Causes of tuberculosis

The family Mycobacteriaceae of the order Actinomycetales contains the single genus Mycobacterium. In 1975, this genus numbered about 30 species, and by 2000 this number was close to 100. Most species of mycobacteria are classified as saprophyte microorganisms widely distributed in the environment.

The group of obligate parasites is insignificant, however its practical significance is great and is determined by species causing tuberculosis in humans and animals. There is an opinion that the ancestors of human pathogenic mycobacteria were ancient soil mycobacteria.

[1], [2], [3], [4],

Taxonomy of mycobacteria

All mycobacteria are divided into pathogenic for humans and conditionally pathogenic.

In clinical microbiology, several approaches are used to classify mycobacteria:

• by speed and optimal growth temperature, ability to form pigment;
• on clinically significant complexes.

Tuberculosis-causing species of mycobacteria are combined into a complex of M. Tuberculosis, including M. Tuberculosis, M. Bovis. M. Bovis BCG, M. Africanum, M. Microti, M. Canettii. Recently, M. Pinnipedii, M. Sargae, phylogenetically related to M. Microti and M. Bovis have been assigned to him.

The rest of the mycobacteria that cause various mycobacteriosis are classified as non-tuberculous mycobacteria. From this group the following complexes are distinguished: M. Avium, consisting of M. Avium, M. Intracellulare, M. Scrofulaceum; M.fortuitum including subspecies M.fortuitum and M. Chelonae, and M. Terrae, including M. Terrae, M. Triviale and M. Nonchromogenicum. The most important groups are the causative agents of M. Leprae leprosy, as well as the causative agents of ulcerative lesions Buruli M. Ulcerans.

This classification combines the types of mycobacteria with the same clinical significance, when the finer their differentiation is insignificant. Biological, biochemical and molecular methods are used to identify species within groups and complexes.

Classification of nontuberculous mycobacteria on the basis of cultural differences was developed by Runion in 1959. According to her. 4 groups of mycobacteria are isolated.

Group I - photochromogenic mycobacteria

This group includes mycobacteria that are not pigmented when grown on darkness, but acquire a bright yellow or yellow-orange pigmentation after exposure to light. Potentially pathogenic strains belonging to this group. - M. Asiaticum, M. Kansasii, M. Marinum, M. Simiae. Among the mycobacteria of this group there are both fast-growing (M. Marinum) and slow growing (M. Asiaticum, M. Kansasii). The optimum growth temperature ranges from 25 ^to C for M. Simiae, 32-33 ^about C for M. Marinum 37 to ^about C for M. Asiaticum.

The greatest clinical significance in our country is the form of M. Kansasii, found in water bodies. The strain of M. Kansasii (M. Luciflavum) causes disease in humans. Egg medium grows as smooth or rough colonies 37 temperature optimum ^of S. Morphologically bacteria moderate length. To date, two variants of M. Kansasii have been described: orange and white. With the introduction of guinea pigs M. Kansasii cause infiltrates and densification of regional lymph nodes.

Group II - scotochromogenic mycobacteria (from the Greek word scotos - darkness)

To this group include mycobacteria, forming a pigment in the dark. The growth rate is 30-60 days. This group includes M. Aquae (M. Gordonae) and M. Scrofulaceum.

M. Scrofulaceum refers to potentially pathogenic species. On the egg medium, the bacteria of this species grow in the form of smooth or rough orange colonies. Morphologically, mycobacteria are rod-shaped, short or long. They grow at a temperature of 25-37 ^o C. Children are affected by lymph nodes and lungs.

M. Aquae (M. Gordonae) is referred to as saprophytic scotochromogenic mycobacteria. On the egg medium grow as orange colonies at a temperature of 25-37 ° C. Morphologically the mycobacterium is rod-shaped and of moderate length (> 5 μm). Found in the reservoirs.

Group III - nonphotochromogenic mycobacteria

This group includes mycobacteria that do not form a pigment or have a pale yellow color, which is not enhanced by light. They grow for 2-3 or 5-6 weeks. To them carry. M. Avium, M. Intracellulare, M. Xenopi, M. Terrae, M. Gastri, M. Hattey, M. Bruiiense.

M. Avium (Mycobacterium avian type) grow on Lowenstein-Jensen medium as a pigmented or slabopigmentirovannyh colonies at 37 ^to C 45 and ^of S. Morphologically - a rod having an average length. They can be pathogenic to humans and a number of laboratory animals, as well as domestic animals (eg, pigs). Found in water and soil.

M. Xenopi is separated from the toad. Young cultures grow in the form of unpigmented colonies. Later, a yellow pigment appears. Morphologically, long threadlike sticks. Grow at a temperature of 40-45 ^o C. Conditionally pathogenic to humans.

M. Terrae was first isolated from radish. They grow on the medium of Levenstein-Jensen and the form of pigmentless colonies. The growth optimum is 37 ^o C. Morphologically they are represented by medium-length rods, saprophytes.

Group IV - fast-growing mycobacteria

Mycobacteria belonging to this group are characterized by rapid growth (up to 7-10 days). Grow in the form of pigment or pigmentless colonies, often in the form of R-form. Good growth allowed for 2-5 days at 25 ^on C. This group includes potentially pathogenic mycobacteria M.fortuitum, and saprophytic mycobacteria such as M. Phlei, M. Smegmatis, and others. M. Fortuitum gives visible growth on the egg medium on the 2nd-4th day in the form of a "rosette." Morphologically, mycobacteria are represented by short rods. On the Lowenstein-Jensen medium, they can absorb malachite greens and be colored green. Widespread in nature.

The Runyon classification proved to be very convenient for identifying the most common species of mycobacteria. However, the identification of new species and the appearance of an increasing number of intermediate forms of mycobacteria causes difficulties in their registration in a certain group of Runyon.

M. Tuberculosis is a young evolutionary formation. Recently, there has been a tendency to divide M. Tuberculosis into clusters or families. The most important are strains belonging to the Beijing family, which are characterized by clonal behavior and the ability to cause microtubes of tuberculosis.

Morphology of mycobacteria

Mycobacteria - thin rod-shaped cells with a characteristic property of acid and alcohol resistance (at one of the stages of growth), aerobic. Gram staining is poorly gram-positive. Mycobacteria are immobile, they do not form a spore. Conidia or capsules are absent. Grow on dense nutrient media slowly or very slowly: at optimal temperature, the visible colonies appear after 2-60 days. Colonies pink, orange or yellow, especially with growth in the light. The pigment does not diffuse. The surface of the colonies is usually matte (S-type) or rough (R-type). Often mycobacteria grow in the form of mucous or wrinkled colonies. On liquid media, mycobacteria grow on the surface. A gentle dry film thickens over time, becomes bumpy-wrinkled and acquires a yellowish hue. The broth remains transparent and it is possible to achieve diffuse growth in the presence of detergents. In microcolonies of M. Tuberculosis (ie in the early stages), structures resembling bundles are formed - a sign that is associated with the cord-factor.

When staining with carbolic fuchsin, mycobacterium tuberculosis is revealed as thin, slightly curved rods of crimson-red color, containing a different number of granules.

The length of mycobacteria is about 1-10 μm. And the width is 0.2-0.7 μm. Sometimes it is possible to find curved or convoluted variants. Microorganisms located singly, in pairs or in groups, stand out well against the blue background of the other components of the preparation. Often, the bacterial cells can be arranged in the form of a Roman numeral "V".

In the preparation, it is also possible to detect altered coccoid acid-resistant forms of the pathogen, rounded spherical or mycelial-like structures. In this case, the positive answer must be confirmed by additional methods.

Structure of the cell wall of mycobacteria

The cell wall of mycobacteria is the most complex in comparison with the rest of the prokaryotes.

While gram-negative bacteria have two membranes, the cell wall of mycobacteria consists of several layers, some of which contain sugars and are characterized by a relatively constant composition. The outer layers have a variable chemical composition and are mainly represented by lipids, most of which are mycolic acids and their derivatives. As a rule, these layers are not visible in electron microscopy. The primary framework of the cell wall is cross-linked peptidoglycans - the electron-dense layer. The layer of arabinogalactans repeats a layer of peptidoglycans, forming a polysaccharide stroma of the cell wall. It has points of connection with the peptidoglycan layer and structures for fixing mycolic acids and their derivatives.

Mycolic acids are present in the form of free sulfolipids and cord-factor, the presence of which on the cell surface is associated with the characteristic formation of M colonies. Tuberculosis in the form of harnesses. The uniqueness and key role of mycolic acids in the structural organization and physiology of mycobacteria make them an excellent target for etiotropic therapy.

A layer of glycolipids is called "mycosides" and is sometimes compared to a microcapsule. Mycosides structurally and functionally resemble the lipopolysaccharides of the outer membrane of gram-negative bacteria, but are devoid of their aggressiveness, yet they are toxic and (like cord-factor and sulfolipids) cause the formation of a granule.

The cell membrane and cell wall layers are permeated with channels or pores, among which passive pores with a short lifetime providing controlled diffusion of substances and channels with a longer lifetime providing energy-dependent transport of substances can be distinguished.

Another component of the cell wall of mycobacteria is lipoarabinomannan. It is anchored on the plasma membrane, permeates the cell wall and emerges on its surface. In this respect, it is similar to lipoteichoic acids of gram-positive bacteria or lipopolysaccharide O-antigen of gram-negative bacteria. Terminal fragments of lipoarabinomannan, especially its mannose radicals, nonspecifically suppress the activation of T-lymphocytes and peripheral blood leukocytes. This leads to a violation of the immune response to mycobacteria.

Variability and forms of mycobacteria

The persistence of bacteria has a particular pathogenetic significance. Laboratory experiments conducted in vitro and in vivo showed that bacterial preparations of isoniazid and pyrazinamide kill mycobacteria only during the reproduction phase. If the mycobacteria are in a phase of low metabolic activity (ie, bacterial growth is almost completely suspended and the bacteria can be called "dormant"), bactericidal preparations do not act on them. This state is usually called dormant, and microorganisms are called persisters. Persisters are not sensitive to chemotherapy, i.e. Behave as resistant microorganisms. In fact, they can remain sensitive to drugs.

A powerful stimulus for the transition of mycobacterial cells to the dormant state is chemotherapeutic agents, as well as the factors of the host's immune system. Persisters can survive in the lesions for months or even years. During persistence, mycobacteria can be transformed into L-forms. In this form, mycobacteria exhibit extremely low metabolic activity, primarily aimed at increasing the thickness of the cell wall and extracellular matrix, which prevents simple diffusion of substances. In addition, in the mycobacteria, there is an accumulation of genetic material, which makes it possible to increase the probability of reconstructing a normally functioning cell under favorable conditions. The detection of L-forms by standard microbiological methods is difficult.

If dormant mycobacteria again acquire metabolic activity and begin to multiply during chemotherapy, they quickly die. If chemotherapy is completed, such "revived" mycobacteria continue to multiply and cause a relapse of the disease. This explains the validity of long courses of chemotherapy and the application of subsequent short prophylactic. As a rule of seasonal, courses of chemoprophylaxis.

Physiology of mycobacteria

In the realm of prokaryotes, mycobacteria are undoubted leaders in the synthesis of the most complex organic compounds. Probably, they possess the most flexible metabolism, providing the necessary variability for survival both in the external environment and in the macroorganism. To date, more than 100 enzymatic reactions have been described, showing the branched and complex character of the metabolism of mycobacteria. For the synthesis of terminal compounds or the provision of necessary physiological functions in the mycobacteria, parallel metabolic pathways may be carried out depending on the availability of the substrate, the chemical environment, and the availability of the necessary components (metal ions, partial pressure of oxygen, carbon dioxide, etc.) in the respiratory cycles.

Biochemical properties of mycobacteria

[5], [6], [7], [8], [9], [10]

Metabolism of lipids

Lipids of the cell wall, which make up to 60% of the dry weight of the cell, determine the non-standard nature of tinctorial, physiological and ecological properties of mycobacteria.

The specific lipids of mycobacteria described so far are structurally divided into 7 main groups:

1. fatty acid derivatives of carbohydrates (mainly trehalose - cord-factor):
2. mannosides of phosphatidylmiosine:
3. fatty acid derivatives of peptides;
4. glycosides of N-acylpeptides - mycosides C;
5. fatty acid esters of fluorothyroles;
6. mycosides A, B. G;
7. Mycols of glycerin.

Lipids of groups 4-6 are found only in mycobacteria.

Among the unique, it should be noted tuberculostearic and tuberculopalmitic acid, which are precursors of mycolic acids.

Mikolovye acid - a group of high molecular fatty acids with a chain length of up to 84 carbon atoms, the structure of the main chain is determined by the systematic position of the microorganism and the conditions of its growth. Their low reactivity provides high chemical stability of the mycobacterium cell wall. Mikolata suppress the enzymatic cleavage of the cell wall and free radical reactions.

Cord factor is attributed to the 1 st group of lipids. It is associated with high toxicity of mycobacteria and virulence.

Surface-active lipids, or sulfolipids, play an important role in intracellular adaptation of mycobacteria. Together with the cord factor, they form cytotoxic membrane -otropic complexes.

Lipoarabinomannan is a heterogeneous mixture of high molecular weight lipopolysaccharides: branched arabinose and mannose polymers with diacylglycerol derivatives of palmitic and tuberculostearic acids.

Mycosides C are peptidoglycolipids. Forming the outer shell of mycobacteria, which can be observed by electron microscopy in the form of a transparent zone on the periphery of the cells. Mycosides are species-specific compounds. Antigenic properties of mycobacteria depend on their type.

The quantitative and qualitative composition of lipid compounds of mycobacteria is dynamic and depends on the age of the cells, the composition of the nutrient media, and the physico-chemical characteristics of the environment. Young cells of mycobacteria begin to form a cell wall from the synthesis of lipopolysaccharides with relatively short aliphatic chains. At this stage, they are quite vulnerable and accessible to the immune system. With the growth of the cell wall and the formation of high-molecular lipids, mycobacteria acquire stability and indifference in the relationship with the immune system.

Metabolism of carbohydrates

The most preferred source of carbon for mycobacteria is glycerol.

The most important carbohydrates are arabinose. Mannose and maltose - account for more than half of all saccharides. In addition, in the life of the cells play the role of trehalose, glucose, fructose, galactose, rhamnose and some other saccharides. At the same time, the synthesis goes along the hydrolase and aldolase pathways. The pyruvate pathway is used for the synthesis of glycogen. Arabinosis and mannose are involved in the formation of important structural compounds. To obtain energy, the pentose phosphate pathway of glucose oxidation is used. It is provided by malate, isocitrate and succinate dehydrogenases, which gives flexibility to the respiratory system.

The glyoxylate pathway is unique, which mycobacteria are used to involve free fatty acids into the tricarboxylic acid cycle, which accumulate during the growth of the mycobacterium. This cycle attracts the attention of researchers as a possible mechanism for the chemotaxis of mycobacteria during persistence.

Metabolism of nitrogen and amino acids

The rate of utilization of mycobacteria nitrates, nitrites, hydroxylamines can serve to identify species. As a source of nitrogen, mycobacteria prefer asparagine. Synthesis of amino acids is a volatile process and is provided by a group of enzymes that allow the use of other amino acid compounds, for example, glutamate.

Nitrite and nitrate reductase activity

Mycobacterium tuberculosis can form adenosine triphosphate (ATP) when transferring electrons through a chain of vectors ending in NO ₃ - rather than O _2. In these reactions, NO ₃ is reduced to NH ₃ in amounts that are necessary for synthesizing amino acids, purine and pyrimidine bases. This is done by the sequential action of nitrate and nitrite reductases.

Catalase and peroxidase activity

Catalase prevents the accumulation of hydrogen peroxide, which is formed during the aerobic oxidation of reconstituted flavoproteins. The enzyme activity depends on the pH of the medium and the temperature. At a temperature of 56 ° C, catalase is not active. There are tests for belonging to the pathogenic complex of mycobacteria, based on the thermal stability of catalase.

It is known that 70% of strains of mycobacterium tuberculosis, resistant to isoniazid, lose its catalase and peroxidase activity.

Peroxidase and catalase activity is carried out by the same enzyme complex.

[11], [12], [13], [14], [15]

Vitamins and coenzymes

The composition of M. Tuberculosis includes vitamins of group B (riboflavin, pyridoxine, cyanocobalamin, thiamine), vitamins C and K. Paraaminobenzoic acid, pantothenic and nicotinic acids, biotin and folic acid.

Metabolism, nutrition and respiration of mycobacteria

In the usual, favorable conditions, mycobacterium tuberculosis - strict aerobes and mesophiles, i.e. They grow in the presence of oxygen and in the temperature range of 30-42 ^{on the} C, preferably at 37 ^for S. Under unfavorable environmental conditions, and (or) oxygen deficiency Mycobacterium tuberculosis manifest themselves as microaerophiles and even anaerobes. At the same time, their metabolism undergoes significant changes.

On consumption of oxygen and development of oxidase systems, mycobacteria are similar to true fungi. As a link between NADH dehydrogenase and cytochrome b in the transfer system of the genus Mycobacterium is vitamin K ₉. This system of cytochromes resembles a mitochondrial system. It is sensitive to dinitrophenol, as well as in higher organisms.

The type of respiration described is not the only source of ATP formation. In addition to O ₂ -terminal. Mycobacteria can use respiratory chains that carry electrons and terminate in nitrates (NO ₃ ^- ). The reserve of the respiratory system of mycobacteria is the glyoxylate cycle.

Anoxic (endogenous) respiration, manifested in an atmosphere with an oxygen concentration of less than 1%, stimulates azide compounds that reduce the oxidation of pyruvate or trehalose.

Growth and reproduction of mycobacteria

Mycobacterium tuberculosis breeds extremely slowly: the period of doubling is 18-24 hours (common bacteria divide every 15 minutes). Therefore, to obtain a visible growth of typical colonies, it takes at least 4-6 weeks. One of the reasons for the slow reproduction of mycobacteria is their pronounced hydrophobicity, which makes diffusion of nutrients more difficult. It is more likely that this is genetically determined and is associated with a more complex device of mycobacteria. It is known, for example, that most bacteria have multiple copies of the ribonucleic ribonucleic acid (rRNA) operon. Slowly growing mycobacteria (M. Tuberculosis, M. Leprae) have one copy of the operon, and fast-growing (M. Smegmatis) have only two copies.

When cultivated on liquid media, mycobacteria grow on the surface. The gentle dry film eventually thickens, becomes bumpy-wrinkled and acquires a yellowish tinge, often compared with the color of ivory. The broth remains transparent, and it is possible to achieve diffuse growth only in the presence of detergents, for example, Tween-80. In microcolonies (ie, in the early stages) structures resembling bundles are formed, a sign that is associated with the cord-factor of M. Tuberculosis.

Genetics of mycobacteria

The genus of mycobacteria is very diverse from a genetic point of view. Unlike many saprophytic and nontuberculous mycobacteria, mycobacterium tuberculosis does not contain extrachromosomal inclusions (for example, plasmids). The whole variety of properties of mycobacterium tuberculosis is determined by its chromosome.

The genome of M. Tuberculosis complex is extremely conservative. Its representatives have DNA homology at 85-100%. While the DNA of other mycobacterial species is homologous to M. Tuberculosis by only 4-26%.

Representatives of the genus Mycobacterium have large genomes in comparison with other prokaryotes - 3.1-4.5x10 ⁹ Da. However, genomes of pathogenic species are less than in other mycobacteria (in M. Tuberculosis - 2.5x10 ⁹ Da). The classical causative agent of human tuberculosis, M. Tuberculosis, has more genes than M. Africanum and M. Bovis, which have lost some of the genetic material during evolution.

In 1998, the nucleotide sequence of the chromosome of strain H37Rv M. Tuberculosis was published. Its length is 4 411529 base pairs. Chromosome Mycobacterium tuberculosis is a ring structure. It contains about 4000 genes encoding proteins, as well as 60. Coding functional components of RNA: unique ribosomal RNA operon, 10Sa RNA. Involved in the degradation of proteins with atypical matrix RNA. 45 transport RNA (tRNA), more than 90 lipoproteins.

More than 20% of the genome is occupied by the genes of fatty acid metabolism of the cell wall, including mycolic acids, glycine-rich acidic polypeptides (PE and PPE families) encoded by the polymorphic regions of the PGRS genome (PEMS) and MPTR (Major polymorphic tandem repeat) , respectively (the fifth and fourth rings of the genomic chromosome map). The variability of these parts of the genome provides differences in antigens and the ability to inhibit the immune response. Genes controlling virulence factors are widely represented in the genome of mycobacterium tuberculosis.

Mycobacterium tuberculosis synthesizes all the components necessary for metabolism: essential amino acids, vitamins, enzymes and cofactors. In comparison with other bacterial species, the activity of lipogenesis enzymes in M. Tuberculosis has been increased. Two genes encode hemoglobin-like proteins, which play the role of antioxidant protectors or traps of excess cellular oxygen. These features contribute to the rapid adaptation of mycobacteria tuberculosis to abrupt changes in environmental conditions.

The peculiarity of the genome of M. Tuberculosis complex is a large number of repeating DNA sequences. So. In the chromosome of M. Tuberculosis H37Rv, there are up to 56 copies of IS-elements (insertion sequences) that provide DNA polymorphism of mycobacterium tuberculosis. Most of them. Except for the IS6110 element. Are unchanged. As a rule, from 5 to 20 copies of IS6110 are present in the chromosome of different strains of mycobacterium tuberculosis, but strains that do not have this element are found. Along with the IS elements, the genome contains several types of short nucleotide repeats (PGRS and MPTR), as well as direct repeats DR (Direct Repeat), located in the DR region and separated by variable sequences - spacers (the sixth ring on the chromosome map). Differences in the number of copies and localization on the chromosome of these genetic elements are used to differentiate strains of mycobacterium tuberculosis in molecular epidemiology. The most perfect genotyping schemes for mycobacteria are based on the detection of genomic polymorphism caused by the element IS6110, as well as DR and their spacers. It is characteristic that the divergence of M. Tuberculosis occurs, as a rule, due to recombinations between copies of the element IS6110. Which flank different genes.

In the genome H37Rv, two prophages are found - phiRv1 and phiRv2. Like the polymorphic Dral site they are. Probably, are associated with pathogenicity factors, since it is these parts of the genome that differ from those of the avirulent strains of M. Tuberculosis H37Ra and M. Bom BCG. Genomic regions (mutT, ogt-genes) are identified that are responsible for increasing the frequency of mutations and adapting mycobacterium tuberculosis in the press conditions. Detection of trigger genes for dermatology of mycobacterium tuberculosis changed the idea of latent tuberculosis infection.

Study of polymorphism of genes encoding catalase, peroxidase and A-subunit of DNA-gyrase. In the M. Tuberculosis complex, three genotypic groups were isolated. The most ancient (from the point of view of evolution) group I: M. Africanum, M. Bovis. M. Tuberculosis and M. Microti. Group II and III include various strains of M. Tuberculosis that have spread in some geographical regions. Clonal behavior is characteristic of groups I and II, and strains of group III extremely rarely cause mass diseases. In various regions of the world, the genetic families of M. Tuberculosis, which have received the names Haarlem, are common. Africa, Filipino.

A special place belongs to the family Beijing, first identified in histological preparations of lung tissue in 1956-1990. From the sick of the suburbs of Beijing. For today, the strains of this family are found in the states of Asia. South Africa, the Caribbean, the United States. The distribution of this genotype in different territories is determined by the ethnic characteristics of the indigenous population and migrants. Recently, data on the distribution of strains of the SI / Beijing genotype in the northwestern European part of Russia (St. Petersburg) and in the regions of Siberia have been obtained.

Stability of mycobacteria

During the evolution of mycobacterium tuberculosis, various mechanisms developed to overcome or inactivate adverse environmental factors. At first. This is a powerful cell wall. Secondly, there are extensive metabolic opportunities. They are able to inactivate many cellular toxins and substances (various peroxides, aldehydes and others) that destroy the cell membrane. Thirdly, it is the morphological plasticity, which consists in the transformation of mycobacteria (formation of L-forms by dormant cells). By their stability, after spore-forming bacteria, they occupy a leading place in the realm of prokaryotes.

The causative agent retains its viability in the dry state for up to 3 years. When the Mycobacterium tuberculosis is heated, it can withstand temperatures well above 80C. To date, it is believed that mycobacterium tuberculosis, which are in sputum, remain viable when the latter is boiled within 5 minutes.

Mycobacterium tuberculosis is resistant to organic and inorganic acids, alkalis, many oxidants, as well as to a number of antiseptic and dehydrating substances, which have a harmful effect on other pathogenic microorganisms. Mycobacteria are resistant to alcohols and acetone.

It is noted that quaternary ammonium-based agents do not exhibit anti-tuberculosis activity. Under certain conditions, concentrations of chlorine and oxygen radicals up to 0.5% also do not have a harmful effect on the mycobacterium tuberculosis. This implies the impossibility of using such a means for sterilization of sputum and other infected biological materials.

Mycobacterium tuberculosis is insensitive to diffused sunlight and can exist for more than a year in the external environment without loss of viability. A shortwave ultraviolet study has a universal bactericidal effect on all microorganisms. However, in real conditions, when mycobacterium tuberculosis is suspended in the form of cellular agglomerates with dust particles, their resistance to ultraviolet radiation increases.

The high survival rate of mycobacterium tuberculosis contributes to the extremely wide spread of this infection among the population irrespective of climatic conditions. However, this not only contributes to the globalization of the problem - mycobacterium tuberculosis can persist for a long time in the human body and can be reactivated at unlimited intervals.

Localization of mycobacterium tuberculosis within macrophages provides sufficient substrate stability, given the "longevity" of mononuclear phagocytes and the duration of replication of mycobacteria, as well as isolation from effectors of humoral immunity. At the same time, the pathogen selects a biotope, which because of potential danger is unacceptable for most microorganisms. This symbiosis is provided by a number of adaptive mechanisms of mycobacteria.

The process of macrophage damage and parasitism in it looks like this: penetration of mycobacteria into a macrophage without its activation; suppression of phagolysosome formation or transformation into a zone that is comfortable for bacteria; breakthrough from phagosomes into the cytoplasm with inactivation of antimicrobial factors; interference in the life of the cell; weakening the sensitivity of macrophages to the activating signals of T-lymphocytes; a decrease in the antigen-presenting function of macrophages and the consequent weakening of the reactions of cytotoxic T-lymphocytes, tuned to the destruction of infected cells.

Undoubtedly, in the provision of this important role play the features of the cell wall. As well as metabolic and functional capabilities. At the first contact with mycobacteria, the immune systems of the macroorganism are not able to connect humoral immunity, quickly disinfect and eliminate the cell from the body, since the mobile aliphatic chains of the mycobacterial wall do not allow evaluation of the surface structures of the pathogen and transmit relevant information for the synthesis of the required set of antibodies.

High hydrophobicity of mycobacteria provides nonspecific, i.e. Independent of receptors, contacts with macrophages. Forming around the mycobacterium cell phagosome, the macrophage places it inside itself. Surface mycosidic and lipoarabinomannan complexes can be recognized by receptors, however the signals triggered through them do not activate or weakly activate macrophages. As a consequence, phagocytosis is not accompanied by the release of free radical forms of oxygen and nitrogen. It is believed that this is more typical for virulent strains of M. Tuberculosis, which, due to the structural features of lipoarabinomannan, initiate a "non-aggressive" phagocytosis. In the recognition of M. Tuberculosis, other macrophage receptors, in particular CD 14 and receptors of the complement C3 component (CR1-CR3), also participate.

Penetrating into the macrophage, the mycobacterium includes a number of mechanisms that prevent the formation of phagolysosomes: the production of ammonium, which alkalizes the environment inside the phagosome, the synthesis of sulfolipids, leading to the formation of a negative charge on the surface of the phagosome. Which prevents the fusion of the phagosome and lysosome.

If a phagolysosome is formed, the mycobacterium, thanks to a powerful waxy membrane, can extinguish the free radical reactions caused by the bactericidal substances of the phagocytes. Ammonium alkalizes the medium, blocking the activity of lysosomal enzymes, and sulfolipids neutralize membrane-cationic proteins. In addition, mycobacteria tuberculosis produce highly active enzymes with catalase and peroxidase activity, which compete with peroxidase systems of macrophages, and simultaneously inactivate hydroperoxides of lysosomes. All this increases the resistance of mycobacteria to oxidative stress.

Further adaptation of mycobacteria consists in using iron-containing macrophages for their enzyme systems and blocking the immunospecific functions of macrophages. Macrophages are one of the main reservoirs of iron, the excess of which accumulates in the form of ferritin. The iron content in the alveolar macrophages is 100 times higher than in the blood monocytes, which certainly contributes to their colonization by mycobacteria tuberculosis.

The toxic effect on macrophages of mycobacterium is carried out by means of endotoxins and nonspecific factors. Both those and others primarily affect the respiratory system of macrophages - mitochondria. Endotoxins include mycolic arabinolipids, which inhibit the respiration of mitochondria. To non-specific toxins include the products of the synthesis of the lipid part of the mycobacterium cell - phthiene and phthionic acids, which cause the dissociation of oxidative phosphorylation. The intensification of metabolic processes under these conditions is not accompanied by the proper synthesis of ATP. Host cells begin to experience energy hunger, which leads to the inhibition of their vital activity, and in the future to cytolysis and apoptosis.

It is possible that some factors of pathogenicity are formed only inside infected cells, as occurs in other bacteria that prefer an intracellular lifestyle. For example, salmonella, parasitizing inside macrophages, additionally express more than 30 genes. Despite the full description of the genome of mycobacterium tuberculosis. 30% of codons are related to proteins with unknown properties.

Drug resistance of mycobacteria

From the clinical point of view, the drug sensitivity of the microorganism determines the possibility of using standard chemotherapy with the indicated drug for treating the disease caused by the isolated strain. Stability "predicts the failure of treatment with a chemotherapeutic drug tested." In other words, the use of standard chemotherapy resulting in achieving a systemic drug concentration, usually effective under normal conditions, does not suppress the reproduction of "resistant microorganisms".

In microbiology, a population approach is based on the definition of drug susceptibility or drug resistance, implying a different degree of stability of the pool (a heterogeneous population) of microbial cells. Drug resistance is assessed in quantitative characteristics, such as "minimal inhibitory concentration" (MIC). For example, with MIK-90, 90% of microorganisms die (bacteriostatic concentration). Thus, resistance should be understood as its degree in a part of the microbial population, which predetermines the failure of treatment in most cases. It is generally accepted that 10% of resistant strains among the entire microbial population of the patient can have a pathogenic effect. In phthisiobacteriology for first-line antituberculosis drugs, it is 1%. Or 20 colony forming units - CFU). This part of the microbial population in a month is able to displace the original and form a lesion focus. For anti-TB drugs of the second series, the stability criterion is a 10% increase in the microbial population.

The development of drug resistance of microorganisms is associated with selection (selection) in the presence of an antibiotic and with the predominant survival of a portion of a microbial population that has protection mechanisms against an antibacterial agent. In each population there is an insignificant amount of mutant cells (usually 10 ⁶ -10 ⁹ ) resistant to this or that drug. During the chemotherapy, sensitive microbial cells die, and the resistant ones multiply. As a result, the sensory cells are replaced by stable cells.

Mycobacteria initially have a high natural resistance to many antibacterial drugs of a wide spectrum of action, but different species have different spectrum and degree of this sensitivity.

True natural resistance is understood as a permanent species characteristic of microorganisms associated with the lack of a target for the action of the antibiotic or the inaccessibility of the target due to the initially low permeability of the cell wall, the enzymatic inactivation of the substance or other mechanisms.

Acquired resistance is the property of individual strains to remain viable at those concentrations of antibiotics that inhibit the growth of the main part of the microbial population. The acquisition of resistance in all cases is caused genetically: the emergence of new genetic information or a change in the level of expression of their own genes.

At the present time, various molecular mechanisms of resistance of mycobacteria of tuberculosis have been discovered:

• inactivation of the antibiotic (enzymatic inactivation), for example, β-lactamases;
• modification of the target of action (change in the spatial configuration of the protein due to mutation of the corresponding region of the genome):
• hyperproduction of the target, leading to a change in the ratio of the target agent to the release of a portion of the life-supporting proteins of the bacterium;
• active removal of the drug from the microbial cell (efflux) due to the inclusion of stress protective mechanisms:
• change the parameters of permeability of external microbial cell structures that block the ability of the antibiotic to penetrate the interior of the cell;
• Inclusion of the "metabolic shunt" (bypass exchange path).

In addition to direct effects on the metabolism of microbial cells, many antibacterial drugs (benzylpenicillin streptomycin, rifampicin) and other adverse factors (biocides of the immune system) lead to the appearance of altered forms of mycobacteria (protoplasts, L-forms). As well as transfer cells into a dormant state: the intensity of cell exchange decreases and the bacterium becomes immune to the action of the antibiotic.

All mechanisms form a different degree of resistance, providing resistance to different concentrations of chemotherapy drugs, so the emergence of resistance in bacteria is not always accompanied by a decrease in the clinical effectiveness of the antibiotic. To assess the effectiveness and prognosis of treatment, it is important to know the degree of resistance.

At present, at least one gene is determined for each antituberculous drug of the first series and for most of the reserve preparations. Specific mutations in which lead to the development of resistant variants of mycobacteria. In the wide spread of drug resistance in mycobacteria, the high incidence of mutations in vivo is more important than in vitro.

[16], [17], [18], [19], [20], [21], [22], [23], [24], [25],

Types of drug resistance of mycobacteria

Distinguish between primary and acquired drug resistance. The microorganisms with primary resistance include strains isolated from patients who did not receive specific therapy or who received drugs for a month or less. If it is impossible to clarify the fact of using anti-tuberculosis drugs, the term "initial resistance" is used.

Primary drug resistance is of great clinical and epidemiological importance, therefore, in order to correctly evaluate it, it is necessary not to carry out chemotherapy for the first time detected by the tuberculosis patient before microbiological examination of the diagnostic material. The frequency of primary drug resistance is calculated as the ratio of the number of newly diagnosed tuberculosis patients with primary resistance to the number of all newly diagnosed patients who were tested for drug sensitivity during the goal. If a stable strain is isolated in the patient against the background of antituberculous therapy conducted for a month or more, regard as acquired. The frequency of primary drug resistance characterizes the epidemiological state of the TB pathogen population.

Acquired drug resistance among newly diagnosed patients is the result of unsuccessful treatment (incorrect selection of drugs, failure to adhere to the regimen, lower dosages of drugs, unstable supplies and poor quality of drugs). These factors lead to a decrease in the systemic concentration of drugs in the blood and their effectiveness, while simultaneously "triggering" protective cells in mycobacterial cells.

For epidemiological purposes, the frequency of previously treated cases is calculated. For this purpose, patients recruited for repeated treatment after an unsuccessful course of chemotherapy or relapses are taken into account. The ratio of the number of resistant cultures of mycobacterium tuberculosis is calculated to the number of all strains examined for the presence of drug resistance during the year among patients of this group at the time of their registration.

In the structure of drug resistance, mycobacteria tuberculosis is distinguished:

Monoresistance - resistance to one of the antituberculous drugs, sensitivity to other drugs is preserved. When using complex therapy, mono-resistance is rarely detected and. As a rule, to streptomycin (in 10-15% of cases among newly diagnosed patients).

Multidrug resistance - resistance to two or more drugs.

Multiple drug resistance - resistance to isoniazid and rifampicin simultaneously (regardless of the availability of resistance to other drugs). It is accompanied, as a rule, by resistance to streptomycin, etc. At present, MDR of tuberculosis pathogens has become an epidemiologically dangerous phenomenon. The calculations show that detection of pathogens with MDR in more than 6.6% of cases (among newly diagnosed patients) requires a change in the strategy of the National Tuberculosis Program. According to the monitoring of drug resistance, the frequency of MDR among newly diagnosed patients is 4 to 15%, among relapses - 45-55%, and among the cases of unsuccessful treatment - up to 80%.

Superstability - multiple drug resistance combined with resistance to fluoroquinolones and one of the injectable drugs (kanamycin, amikacin, capreomycin). Tuberculosis, caused by strains with superstability, poses a direct threat to the life of patients, as the other anti-tuberculosis drugs of the second row do not have a pronounced antibacterial effect. Since 2006, in some countries, the distribution of strains of mycobacteria with superstability has been monitored. Abroad it is customary to designate this version of MDR as XDR.

Cross-resistance - when the emergence of resistance to a single drug entails resistance to other drugs. In M. Tuberculosis, as a rule, the mutations associated with resistance are not interrelated. The development of cross-resistance is due to the similarity of the chemical structure of some anti-tuberculosis drugs. Particularly often cross resistance is detected within one group of drugs, for example aminoglycosides. For the prediction of cross-resistance, studies of the culture of mycobacteria at the genetic level in combination with a microbiological study of resistance are needed.

Non-tuberculous mycobacteria

Nontuberculous mycobacteria are transmitted from person to person very rarely. The frequency of allocation of some of their species from the material from patients is comparable with the frequency of these species from the objects of the external environment. Sources of infection can be farm animals and birds, unprocessed foods. Mycobacteria are found in post-mortem material and in milk of cattle.

According to bacteriological laboratories, the prevalence of non-tuberculosis mycobacteria in 2004-2005 was 0.5-6.2% among all mycobacteria in newly diagnosed patients. Probably, the frequency can be somewhat higher, since the method used to process the diagnostic material is not optimal for non tuberculous mycobacteria. Saprophytic mycobacteria can be present in the diagnostic material if the collection rules are not followed, or because of the material's specificity (for example, M. Smegmatis may be excreted from the urine of male patients).

In this regard, it is important to repeatedly confirm the detected species of mycobacteria from the material from the patient.

Mycobacteria affect the skin, soft tissue, and can also cause mycobacteriosis of the lungs, which is especially common in immunodeficient conditions. With pulmonary localization is more often detected in older men, whose history has chronic pulmonary diseases, including those with fungal lesions.

Of all mycobacteria, the M. Avium-intracellularae complex is the most common causative agent of lung mycobacteriosis in man. It causes diseases of the lungs, peripheral lymph nodes and disseminated processes. In the north of the European region, about 60% of mycobacteriosis of the lungs. Fibrous-cavernous and infiltrative processes predominate taking chronic course because of high resistance to anti-tuberculosis drugs.

M. Kansasii are the causative agents of chronic lung disease, resembling tuberculosis. Chemotherapy is more effective, due to the higher sensitivity of M. Kansasii to antibacterial drugs. M. Xenopi and M. Malmoense cause, mainly, chronic lung diseases. They can pollute the water system of hot and cold water. The habitat of M. Malmoens is not fully established. M. Xenopi show quite good sensitivity to anti-tuberculosis therapy. M. Malmoense show a sufficiently high sensitivity to antibiotics in vitro, but conservative treatment is often ineffective until death. M. Fortuitum and M. Chelonae are recognized as causative agents of diseases of bones and soft tissues due to direct infection of the wound with trauma, surgical intervention and penetrating injury. They cause up to 10% of mycobacteriosis of the lungs. It flows like a chronic, destructive, bilateral defeat, often lethal. Antituberculosis drugs and broad-spectrum antibiotics are not active or are not very active against these types of mycobacteria.

In the southern regions, mycobacteriosis of the skin and soft tissues caused by M. Leprae, M. Ulceranse. Detection of nontuberculous mycobacteria is carried out in laboratories of the leading anti-tuberculosis institutions of the country. This requires high qualification and good equipment of laboratories.