Causes of tuberculosis

The Mycobacteriaceae family of the Actinomycetales order contains the single genus Mycobacterium. In 1975, this genus contained about 30 species, and by 2000, this number had already approached 100. Most mycobacteria species are classified as saprophytic microorganisms, widespread in the environment.

The group of obligate parasites is insignificant, but its practical significance is great and is determined by the species that cause tuberculosis in humans and animals. There is an opinion that the predecessors of mycobacteria pathogenic for humans were ancient soil mycobacteria.

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Taxonomy of mycobacteria

All mycobacteria are divided into those pathogenic for humans and opportunistic.

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

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

The mycobacteria species that cause tuberculosis are combined into the M. tuberculosis complex, which includes M. tuberculosis, M. bovis. M. bovis BCG, M. africanum, M. microti, M. canettii. Recently, M. pinnipedii and M. sarrae, which are phylogenetically related to M. microti and M. bovis, have been added to it.

The remaining mycobacteria causing various mycobacterioses are classified as non-tuberculous mycobacteria. The following complexes are distinguished from this group: M. avium, consisting of M. avium, M. intracellulare, M. scrofulaceum; M.fortuitum including the subspecies M.fortuitum and M. chelonae, and M. terrae, including M. terrae, M. triviale and M. nonchromogenicum. The most important groups are the leprosy pathogen M. leprae, as well as the ulcerative lesion pathogen Buruli M. ulcerans.

This classification unites mycobacteria species with the same clinical significance, when their finer differentiation is not essential. Biological, biochemical and molecular methods are used to identify species within groups and complexes.

The classification of non-tuberculous mycobacteria based on cultural differences was developed by Runyon in 1959. According to it, 4 groups of mycobacteria are distinguished.

Group I - photochromogenic mycobacteria

This group includes mycobacteria that are not pigmented when grown in the dark, but acquire bright yellow or yellow-orange pigmentation after exposure to light. Potentially pathogenic strains belonging to this group are 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 optimal growth temperature varies from 25 ^° C for M. simiae, 32-33 ^° C for M. marinum to 37 ^° C for M. asiaticum.

The most clinically significant species in our country is M. kansasii, which is found in water bodies. The M. kansasii strain (M. luciflavum) causes diseases in humans. It grows in an egg medium as rough or smooth colonies, with a temperature optimum of 37 ^° C. Morphologically, the bacteria are of moderate length. Two variants of M. kansasii have been described to date: orange and white. When introduced to guinea pigs, M. kansasii causes infiltrates and compaction of regional lymph nodes.

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

This group includes mycobacteria that form pigment in the dark. The growth rate is 30-60 days. This group includes M. aquae (M. gordonae) and M. scrofulaceum.

M. scrofulaceum is considered a potentially pathogenic species. On egg medium, bacteria of this species grow as smooth or rough colonies of orange color. Morphologically, mycobacteria are rod-shaped, short or long. They grow at a temperature of 25-37 ^o C. In children, they cause damage to the lymph nodes and lungs.

M. aquae (M. gordonae) are classified as saprophytic scotochromogenic mycobacteria. They grow in egg medium as orange colonies at a temperature of 25-37 °C. Morphologically, the mycobacteria are rod-shaped and moderately long (>5 μm). They are found in water bodies.

Group III - non-photochromogenic mycobacteria

This group includes mycobacteria that do not form pigment or have a pale yellow color that does not intensify in the light. They grow for 2-3 or 5-6 weeks. They include: M. avium, M. intracellulare, M. xenopi, M. terrae, M. gastri, M. hattey, M. bruiiense.

M. avium (avian mycobacteria) grow on the Lowenstein-Jensen medium as pigmented or weakly pigmented colonies at 37 ^° C and 45 ^° C. Morphologically, they are medium-length rods. They can be pathogenic for humans and a number of laboratory and domestic animals (e.g., pigs). They are found in water and soil.

M. xenopi is isolated from a toad. Young cultures grow as unpigmented colonies. Later, a yellow pigment appears. Morphologically, they are long filiform rods. They grow at a temperature of 40-45 ^o C. They are conditionally pathogenic for humans.

M. terrae were first isolated from radish. They grow on Lowenstein-Jensen medium and as pigment-free colonies. The optimum growth temperature is 37 ^° C. Morphologically, they are represented by rods of moderate length, saprophytes.

Group IV - fast growing mycobacteria

Mycobacteria belonging to this group are characterized by rapid growth (up to 7-10 days). They grow in the form of pigmented or non-pigmented colonies, more often in the form of R-form. Good growth is given for 2-5 days at a temperature of 25 ^o C. This group includes potentially pathogenic mycobacteria M.fortuitum, as well as saprophytic mycobacteria, such as M. phlei, M. smegmatis, etc. M. fortuitum gives visible growth on the egg medium on the 2-4th day in the form of a "rosette". Morphologically, mycobacteria are represented by short rods. On the Lowenstein-Jensen medium, they can absorb malachite green and turn green. They are widespread in nature.

The Runyon classification has proven to be very convenient for identifying the most common types of mycobacteria. However, the discovery of new species and the emergence of an increasing number of intermediate forms of mycobacteria causes difficulties in registering them in one or another Runyon group.

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

Morphology of mycobacteria

Mycobacteria are thin rod-shaped cells with the characteristic property of acid- and alcohol-fastness (at one of the growth stages), aerobic. When stained according to Gram, they are weakly gram-positive. Mycobacteria are immobile, do not form spores. Conidia or capsules are absent. They grow on dense nutrient media slowly or very slowly: at the optimal temperature, visible colonies appear after 2-60 days. Colonies are pink, orange or yellow, especially when growing in the light. The pigment does not diffuse. The surface of the colonies is usually matte (S-type) or rough (R-type). Mycobacteria often grow in the form of mucous or wrinkled colonies. On liquid media, mycobacteria grow on the surface. The delicate dry film thickens over time, becomes bumpy-wrinkled and acquires a yellowish tint. The broth remains transparent and diffuse growth can be achieved in the presence of detergents. In the microcolonies of M. tuberculosis (i.e. in the early stages), structures resembling cords are formed - a feature that is associated with the cord factor.

When stained with carbol fuchsin, mycobacteria tuberculosis appear as thin, slightly curved rods of a raspberry-red color, containing a varying number of granules.

The length of mycobacteria is approximately 1-10 µm and the width is 0.2-0.7 µm. Sometimes curved or twisted variants can be found. Microorganisms located singly, in pairs or in groups stand out well against the blue background of other components of the preparation. Bacterial cells can often be arranged in the form of the Roman numeral "V".

The preparation can also reveal altered coccoid acid-resistant forms of the pathogen, rounded spherical or mycelium-like structures. In this case, a positive answer must be confirmed by additional methods.

The structure of the cell wall of mycobacteria

The cell wall of mycobacteria is the most complex compared to other prokaryotes.

While gram-negative bacteria have two membranes, the mycobacterial cell wall consists of several layers, some of which contain sugars and are characterized by a relatively constant composition. The outer layers have a changing 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 under electron microscopy. The primary framework of the cell wall is cross-linked peptide glycans - an electron-dense layer. The arabinogalactan layer repeats the peptide glycan layer, forming a polysaccharide stroma of the cell wall. It has points of connection with the peptide glycan layer and structures for the attachment of 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. tuberculosis colonies in the form of flagella. The uniqueness and key role of mycolic acids in the structural organization and physiology of mycobacteria make them an excellent target for etiotropic therapy.

The glycolipid layer is called "mycosides" and is sometimes compared to a microcapsule. Mycosides are structurally and functionally similar to the lipopolysaccharides of the outer membrane of gram-negative bacteria, but lack their aggressiveness; nevertheless, they are toxic and (like cord factor and sulfolipids) cause the formation of granulomas.

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

Another component of the mycobacterial cell wall is lipoarabinomannan. It is anchored to the plasma membrane, penetrates the cell wall and comes out on its surface. In this respect, it is similar to the lipoteichoic acids of gram-positive bacteria or the lipopolysaccharide O-antigen of gram-negative bacteria. The terminal fragments of lipoarabinomannan, primarily its mannose radicals, non-specifically suppress the activation of T-lymphocytes and leukocytes in the peripheral blood. This leads to a disruption of the immune response to mycobacteria.

Variability and forms of existence of mycobacteria

Persistence of bacteria has a special pathogenetic significance. Laboratory experiments conducted in vitro and in vivo have shown that the bactericidal drugs isoniazid and pyrazinamide kill mycobacteria only in the reproduction phase. If mycobacteria are in the phase of low metabolic activity (i.e. bacterial growth is almost completely suspended and the bacteria can be called "dormant"), bactericidal drugs do not affect them. This state is usually called dormant, and the microorganisms are called persisters. Persisters are not sensitive to chemotherapeutic drugs, i.e. they behave like resistant microorganisms. In fact, they can retain sensitivity to drugs.

A powerful stimulus for the transition of mycobacterial cells to a dormant state is chemotherapeutic drugs, as well as factors of the host immune system. Persisters are able to remain in the lesions for months or even years. During persistence, mycobacteria can transform into L-forms. In this form, mycobacteria exhibit extremely low metabolic activity, aimed primarily at increasing the thickness of the cell wall and extracellular matrix, which prevents simple diffusion of substances. In addition, mycobacteria accumulate genetic material, which increases the likelihood of recreating a normally functioning cell when favorable conditions occur. Detection of L-forms by standard microbiological methods is difficult.

If dormant mycobacteria regain 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 justification for long courses of chemotherapy and the use of subsequent short prophylactic, usually seasonal, courses of chemoprophylaxis.

Physiology of mycobacteria

In the kingdom of prokaryotes, mycobacteria are the undisputed leaders in the field of synthesis of complex organic compounds. They probably have 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 nature of mycobacterial metabolism. To synthesize final compounds or provide the necessary physiological functions in mycobacteria, parallel metabolic pathways can be carried out depending on the availability of the substrate, chemical environment, provision of respiratory cycles with the necessary components (metal ions, partial pressure of oxygen, carbon dioxide, etc.).

Biochemical properties of mycobacteria

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Lipid metabolism

Cell wall lipids, which make up to 60% of the dry mass of the cell, determine the non-standard tinctorial, physiological and ecological properties of mycobacteria.

The specific lipids of mycobacteria described to date are divided into 7 main groups according to structural features:

1. fatty acid derivatives of carbohydrates (mainly trehalose - cord factor):
2. phosphatidyl myoinositol mannosides:
3. fatty acid derivatives of peptides;
4. N-acylpeptide glycosides - mycosides C;
5. fatty acid esters of phthiocerols;
6. mycosides A, B. G;
7. glycerol mycolates.

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

Among the unique ones, it is worth noting tuberculostearic and tuberculopalmitic acids, which are precursors of mycolic acids.

Mycolic acids are a group of high-molecular fatty acids with a chain length of up to 84 carbon atoms, the structure of the main chain of which is determined by the systematic position of the microorganism and the conditions of its growth. Their low reactivity ensures high chemical resistance of the cell wall of mycobacteria. Mycolates suppress enzymatic cleavage of the cell wall and free-radical reactions.

Cord factor is classified as a lipid group 1. It is associated with high toxicity of mycobacteria and virulence.

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

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

Mycosides C are peptide glycolipids that form the outer membrane of mycobacteria, which can be observed under electron microscopy as a transparent zone on the periphery of the cells. Mycosides are species-specific compounds. The 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 physicochemical characteristics of the environment. Young mycobacterial cells begin to form a cell wall by synthesizing lipopolysaccharides with relatively short aliphatic chains. At this stage, they are quite vulnerable and accessible to the immune system. As the cell wall grows and high-molecular lipids are formed, mycobacteria acquire resistance and indifference in their interactions with the immune system.

Carbohydrate metabolism

The most preferred carbon source for mycobacteria is glycerol.

The most important carbohydrates are arabinose, mannose and maltose, which make up more than half of all saccharides. In addition, trehalose, glucose, fructose, galactose, rhamnose and some other saccharides play a role in the cell's vital activity. In this case, synthesis occurs along the hydrolase and aldolase pathways. The pyruvate pathway is used to synthesize glycogen. Arabinose and mannose participate in the formation of important structural compounds. The pentose phosphate pathway of glucose oxidation is used to obtain energy. It is provided by the enzymes malate, isocitrate and succinate dehydrogenases, which gives flexibility to the respiratory system.

The glyoxylate pathway, which mycobacteria use to incorporate free fatty acids that accumulate during mycobacterial growth into the tricarboxylic acid cycle, is unique. This cycle has attracted the attention of researchers as a possible mechanism for mycobacterial chemotaxis during persistence.

Nitrogen and amino acid metabolism

The rate of utilization of nitrates, nitrites, and hydroxylamines by mycobacteria can be used to identify species. Mycobacteria prefer asparagine as a source of nitrogen. The synthesis of amino acids is an energy-dependent process and is provided by a group of enzymes that allow the use of other amino acid compounds, such as glutamate.

Nitrite and nitrate reductase activity

Mycobacterium tuberculosis can form adenosine triphosphate (ATP) by transferring electrons along a chain of carriers ending in NO ₃ - rather than O _2. These reactions reduce NO ₃ to NH ₃ in quantities that are necessary for the synthesis of amino acids, purine and pyrimidine bases. This is accomplished through the sequential action of nitrate and nitrite reductases.

Catalase and peroxidase activity

Catalase prevents the accumulation of hydrogen peroxide, which is formed during aerobic oxidation of reduced flavoproteins. The enzyme activity depends on the pH of the medium and 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 thermolability of catalase.

It is known that 70% of Mycobacterium tuberculosis strains resistant to isoniazid lose their catalase and peroxidase activity.

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

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Vitamins and coenzymes

M. tuberculosis contains B vitamins (riboflavin, pyridoxine, cyanocobalamin, thiamine), vitamins C and K, para-aminobenzoic acid, pantothenic and nicotinic acids, biotin and folic acid.

Metabolism, nutrition and respiration of mycobacteria

In normal, favorable conditions, mycobacteria tuberculosis are strict aerobes and mesophiles, i.e. they grow in the presence of oxygen and in the temperature range of 30-42 ^o C, best of all at 37 ^o C. Under unfavorable external conditions and/or oxygen deficiency, mycobacteria tuberculosis manifest themselves as microaerophiles and even as anaerobes. In this case, their metabolism undergoes significant changes.

_{In terms of oxygen consumption and the development of oxidase systems, mycobacteria are similar to true fungi. Vitamin K 9} serves as a link between NADH dehydrogenase and cytochrome b in the transfer system of the genus Mycobacterium. This cytochrome system resembles the mitochondrial one. It is sensitive to dinitrophenol, just as in higher organisms.

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

Anoxic (endogenous) respiration, which occurs in an atmosphere with an oxygen concentration of less than 1%, is stimulated by azide compounds, which reduce the oxidation of pyruvate or trehalose.

Growth and reproduction of mycobacteria

Mycobacterium tuberculosis reproduces extremely slowly: the doubling period is 18-24 hours (normal bacteria divide every 15 minutes). Therefore, to obtain visible growth of typical colonies, at least 4-6 weeks are required. One of the reasons for the slow reproduction of mycobacteria is considered to be their pronounced hydrophobicity, which complicates the diffusion of nutrients. It is more likely that this is genetically determined and is associated with a more complex structure of mycobacteria. It is known, for example, that most bacteria have multiple copies of the ribosomal ribonucleic acid (rRNA) operon. Slowly growing mycobacteria (M. tuberculosis, M. leprae) have one copy of the operon, and fast-growing (M. smegmatis) - only two copies.

When cultured in liquid media, mycobacteria grow on the surface. The delicate dry film thickens over time, becomes bumpy and wrinkled, and acquires a yellowish tint, often compared to the color of ivory. The broth remains transparent, and diffuse growth can only be achieved in the presence of detergents, such as Tween-80. In microcolonies (i.e., in the early stages), structures resembling bundles are formed - a feature associated with the cord factor of M. tuberculosis.

Genetics of mycobacteria

The genus Mycobacterium is genetically very diverse. Unlike many saprophytic and non-tuberculous mycobacteria, Mycobacterium tuberculosis does not contain extrachromosomal inclusions (e.g. plasmids). All the diversity of properties of Mycobacterium tuberculosis is determined by its chromosome.

The genome of the M. tuberculosis complex is extremely conservative. Its representatives have DNA homology at the level of 85-100%, while the DNA of other species of mycobacteria is homologous to M. tuberculosis by only 4-26%.

Representatives of the genus Mycobacteria have large genomes compared to other prokaryotes - 3.1-4.5x10 ⁹ Da. However, the genomes of pathogenic species are smaller than those of other mycobacteria (in M. tuberculosis - 2.5x10 ⁹ Da). The classic causative agent of human tuberculosis, M. tuberculosis, has more genes than M. africanum and M. bovis, which have lost some of their genetic material during evolution.

In 1998, the nucleotide sequence of the chromosome of the H37Rv strain of M. tuberculosis was published. Its length is 4,411,529 base pairs. The chromosome of the tuberculosis mycobacterium is a ring structure. It contains about 4,000 genes encoding proteins, as well as 60 encoding functional RNA components: a unique ribosomal RNA operon, 10Sa RNA. participating 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 genes of cell wall fatty acid metabolism, including mycolic acids, glycine-rich acidic polypeptides (PE and PPE families), encoded by polymorphic regions of the PGRS (Polymorphic GC-rich repetitive sequence) and MPTR (Major polymorphic tandem repeat) genome, respectively (the fifth and fourth rings of the genomic chromosome map). The variability of these genome regions ensures differences in antigens and the ability to inhibit the immune response. The genome of Mycobacterium tuberculosis widely contains genes that control virulence factors.

Mycobacterium tuberculosis synthesizes all the components necessary for metabolism: essential amino acids, vitamins, enzymes and cofactors. Compared to other types of bacteria, M. tuberculosis has increased activity of lipogenesis enzymes. Two genes encode hemoglobin-like proteins that act as antioxidant protectors or traps for excess cellular oxygen. These features facilitate rapid adaptation of mycobacterium tuberculosis to abrupt changes in environmental conditions.

A distinctive feature of the M. tuberculosis complex genome is a large number of repeating DNA sequences. Thus, the chromosome of M. tuberculosis H37Rv contains up to 56 copies of IS elements (insertion sequences), which provide DNA polymorphism of Mycobacterium tuberculosis. Most of them, with the exception of the IS6110 element, are unchanged. The chromosomes of various strains of Mycobacterium tuberculosis usually contain from 5 to 20 copies of IS6110, but there are strains that do not have this element. Along with 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 advanced schemes for genotyping mycobacteria are based on the detection of genomic polymorphism caused by the IS6110 element, as well as DR and their spacers. It is characteristic that the divergence of the M. tuberculosis species occurs, as a rule, due to recombinations between copies of the IS6110 element, which flank different genes.

Two prophages, phiRv1 and phiRv2, were found in the H37Rv genome. Like the Dral polymorphic site, they are probably associated with pathogenicity factors, since these regions of the genome differ from similar regions of the avirulent strains of M. tuberculosis H37Ra and M. bom BCG. Regions of the genome (mutT, ogt-genes) responsible for an increase in the mutation rate and adaptation of mycobacteria tuberculosis under press conditions were identified. The discovery of trigger genes for dormancy of mycobacteria tuberculosis changed the concept of latent tuberculosis infection.

Study of polymorphism of genes encoding catalase, peroxidase and A-subunit of DNA gyrase. Three genotypic groups were identified in M. tuberculosis complex. The most ancient (from the point of view of evolution) is group I: M. africanum, M. bovis. M. tuberculosis and M. microti. Groups II and III include different strains of M. tuberculosis, which have become widespread in some geographic regions. Clonal behavior is characteristic of groups I and II, and strains of group III extremely rarely cause mass diseases. Genetic families of M. tuberculosis, which received the names Haarlem. Africa, Filipino, are widespread in different regions of the world.

A special place is occupied by the Beijing family, first identified in histological preparations of lung tissue from patients in the suburbs of Beijing in 1956-1990. To date, strains of this family have been found in Asian countries, South Africa, the Caribbean, and the United States. The spread of this genotype in different territories is determined by the ethnic characteristics of the indigenous population and migrants. Recently, data have been obtained on the spread of strains of the SI/Beijing genotype in the northwest of the European part of Russia (St. Petersburg) and in the regions of Siberia.

Mycobacterial resistance

During the evolution, tuberculosis mycobacteria have developed various mechanisms to overcome or inactivate unfavorable environmental factors. Firstly, this is a powerful cell wall. Secondly, these are extensive metabolic capabilities. They are capable of inactivating many cellular toxins and substances (various peroxides, aldehydes, and others) that destroy the cell membrane. Thirdly, this is morphological plasticity, which consists in the transformation of mycobacteria (the formation of L-forms of dormant cells). In terms of their stability, after spore-forming bacteria, they occupy a leading place in the kingdom of prokaryotes.

The pathogen remains viable in a dry state for up to 3 years. When heated, tuberculosis mycobacteria can withstand temperatures significantly higher than 80C. Today, it is believed that tuberculosis mycobacteria found in sputum remain viable when the latter is openly boiled for 5 minutes.

Mycobacterium tuberculosis is resistant to organic and inorganic acids, alkalis, many oxidizers, as well as a number of antiseptic and dehydrating substances that have a detrimental effect on other pathogenic microorganisms. Mycobacterium exhibits resistance to the effects of alcohols and acetone.

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

Mycobacterium tuberculosis is insensitive to diffuse sunlight and can exist in the external environment for more than a year without losing viability. Short-wave ultraviolet radiation 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 tuberculosis mycobacteria contributes to the extremely wide spread of this infection among the population regardless of climatic conditions. However, this is not the only factor that contributes to the globalization of the problem - tuberculosis mycobacteria can persist in the human body for a long time and reactivate at unlimited intervals.

Localization of the tuberculosis mycobacterium inside macrophages provides sufficient substrate stability, taking into account the "longevity" of mononuclear phagocytes and the duration of mycobacterial replication, as well as isolation from humoral immunity effectors. At the same time, the pathogen selects a biotope that is unacceptable for most microorganisms due to its potential danger. 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 the macrophage without its activation; suppression of the formation of phagolysosomes or their transformation into a zone comfortable for bacteria; breakthrough from phagosomes into the cytoplasm with inactivation of antimicrobial factors; interference in the vital activity of the cell; weakening of the sensitivity of macrophages to activating signals of T-lymphocytes; reduction of the antigen-presenting function of macrophages and the associated weakening of the reactions of cytotoxic T-lymphocytes configured to destroy infected cells.

Of course, the cell wall features play an important role in ensuring this, as well as metabolic and functional capabilities. At the first contact with mycobacteria, the immune systems of the macroorganism are not able to activate humoral immunity, quickly neutralize and eliminate the cell from the body, since the mobile aliphatic chains of the mycobacterial wall do not allow an assessment of the surface structures of the pathogen and transmit the relevant information for the synthesis of the necessary set of antibodies.

The high hydrophobicity of mycobacteria ensures non-specific, i.e. receptor-independent, contacts with macrophages. By forming a phagosome around the mycobacterium cell, the macrophage places it inside itself. Surface mycoside and lipoarabinomannan complexes can be recognized by receptors, but the signals triggered through them do not activate or weakly activate macrophages. As a result, phagocytosis is not accompanied by the release of free-radical forms of oxygen and nitrogen. It is believed that this is more characteristic of virulent strains of M. tuberculosis, which, due to the structural features of lipoarabinomannan, initiate "non-aggressive" phagocytosis. Other macrophage receptors, in particular CD 14 and receptors of the complement component C3 (CR1-CR3), also participate in the recognition of M. tuberculosis.

Having penetrated inside the macrophage, the mycobacterium includes a number of mechanisms that prevent the formation of the phagolysosome: 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 its powerful waxy shell, is able to quench free-radical reactions caused by bactericidal substances of phagocytes. Ammonium alkalizes the environment, blocking the activity of lysosomal enzymes, and sulfolipids neutralize membranotropic cationic proteins. In addition, tuberculosis mycobacteria produce highly active enzymes with catalase and peroxidase activity, which compete with the peroxidase systems of macrophages, and simultaneously inactivate lysosome hydroperoxides. All this increases the resistance of mycobacteria to oxidative stress.

Further adaptation of mycobacteria consists of using iron-containing compounds of 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 alveolar macrophages is 100 times higher than in blood monocytes, which certainly contributes to their colonization by tuberculosis mycobacteria.

Mycobacteria exert toxic effects on macrophages by means of endotoxins and non-specific factors. Both of them primarily affect the respiratory system of macrophages - mitochondria. Endotoxins include mycolic arabinolipids, which inhibit mitochondrial respiration. Non-specific toxins include products of the synthesis of the lipid part of the mycobacterial cell - phthiene and phthionic acids, which cause uncoupling of oxidative phosphorylation. Increased metabolic processes under these conditions are not accompanied by proper ATP synthesis. The host cells begin to experience energy starvation, which leads to inhibition of their vital activity, and subsequently to cytolysis and apoptosis.

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

Drug resistance of mycobacteria

From a clinical point of view, drug susceptibility of a microorganism determines whether standard chemotherapy with the indicated drug can be used to treat the disease caused by the isolated strain. Resistance "predicts treatment failure with the drug being tested." In other words, using standard chemotherapy that results in a systemic drug concentration that is usually effective under normal conditions does not suppress the proliferation of "resistant microorganisms."

In microbiology, the definition of drug sensitivity or drug resistance is based on the population approach, which implies different degrees of resistance of a pool (heterogeneous set) of microbial cells. Drug resistance is assessed in quantitative characteristics, such as the "minimum inhibitory concentration" (MIC). For example, at MIC-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 a patient can have a pathogenic effect. In phthisiobacteriology, for first-line anti-tuberculosis drugs, it is 1%. or 20 colony-forming units - CFU). Such a part of the microbial population is able to displace the original one in a month and form a lesion. For second-line anti-tuberculosis drugs, the criterion for resistance is a 10% increase in the microbial population.

The development of drug resistance in microorganisms is associated with selection in the presence of an antibiotic and with the preferential survival of a portion of the microbial population that has mechanisms of protection against the antibacterial agent. Each population contains a small number of mutant cells (usually 10 ⁶ -10 ⁹ ) that are resistant to a particular drug. During chemotherapy, sensitive microbial cells die, and resistant ones multiply. As a result, sensitive cells are replaced by resistant ones.

Mycobacteria initially have high natural resistance to many broad-spectrum antibacterial drugs, but different species have different spectrums and degrees of this sensitivity.

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

Acquired resistance is the ability of individual strains to remain viable at antibiotic concentrations that suppress the growth of the main part of the microbial population. The acquisition of resistance in all cases is genetically determined: the appearance of new genetic information or a change in the expression level of one's own genes.

Currently, various molecular mechanisms of resistance of Mycobacterium tuberculosis have been discovered:

• antibiotic inactivation (enzyme inactivation), for example, by 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 agent-target ratio and the release of part of the bacteria's life-support proteins;
• active removal of the drug from the microbial cell (efflux) due to the activation of stress defense mechanisms:
• changes in the permeability parameters of the external structures of the microbial cell, blocking the ability of the antibiotic to penetrate into the cell;
• inclusion of a "metabolic shunt" (bypass metabolic pathway).

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

All mechanisms form different degrees 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, for each first-line anti-TB drug and for most reserve drugs, at least one gene has been identified. Specific mutations in which lead to the development of resistant variants of mycobacteria. In the widespread distribution of drug resistance in mycobacteria, a high mutation rate in vivo is important, greater than in vitro.

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

Types of drug resistance of mycobacteria

A distinction is made between primary and acquired drug resistance. Microorganisms with primary resistance include strains isolated from patients who have not received specific therapy or have received drugs for a month or less. If it is impossible to clarify the fact of the use of anti-tuberculosis drugs, the term "initial resistance" is used.

Primary drug resistance is of great clinical and epidemiological importance, therefore, for its correct assessment, it is necessary not to administer chemotherapy to a newly diagnosed patient with tuberculosis before microbiological examination of the diagnostic material. The frequency of primary drug resistance is calculated as the ratio of the number of newly diagnosed patients with primary resistance to the number of all newly diagnosed patients who were tested for drug susceptibility during the year. If a resistant strain is isolated from a patient during anti-tuberculosis therapy administered for a month or more, the resistance is considered acquired. The frequency of primary drug resistance characterizes the epidemiological state of the tuberculosis pathogen population.

Acquired drug resistance among newly diagnosed patients is the result of unsuccessful treatment (incorrect selection of drugs, non-compliance with the regimen, reduction in drug dosages, inconsistent supply 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" defense mechanisms in mycobacterial cells.

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

In the structure of drug resistance of Mycobacterium tuberculosis, the following are distinguished:

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

Polyresistance is resistance to two or more drugs.

Multiple drug resistance is resistance to isoniazid and rifampicin simultaneously (regardless of the presence of resistance to other drugs). It is usually accompanied by resistance to streptomycin, etc. Currently, MDR of tuberculosis pathogens has become an epidemiologically dangerous phenomenon. Calculations show that the 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 Anti-Tuberculosis Program. According to drug resistance monitoring data, the frequency of MDR among newly diagnosed patients ranges from 4 to 15%, among relapses - 45-55%, and among cases of unsuccessful treatment - up to 80%.

Super-resistance is multiple drug resistance combined with resistance to fluoroquinolones and one of the injectable drugs (kanamycin, amikacin, capreomycin). Tuberculosis caused by strains with super-resistance poses a direct threat to the lives of patients, since other second-line anti-TB drugs do not have a pronounced antibacterial effect. Since 2006, some countries have organized surveillance for the spread of mycobacteria strains with super-resistance. Abroad, this MDR variant is usually designated as XDR.

Cross-resistance is when resistance to one drug leads to resistance to other drugs. In M. tuberculosis, mutations associated with resistance are usually not interrelated. The development of cross-resistance is due to the similarity of the chemical structure of some anti-tuberculosis drugs. Cross-resistance is especially often detected within one group of drugs, such as aminoglycosides. To predict cross-resistance, genetic studies of mycobacterial cultures are necessary in combination with microbiological studies of resistance.

Non-tuberculous mycobacteria

Non-tuberculous mycobacteria are transmitted from person to person extremely rarely. The frequency of isolation of some of their species from material from patients is comparable to the frequency of isolation of these species from environmental objects. Sources of infection can be farm animals and birds, unprocessed products. Mycobacteria are found in post-slaughter material and milk of cattle.

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

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

Mycobacteria affect the skin, soft tissues, and can also cause mycobacteriosis of the lungs, which is especially common in immunodeficiency states. With pulmonary localization, it is more often detected in elderly men with a history of chronic pulmonary diseases, including fungal lesions.

Of all mycobacteria, the M. avium-intracellularae complex is the most common causative agent of pulmonary mycobacteriosis in humans. It causes diseases of the lungs, peripheral lymph nodes and disseminated processes. In the north of the European region, about 60% of pulmonary mycobacterioses. Fibro-cavernous and infiltrative processes predominate, taking a chronic course due to 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 contaminate hot and cold water supply systems. The habitat of M. malmoens is not fully established. M. xenopi exhibit fairly good sensitivity to antituberculosis therapy. M. malmoense exhibit fairly high sensitivity to antibiotics in vitro, but conservative treatment is often ineffective and even fatal. M. fortuitum and M. chelonae are recognized as causative agents of bone and soft tissue diseases due to direct contamination of a wound during trauma, surgery, and penetrating injury. They cause up to 10% of pulmonary mycobacterioses. It occurs as a chronic destructive bilateral lesion, often fatal. Anti-tuberculosis drugs and broad-spectrum antibiotics are not active or have little activity against these types of mycobacteria.

In the southern regions, mycobacterioses of the skin and soft tissues caused by M. leprae, M. ulceranse are widespread. Identification of non-tuberculous mycobacteria is carried out in the laboratories of the leading anti-tuberculosis institutions of the country. This requires high qualifications and good equipment of laboratories.