible to calculate how many bacteria are contained in the microbiota. Therefore, for a more complete picture, the data on metabolites are accompanied by the results of metagenomic analyzes. This approach is sometimes used to study how the 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 a microbiota metagenomic study to assess the composition of intestinal bacteria.
We warn users that the Genetics of Microbiota test is only suitable for educational purposes and is designed for healthy people who are interested in meeting their bacteria. If a person is sick, 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 recommend research for children, because there is much less data on their microbiota. And unnecessary intervention and restriction of the diet of children according to the results of the study is potentially dangerous, since the child may receive less nutrients or suffer from overdiagnosis.
Today, residents of Russia and the CIS countries are offered a study of microbiota metabolites for children and adults using blood or saliva samples using 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 inflammations and diseases 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 on the intestinal microbiota:
Illustration by Rentonorama
What began the study of microbiota
The intestinal microbiota is called the new organ in the human body. We more or less knew about other organs for a long time, but the fact that bacteria perform important functions for a person became known only at the end of the 20th century. The study of microbiota began in the XVII century. The creator of the microscope and the "father of microbiology," Anthony Van Leeuwenhoek, first examined and described the bacteria of the oral cavity and feces.
In 1828, Christian Ehrenberg introduced the new term Bacterium. At that moment, he was studying E. coli (Escherichia coli) - a kind 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 related to bacteria. However, it became possible to fully study the microbiota after the discovery of gene sequencing technology by Frederick Sanger. 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.
Simultaneously with the development of technologies in the 70s, microbiologist Karl Wöse proposed classifying microorganisms based on sequencing of the 16s rRNA molecule, which makes it convenient to compare nucleotide sequences and determine the degree of relatedness. 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, while bacteria and archaea do not have it. Archaea are simple single-celled microorganisms that live in extreme conditions (in geysers, at the bottom of seas and oceans). And they are the most ancient: archaea has existed on Earth for about 4 billion years. Also, there are no parasitic and pathogenic microorganisms among them, and among bacteria there are, although not so much as it seems to us - about 1%.
In the human intestine, archaea produces methane. Also, the more of them, the lower the risk of obesity, but the causal relationship remains unclear. Archaea is not all and rarely go beyond 1–2%.
Bacteria live in a wide variety of environments and we are in 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, but archaeans do not.
How to study the microbiota
Before diving into research, let's first refresh our knowledge of DNA, RNA and protein synthesis.
The body needs proteins to work properly. Skin tissues, in order to maintain a protective function, are required alone; eye cells, so that the organ worked properly, - others. Sometimes different cells and tissues require the same protein. To create proteins, cells use DNA as an instruction.
First, determine the area that contains the necessary information. The double-stranded DNA is unwound and only one side of it is copied. Then the DNA is rolled back, and a copy of its one side (RNA) picks up the ribosome. It reads the sequence and builds on it a chain of amino acids, which then takes the form and becomes a protein.
This can be compared to cooking using an old recipe book. First, we write a prescription not to use the once again fragile but valuable book. Further, according to the recipe, we combine different foods, like an 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.
Four approaches are generally used to study microorganisms: metagenomic - DNA research, metatranscriptome - RNA research, metaproteomics - protein research, metabolomics - metabolite research.
Metagenomic sequencing
Metagen - a set of genes of all organisms in the studied environment. The essence of such an analysis is in the sequencing of the 16s rRNA gene, which is responsible for the operation of ribosomal RNA, or in 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 Genetics of Microbiota test is built on it.
Usually under the study of microbiota, this type of analysis is meant, because it was first 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 microorganism database has grown exponentially. Sequencing allows you to study bacteria in the natural environment, whereas in the laboratory, many of them die.
In 2007, US researchers began a project to study the human microbiome of the Human Microbiome Project . It was the impetus for a large-scale study of the composition of intestinal bacteria based on metagenomic data. Following HMP in Europe in 2008 launched a similar project to study the human microbiota - MetaHit .
The essence of metagenomic research is to understand which microorganisms live in the sample, how many are there and what functions they perform. The analysis does not allow to directly evaluate what compounds the bacterium community produces. However, thanks to many metagenomic studies, we can predict this indirectly. For example, if a person has more bacteria producing butyric acid, his microbiota probably produces it well.
Metagenomic studies are widespread because they are easier to conduct in comparison with other methods. The study of RNA, proteins and metabolites requires complex sample cleaning and more time-consuming analyzes.
According to metagenomic data, we have the most results. This is clearly seen in the database of all scientific articles and clinical studies PubMed. A search for a metagenomic microbiome results in about 4,500 different articles, a metatranscriptomic microbiome query - a total of 225, a metaproteomic microbiome - 100, a metabolomic microbiome - 1600.
Metatranscriptome sequencing
A transcript is a collection of all molecules of messenger RNA (mRNA) that are synthesized by a single cell or group of microorganisms. In metatranscriptome analysis, the 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 an RNA molecule. Metatranscriptome studies allow us to estimate the active part of the microbiota. However, the RNA molecule is not as stable as DNA, and quickly disintegrates. Therefore, it is more difficult and expensive to isolate and save it for analysis.
Often, transcriptome studies are used to study certain functions of genes. In this case, the results of the study of RNA are compared with the 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 about the structure, functions and dynamics of the microbial community. Scientists will learn more about how organisms interact with each other, compete for food, produce metabolites.
First, proteins are isolated from the sample. Often, liquid chromatography is used for this. Then conduct an additional analysis to determine the molecular weight - mass spectrometry. This is how we get information about protein fragments (peptides), but not about the whole protein. To assemble the fragments into a coherent whole, special programs are used, and scientists obtain ready-made data.
Metoproteomics is currently less popular than DNA and RNA research. This is due to the complexity of the research and the high probability of error. There may be a lot of human protein or food in the sample. However, metaproteomics can help scientists shed light on the interactions between bacteria and microbiota disruption in people with diseases.
Metabolomics
With this type of analysis, metabolites are studied - substances that bacteria produce. It can be amino acids, lipids, sugars, fatty acids (including butyric) and other compounds. About 40,000 metabolites of the human body are now described, and all of them are recorded in a large database .
As a sample for the study of metabolites, you can use any liquid from the human body: blood, saliva, urine, intestinal lavage (flush), and even cerebrospinal fluid. On average, plasma contains about 4,200 metabolites, 3000 in urine, 500 in cerebrospinal fluid, and 400 in saliva. However, lavage is used as a biomaterial to study the microbiota.
The study of metabolites 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 cannot find out exactly which metabolites the gut microbiota secretes, and which we received from food.
Also, it is impossible to calculate how many bacteria are contained in the microbiota. Therefore, for a more complete picture, the data on metabolites are accompanied by the results of metagenomic analyzes. This approach is sometimes used to study how the microbiota and its metabolites are involved in the development of diseases.
What you need to remember
So far, no microbiota research method has been used in regular clinical practice. Sometimes, for a complete picture, a doctor may recommend a microbiota metagenomic study to assess the composition of intestinal bacteria.
We warn users that the Genetics of Microbiota test is only suitable for educational purposes and is designed for healthy people who are interested in meeting their bacteria. If a person is sick, 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 recommend research for children, because there is much less data on their microbiota. And unnecessary intervention and restriction of the diet of children according to the results of the study is potentially dangerous, since the child may receive less nutrients or suffer from overdiagnosis.
Today, residents of Russia and the CIS countries are offered a study of microbiota metabolites for children and adults using blood or saliva samples using 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 inflammations and diseases 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 on the intestinal microbiota:
Source: https://habr.com/ru/post/459530/
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