Mycoplasmas do not have a cell wall. Mycoplasmas. Synthetic Mycoplasma Genome
Anthroponotic bacterial infections of humans affecting the respiratory or genitourinary tract.
Mycoplasmas belong to the class Mollicutes, which includes 3 orders: Acholeplasmatales, Mycoplasmatales, Anaeroplasmatales.
Mycoplasmas were first discovered by French scientists Nocard and Roux in 1898 in the filtrate of the pleural fluid of cows suffering from pleuropneumonia. Therefore, they were originally called the causative agents of pleuropneumonia - PPO (pleuropneumonia organisms). Subsequently, organisms similar to PPO were found in humans and animals in various pathological conditions: rheumatic diseases, respiratory tract infections, inflammation of the genitourinary system. All of them were designated as PPLO - organisms similar to pathogens of pleuropneumonia (pleuropneumonia like organisms).
Mycoplasmas have different shapes: spherical, oval, thin filaments and stars. In size, the largest of them are close to bacteria, the smallest (125-150 nm) can, like viruses, pass through the pores of porcelain filters.
Mycoplasmas do not have a cell wall and are surrounded by a thin three-layer cytoplasmic membrane consisting of lipoproteins. Unlike viruses, they contain both DNA and RNA; they can be grown on artificial nutrient media with the addition of horse serum. According to Bergey's classification, mycoplasmas are classified in group 19. Mycoplasmas are widespread in nature. They are isolated from soil, wastewater, animals and humans. Mycoplasmas are known to live on the mucous membranes of the mouth and genital tract.
Mycoplasmas are similar in their morphology and biology to stable L-forms of bacteria. Therefore, it is assumed that mycoplasmas arose as a result of genetic mutations from bacteria that have lost their cell wall. Morphology:
Absence of a rigid cell wall, cell polymorphism, plasticity, osmotic sensitivity, resistance to various agents that suppress cell wall synthesis, including penicillin and its derivatives. Gram “-”, better stained according to Romanovsky-Giemsa; distinguish between mobile and immobile species. The cell membrane is in a liquid crystalline state; includes proteins embedded in two lipid layers, the main component of which is cholesterol.. Chemoorganotrophs, the main source of energy is glucose or arginine. They grow at a temperature of 30C. Most species are facultative anaerobes; extremely demanding on nutrient media and cultivation conditions. Nutrient media (beef heart extract, yeast extract, peptone, DNA, glucose, arginine).
Cultivate on liquid, semi-liquid and solid nutrient media.
Biochemical activity: Low. There are 2 groups of mycoplasmas: 1. decomposing glucose, maltose, mannose, fructose, starch and glycogen with the formation of acid; 2. oxidizing glutamate and lactate, but not fermenting carbohydrates. All species do not hydrolyze urea.
Antigenic structure: Complex, has species differences; the main antigens are represented by phospho- and glycolipids, polysaccharides and proteins; The most immunogenic are surface antigens, including carbohydrates as part of complex glycolipid, lipoglycan and glycoprotein complexes.
Pathogenicity factors: adhesins, toxins, aggression enzymes and metabolic products. Adhesins are part of surface Ags and determine adhesion to host cells. Suggested presence of neurotoxin in some strains M. pneumoniae, since respiratory tract infections often accompany damage to the nervous system. Endotoxins have been isolated from many pathogenic mycoplasmas. Hemolysins are found in some species. Among the aggression enzymes, the main pathogenicity factors are phospholipase A and aminopeptidases, which hydrolyze cell membrane phospholipids. Proteases that cause degranulation of cells, including fat cells, breakdown of AT molecules and essential amino acids.
Epidemiology: M. pneumoniae colonizes the mucous membrane of the respiratory tract; M. hominis, M. genitalium u U. urealyticum- “urogenital mycoplasmas” - live in the urogenital tract.
The source of infection is a sick person. The transmission mechanism is aerogenic, the main transmission route is airborne.
Pathogenesis: They penetrate the body, migrate through mucous membranes, and attach to the epithelium through glycoprotein receptors. Microbes do not exhibit a pronounced cytopathogenic effect, but cause disturbances in the properties of cells with the development of local inflammatory reactions.
Clinic: Respiratory mycoplasmosis - in the form of upper respiratory tract infection, bronchitis, pneumonia. Extra-respiratory manifestations: hemolytic anemia, neurological disorders, cardiovascular complications.
Immunity: Respiratory and urogenital mycoplasmosis are characterized by cases of re-infection.
Microbiological diagnostics
Nasopharyngeal swabs, sputum, bronchial washings. For urogenital infections, urine, scrapings from the urethra, and vagina are examined.
For laboratory diagnosis of mycoplasma infections, cultural, serological and molecular genetic methods are used.
In serodiagnosis, the material for research is tissue smears, scrapings from the urethra, vagina, in which antigens of mycoplasmas can be detected in direct and indirect RIF. Mycoplasmas and ureaplasmas are detected in the form of green granules.
Mycoplasma antigens can also be detected in the blood serum of patients. For this purpose, ELISA is used.
For serodiagnosis of respiratory mycoplasmosis, specific ATs are determined in paired patient sera. In some cases, serodiagnosis is carried out for urogenital mycoplasmosis; AT is most often determined by RPGA and ELISA.
Treatment
Antibiotics. Causal chemotherapy.
Prevention
Non-specific.
Mycoplasmas- small prokaryotes, lacking a true cell wall and unable to synthesize its components. Functions of the cell wall in mycoplasmas performed by a three-layer CPM.
That's why mycoplasma belong to the department Tenericuies (literally “tender-skinned”) of the family Mycoplasmataceae of the class Mollicutes (literally “soft-skinned”), which unites mycoplasmas, acholeplasmas, spiroplasmas, anaeroplasmas and ureaplasmas.
Mycoplasmas distinguished by polymorphism due to the absence of a rigid cell wall. Mycoplasmas form coccoid, branching, large multinuclear forms, as well as pseudomycelium, which determines their name [from the Greek. mykes, mushroom, + plasma, something having a shape].
Mycoplasmas reproduce by binary fission, like most bacteria, especially after the formation of small coccoid formations (elementary bodies, EB) in filamentous structures.
Mycoplasmas capable of budding and segmentation. The minimum reproducing unit is considered to be the ET (0.7-0.2 µm). The main component of the cell membrane is cholesterol.
Mycoplasmas are not capable of forming cholesterol and utilize it from tissues or nutrient media supplemented with their addition. The Gram stain is negative, but the best results are obtained by Romanowsky-Giemsa stain.
Mycoplasmas are demanding regarding cultivation conditions: native serum, cholesterol, nucleic acids, carbohydrates, vitamins and various salts must be added to the nutrient media. On dense media they form characteristic small translucent colonies with a raised granular center, giving them a “fried egg” appearance.
On media with blood, some mycoplasma species give a- and beta-hemolysis. In semi-liquid media mycoplasmas grow along the injection line, forming dispersed, crumbly colonies. In liquid media they lead to slight turbidity or opalescence; some strains are capable of forming a thin oily film.
In humans, representatives of the genera are distinguished Mycoplasma, Ureaplasma and Acholeplasma, including pathogenic and saprophytic species.
Mycoplasmas are characterized by extremely pronounced polymorphism, due primarily to the absence of a solid cell wall inherent in bacteria, as well as a complex development cycle. The smallest structural elements capable of reproduction in artificial nutrient media are usually called minimal reproductive units. The shape and size of the minimum reproductive units, as well as cellular elements at different stages of development, are significantly influenced by cultivation conditions, physicochemical properties of nutrient media, characteristics of the strain and the number of passages on the media, techniques for preparing, fixing and staining preparations and other factors.
Due to the fact that mycoplasmas do not have a cell wall, their membrane and cytoplasm are easily damaged by chemical reagents used for fixing and staining preparations. Mycoplasma cells are especially sensitive to the effects of environmental factors in the early stages of development.
In smears from affected organs and from cultures grown in the medium, mycoplasmas are presented as round, oval and ring-shaped formations. Coccobacillary and bacteria-like forms are sometimes found. Certain types of mycoplasmas (M. mycoides var. mycoides, M. mycoides var. capri, M. agalacliae) form filamentous mycelial forms in organs and in nutrient media.
Electron microscopic studies and by filtering grown cultures through membrane filters with a known burrow diameter showed that in the same culture there are formations of different shapes and sizes that are capable of reproduction (Fig. 1). When studying various types of mycoplasmas isolated from animal and human organs, as well as environmental objects, it was found that the size of elementary particles ranges from 125 to 600 nm. In Burge's determinant, the size of mycoplasma cells is estimated at 125-200 nm. According to E. Freundt, the size of the minimum reproductive units of mycoplasmas ranges between 250-300 nm. Other authors determined their size in the range of 200-500-700 nm, and G. Wildfur, using the ultrafiltration method. - 100-150 nm. It should be noted that the size of mycoplasma cells depends not only on the species and strain, but also on other factors affecting the cell.
Thus, the size of the minimum reproductive units in mycoplasma cultures varies widely.
Mycoplasmas belong to the class Mollicutes, which includes 3 orders (Fig. 16.2): Acholeplasmatales, Mycoplasmatales, Anaeroplasmatales. The order Acholeplasmatales includes the family Acholeplasmataceae single gender Acholeplasma. The order Mycoplasmatales consists of 2 families: Spiroplasmataceae single gender Spiroplasma And Mycoplasmataceae, including 2 types: Mycoplasma And Ureaplasma. The newly recognized order Anaeroplasmatales consists of the family Anaeroplasmataceae, including 3 types: Anaeroplasma, Asteroplasma, Termoplasma. The term “mycoplasma” usually refers to all microbes of the families Mycoplasmataceae and Acholeplasmataceae.
Morphology. A distinctive feature is the absence of a rigid cell wall and its precursors, which determines a number of biological properties: cell polymorphism, plasticity, osmotic sensitivity, and the ability to pass through pores with a diameter of 0.22 microns. They are not capable of synthesizing peptidoglycan precursors (muramic and diaminopimelic acids) and are surrounded only by a thin three-layer membrane 7.5-10.0 nm thick. Therefore, they were allocated to a special department Tenericutes, class Mollicutes (“tender skin”), order Mycoplasmatales. The latter includes a number of families, including Mycoplasmataceae. This family includes pathogenic mycoplasmas (cause diseases in humans, animals and birds), opportunistic mycoplasmas (very often asymptomatic carriers of them are cell cultures) and saprophytic mycoplasmas. Mycoplasmas are the smallest and most simply organized prokaryotes, capable of autonomous reproduction, and the minimal elementary bodies, for example Acholeplasma laidlawii, are comparable in size to the minimal initial progenote cell. According to theoretical calculations, the simplest hypothetical cell capable of autonomous reproduction should have a diameter of about 500 angstroms, contain DNA with a mass of 360,000 D and about 150 macromolecules. The elementary body of A. laidlawii has a diameter of about 1000 angstroms, i.e., only 2 times larger than a hypothetical cell, contains DNA with a mass of 2,880,000 D, i.e., it carries out much more metabolic processes, and contains no 150, and about 1200 macromolecules. It can be assumed that mycoplasmas are the closest descendants of the original prokaryotic cells.
Rice. . Formation of a mycoplasma colony on a solid medium (Prokaryotes. 1981, vol. II)
A. Vertical section of agar before sowing (a – film of water, b – agar filaments). B. A drop containing viable mycoplasma is applied to the surface of the agar.
B. After 15 minutes. after inoculation, the drop is adsorbed by agar.
D. Approximately 3-6 hours after sowing. A viable particle has penetrated the agar.
D. Approximately 18 hours after sowing. A small spherical colony formed below the surface of the agar. E. Approximately 24 hours after sowing. The colony has reached the surface of the agar.
G. Approximately 24-48 hours after sowing. The colony has reached a free water film, forming a peripheral zone (d - central zone, c - peripheral zone of the colony)
Resistance to various agents that suppress cell wall synthesis, including penicillin and its derivatives, multiple reproductive pathways (binary fission, budding, fragmentation of filaments, chain forms and spherical formations). Cells are 0.1-1.2 µm in size, gram-negative, but stain better according to Romanovsky-Giemsa; distinguish between mobile and immobile species. The minimum reproducing unit is an elementary body (0.7 - 0.2 µm), spherical or oval, which later lengthens to branched filaments. The cell membrane is in a liquid crystalline state; includes proteins mosaically embedded in two lipid layers, the main component of which is cholesterol. The genome size is the smallest among prokaryotes (accounts for "/16 of the rickettsia genome); they have a minimal set of organelles (nucleoid, cytoplasmic membrane, ribosomes). The ratio of GC pairs in DNA in most species is low (25-30 mol.%), with the exception of M. pneumoniae (39 – 40 mol.%). The theoretical minimum GC content required to encode proteins with a normal set of amino acids is 26%, therefore, mycoplasmas are at this limit. The simplicity of organization and limited genome determine the limitations of their biosynthetic capabilities.
Cultural properties. Chemoorganotrophs, most species have fermentative metabolism; the main source of energy is glucose or arginine. They grow at temperatures of 22 – 41 °C (optimum – 36-37 °C); optimum pH is 6.8-7.4. Most species are facultative anaerobes; extremely demanding on nutrient media and cultivation conditions. Nutrient media must contain all the precursors necessary for the synthesis of macromolecules and provide mycoplasmas with sources of energy, cholesterol, its derivatives and fatty acids. For this, beef heart and brain extract, yeast extract, peptone, DNA, and NAD are used as a source of purines and pyrimidines, which mycoplasmas cannot synthesize. Additionally, the following are added to the medium: glucose - for species that ferment it, urea - for ureaplasmas, and arginine - for species that do not ferment glucose. The source of phospholipids and styrene is animal blood serum; for most mycoplasmas, horse blood serum.
The osmotic pressure of the medium should be in the range of 10 - 14 kgf/cm2 (optimal value - 7.6 kgf/cm2), which is ensured by the introduction of K + and Na + ions. Species that ferment glucose grow better at lower pH values (6.0-6.5). Aeration requirements vary among species; most species grow best in an atmosphere of 95% nitrogen and 5% carbon dioxide.
Mycoplasmas reproduce on cell-free nutrient media, but for their growth, most of them require cholesterol, which is a unique component of their membrane (even in mycoplasmas that do not require sterols for their growth), fatty acids and native protein. Liquid and solid nutrient media can be used to isolate cultures. Growth in liquid media is accompanied by barely visible turbidity; on solid media with yeast extract and horse serum, colony formation occurs as follows (see figure). Due to their small size and the absence of a rigid cell wall, mycoplasmas are able to penetrate from the surface of the agar and multiply inside it - in the spaces between the agar strands. When a drop of material containing mycoplasmas is applied, it penetrates the aqueous film present on the surface of the agar and is adsorbed by the agar, forming a small compaction between its threads. As a result of mycoplasma multiplication, after approximately 18 hours, a small spherical colony forms under the surface of the agar within the intertwined agar strands; it grows, and after 24-48 hours of incubation it reaches the surface water film, as a result of which two growth zones are formed - a cloudy granular center growing into the medium, and a flat openwork semi-translucent peripheral zone (fried egg type). Colonies are small, ranging from 0.1 to 0.6 mm in diameter, but can be smaller (0.01 mm) or larger (4.0 mm) in diameter. On blood agar, zones of hemolysis are very often observed around the colonies, caused by the action of the resulting H 2 O 2. Colonies of some types of mycoplasmas are capable of adsorbing on their surface erythrocytes, epithelial cells of various animals, tissue culture cells, human and some animal sperm. Adsorption occurs better at 37 °C, less intensely at 22 °C and is specifically inhibited by antisera. The optimum temperature for the growth of mycoplasmas is 36-37 ° C (range 22-41 ° C), the optimal pH is 7.0, either slightly acidic or slightly alkaline. Most species are facultative anaerobes, although they grow better under aerobic conditions; some species are aerobes; few grow better in anaerobic conditions. Mycoplasmas are immobile, but some species have gliding activity; are chemoorganotrophs; they use either glucose or arginine as the main source of energy, rarely both substances, sometimes neither one nor the other. They are capable of fermenting galactose, mannose, glycogen, starch with the formation of acid without gas; They do not have proteolytic properties; only some types liquefy gelatin and hydrolyze casein.
Chicken embryos that die after 3–5 passages are suitable for cultivation.
Resistance. Due to the absence of a cell wall, mycoplasmas are more sensitive than other bacteria to the effects of mechanical, physical and chemical factors (UV irradiation, direct sunlight, X-ray irradiation, changes in pH, high temperature, drying). When heated to 50 °C, they die within 10-15 minutes; they are very sensitive to conventional chemical disinfectants.
The Mycoplasma family includes more than 100 species. Humans are a natural carrier of at least 13 types of mycoplasmas that grow on the mucous membranes of the eye, respiratory, digestive and genitourinary tracts. In human pathology, several species of Mycoplasma play the greatest role: M. pneumoniae, M. hominis, M. arthritidis, M. fermentans and, possibly, M. genitalium, and the only species of the genus Ureaplasma - U. urealyticum. The main biochemical difference between the latter and Mycoplasma species is that U. urealyticum has urease activity, which all members of the Mycoplasma genus lack (Table 3)
Mycoplasmas that are pathogenic for humans cause diseases (mycoplasmosis) of the respiratory, genitourinary tract and joints with a variety of clinical manifestations.
Table 3
|
Differential features some mycoplasmas pathogenic for humans |
|||||||||
|
Types of mycoplasmas |
Hydrolysis |
Fermentation |
Phosphatase |
Reduction of tetrazolium aerobically/anaerobically |
Relation to erythromycin |
Amount G+C mol% |
Sterol requirement for growth |
||
|
urea |
arginine |
glucose (k) |
mannose (k) | ||||||
Note, (j) – formation of acid; VR – highly resistant; HF – highly sensitive; (+) – positive sign; (-) – a negative sign.
Biological properties .
Biochemical activity. Low. There are 2 groups of mycoplasmas:
Decomposing glucose, maltose, mannose, fructose, starch and glycogen (“true” mycoplasmas) to form acid;
Reducing tetrazolium compounds that oxidize glutamate and lactate, but do not ferment carbohydrates.
All species do not hydrolyze urea and esculin.
Ureaplasma inert to sugars, do not reduce diazo dyes, catalase negative; exhibit hemolytic activity towards rabbit and guinea pig erythrocytes; produce hypoxanthine. Ureaplasmas secrete phospholipases A p A 2 and C; proteases that selectively act on IgA molecules and urease. A distinctive feature of metabolism is the ability to produce saturated and unsaturated fatty acids.
Antigenic structure. Complex, has species differences; the main antigens are represented by phospho- and glycolipids, polysaccharides and proteins; The most immunogenic are surface antigens, including carbohydrates as part of complex glycolipid, lipoglycan and glycoprotein complexes. The antigenic structure can change after repeated passages on cell-free nutrient media. Characterized by pronounced antigenic polymorphism with a high frequency of mutations.
M. hominis the membrane contains 9 integral hydrophobic proteins, of which only 2 are more or less constantly present in all strains.
Ureaplasma has 16 serovars, divided into 2 groups (A and B); the main antigenic determinants are surface polypeptides.
Pathogenicity factors. Diverse and can vary significantly; the main factors are adhesins, toxins, aggression enzymes and metabolic products. Adhesins are part of surface Ags and cause adhesion to host cells, which is of key importance in the development of the initial phase of the infectious process. Exotoxins have currently been identified only in a few mycoplasmas that are non-pathogenic to humans, in particular in M. neurolyticum And M. gallisepticum ; the targets for their action are astrocyte membranes. The presence of a neurotoxin is suspected in some strains of M. pneumoniae, since respiratory tract infections often accompany lesions of the nervous system. Endotoxins have been isolated from many pathogenic mycoplasmas; their administration to laboratory animals causes a pyrogenic effect, leukopenia, hemorrhagic lesions, collapse and pulmonary edema. In their structure and some properties they are somewhat different from the LPS of gram-negative bacteria. Some species contain hemolysins (M. pneumoniae has the greatest hemolytic activity); Most species cause pronounced β-hemolysis due to the synthesis of free oxygen radicals. Presumably, mycoplasmas not only synthesize free oxygen radicals themselves, but also induce their formation in cells, which leads to the oxidation of membrane lipids. Among the enzymes of aggression, the main factors of pathogenicity are phospholipase A and aminopeptidases, which hydrolyze cell membrane phospholipids. Many mycoplasmas synthesize neuraminidase, which interacts with cell surface structures containing sialic acids; in addition, the activity of the enzyme disrupts the architecture of cell membranes and intercellular interactions. Among other enzymes, mention should be made of proteases that cause degranulation of cells, including mast cells, the breakdown of AT molecules and essential amino acids, RNases, DNases and thymidine kinases, which disrupt the metabolism of nucleic acids in the cells of the body. Up to 20% of the total DNase activity is concentrated in the membranes of mycoplasmas, which facilitates the enzyme’s interference with cell metabolism. Some mycoplasmas (for example, M. hominis) synthesize endopeptidases that cleave IgA molecules into intact monomeric complexes.
Epidemiology. Mycoplasmas are widespread in nature. Currently, about 100 species are known; they are found in plants, mollusks, insects, fish, birds, mammals, some are part of the microbial associations of the human body. 15 types of mycoplasmas are isolated from humans; their list and biological properties are given in table. . A. ladlawii and M. primatum are rarely isolated from humans; 6 types: M.pneumoniae, M. hominis, M. genitalium, M.fermentans (incognitis), M. penetransAndU. urealyticum have potential pathogenicity. M. pneumoniae colonizes the mucous membrane of the respiratory tract; M.hominis, M. genitaliumAndU. urealyticum– “urogenital mycoplasmas” – live in the urogenital tract.
Source of infection- a sick man. The transmission mechanism is aerogenic, the main transmission route is airborne; susceptibility is high. Children and adolescents aged 5–15 years are most susceptible. The incidence in the population does not exceed 4%, but in closed groups, for example in military units, it can reach 45%. The peak incidence is the end of summer and the first autumn months.
Source of infection- a sick man; Ureaplasma infects 25–80% of people who are sexually active and have three or more partners. Transmission mechanism – contact; the main route of transmission is sexual, on the basis of which the disease is included in the group of STDs; susceptibility is high. The main risk groups are prostitutes and homosexuals; Ureaplasma is detected much more often in patients with gonorrhea, trichomoniasis, and candidiasis.
Respiratory mycoplasmosis
Respiratory mycoplasmosis has a global distribution. According to WHO (1985), 8-15 million people get sick every year. Very often it occurs in the form of acute pneumonia (segmental, focal or interstitial). The main pathogen is M. pneumoniae, much less often - M. hominis, and very rarely, respiratory tract disease in children under 1 year of age is caused by U. urealyticum.
M.pneumoniae. The basic biological and morphological properties are typical for the genus Mycoplasma, but there are a number of distinctive features. In shape they are short filaments 2-5 microns long; the cells have gliding motility. They reproduce by binary fission and the release of elementary bodies from the filaments. Freshly isolated colonies often lack a translucent peripheral zone, but have the appearance of a raised ring-shaped granular structure with a diameter of 30-100 µm. They grow more slowly than other mycoplasmas. Colonies appear after 5-10 days of incubation, sometimes later. Several successive reseedings are required for the colonies to acquire the “fried egg” appearance. Colonies on a solid medium adsorb red blood cells, tracheal epithelial cells of monkeys, rats, guinea pigs, chickens, cells of some cultures (HeLa, chicken embryonic tissue, etc.), as well as human and bovine sperm. The erythrocyte and epithelial cell receptors for M. pneumoniae are destroyed by neuraminidase; cell adsorption is prevented if mycoplasmas are pre-treated with neuraminic acid. M. pneumoniae causes hemagglutination of human and animal erythrocytes and has hemolytic activity due to the production of H 2 O 2; mitogenic effect on T - and B-lymphocytes and cytotoxic effect; highly sensitive to erythromycin. Antigenically, the species M. pneumoniae represents a homogeneous group; no type-specific antigens were found.
Resistance to the action of physical and chemical factors is small.
Pathogenicity factors: high affinity for epithelial cells of the respiratory tract, hemadsorption, hemolytic, cytotoxic properties of mycoplasmas, their mobility.
Epidemiology. The source of infection is a sick person, as well as carriers, including those who have had the disease in an asymptomatic form. Infection occurs by airborne droplets, since the pathogen is localized mainly on the cells of the ciliated epithelium of the respiratory tract. Respiratory mycoplasmosis is a low-contagious disease, which is explained by the short-term survival of the pathogen in the external environment, so infection requires close and long-term contact with the source of infection. This determines the higher incidence of illness in organized groups, especially in newly organized ones, where after 2-3 months. up to 50% of its members become infected. People aged from 1 to 30 years are most often affected.
Pathogenesis and clinic. The incubation period lasts 7-14 days, sometimes up to 25 days. The pathogen is adsorbed on the mucous membrane of the upper respiratory tract, multiplies and actively spreads along the mucous membrane of the trachea and bronchi, reaches alveolocytes, and penetrates into the interalveolar septa. As a result of cell damage, peribronchial, perivascular and interstitial infiltrates occur. For mycoplasma pneumonia, which usually develops gradually, a long, debilitating cough with dry or scanty sputum is typical; physical changes in the lungs are weak or absent, so pneumonia is detected by X-ray examination. Inflammatory infiltrates resolve slowly - within 3-4 weeks, sometimes longer. In young children, bilateral lung damage is often observed. The course of the disease is usually benign.
Post-infectious immunity after an acute infection, it persists for 5–10 years, sometimes longer. It is caused by both secretory and humoral antimicrobial antibodies, as well as longer-lasting antibodies that suppress the metabolism of mycoplasmas and cellular elements (macrophages, T-lymphocytes). After asymptomatic and erased forms of infection, immunity is short-lived and weakly expressed.
Laboratory diagnostics. Since atypical pneumonias have different etiologies, laboratory methods play a decisive role in their diagnosis. To diagnose respiratory mycoplasmosis (mycoplasma pneumonia) use bacteriological and serological methods, as well as the detection of mycoplasmas and their identification using immunological techniques. The material for bacteriological examination is sputum, mucus from the pharynx, pleural fluid, lavage (from the French lavage - to wash), washings from the surface of bronchioles and alveolar structures of the lungs obtained during bronchoscopy. To obtain cultures, crops are sown on media containing all the nutrients necessary for the growth of mycoplasmas. To inhibit the growth of accompanying bacteria, penicillin and thallium acetate, to which M. pneumoniae is resistant, are added to the nutrient medium. The isolated culture is identified on the basis of morphological, cultural properties, and characteristics indicated in the table. , as well as using diagnostic immune sera obtained by immunizing rabbits with cytoplasmic membrane preparations of M. pneumoniae.
Guide for doctors
Moscow, 1999
Editorial Board: Doctor of Medical Sciences, Prof. V.G. Nesterenko, Ph.D. V.A. Bekhalo, Ph.D. A.N. Lovenetsky
Introduction
Biology of mycoplasmas
Respiratory mycoplasmosis
Biology of the pathogen
Epidemiology
Clinic
Pathogenesis
Pathogen persistence and immunity
Treatment
Urogenital mycoplasmosis
M. hominis. Biology.
U. urealyticum. Biology.
M. genitalium. Biology.
M. fermentans. Biology.
Epidemiology
Pathogenicity
Treatment
Rehabilitation criteria
Joint diseases of mycoplasma etiology
New mycoplasma infections
Diagnosis of mycoplasma infections
Material under study
Culture methods
The most commonly used serological methods are:
Antigen detection
Antibody detection
Molecular biological methods
Comparison of sensitivity of methods
Interpretation of results
Advantages and disadvantages of diagnostic methods.
Application
Literature
List of abbreviations.
AG - antigens
AT - antibodies
HIV - human immunodeficiency virus
G - guanine
GLA - glutaraldehyde
IL-6 - interleukin-6
CBB - carbonate-bicarbonate buffer
CFU - colony forming unit
PCR - polymerase chain reaction
PA - rheumatoid arthritis
RAHA - hemagglutination aggregate reaction
RIF - immunofluorescence reaction
RPHA - passive hemagglutination reaction
RF - rheumatoid factor
C – cytosine
UTT - urogenital tract
TNF - tumor necrosis factor
PBS - phosphate buffered saline
CNS - central nervous system
CPD - cytopathic effect
CTD - cytotoxic effect
Introduction
Human diseases caused by mycoplasmas are grouped into the group of human mycoplasmosis. The causative agents of this group of infections, mycoplasmas, are the smallest free-living prokaryotes. They attract much attention from researchers for two reasons:1) due to its unique organization
2) due to the fact that they very often contaminate cell cultures, cause diseases of plants, animals and humans, influence the reproduction of a number of viruses, including oncogenic ones and HIV, and are also capable of causing an immunodeficiency state.
According to the modern classification, mycoplasmas belong to the class Mollicutes, division Tenericutes, kingdom Procariotae. The class Mollicutes has 3 orders: Acholeplasmat; Mycoplasmatales, Anaeroplasmatales. 1st order includes one family Acholeplasmataceae with one genus Acholeplasma, 2nd order consists of two families Spiroplasmataceae with one genus Spiroplasma and Mycoplasmataceae with two genera Mycoplasma and Ureaplasma. The newly recognized 3rd order includes the family Anaeroplasmataceae with two genera Anaeroplasma and Asteroplasma.
The term “mycoplasma” usually refers to all microorganisms of the families Mycoplasmataceae and Acholeplasmataceae.
Biology of mycoplasmas (6, 10)
Distinctive features of mycoplasmas unique to prokaryotes are:
1. The absence of a rigid cell wall and its precursors determines a number of biological properties of mycoplasmas: the polymorphism of their cells, plasticity, osmotic sensitivity, the ability to pass through pores with a diameter of 0.22 microns, resistance to various agents that suppress cell wall synthesis, including penicillin, its derivatives and synthetic penicillins. All mycoplasmas are gram-negative.
The polymorphism of mycoplasmas is manifested in the fact that all colonies consist of various elements: rods, coccus-like cells, balls of different optical densities, filaments of different lengths (hence the name “mycoplasma”). The methods of reproduction of these various structures are multiple: budding, segmentation of branched and chain forms, binary fission, disintegration of filaments into individual coccoid elements. Mycoplasmas do not form spores.
2. Small genome size - 500 - 1000 MD, the smallest for prokaryotes (1/16 of the E. coli genome. 1/10 of the rickettsia genome). The simplicity of organization and genome size determine the limited biosynthetic capabilities of mycoplasmas and, consequently, their high requirements for cultivation conditions.
3. The minimum number of organelles is a 3-layer cytoplasmic membrane, a prokaryotic nucleoid and ribosomes.
4. Low ratio of G+C pairs in DNA, in most species 25 - 30%. The exception is M.pneumoniae, in which G+C pairs account for 39 - 40%. The theoretical minimum G+C content required to encode proteins with a normal amino acid set is 26%, so many evolutionists believe that mycoplasmas are on the verge of life.
6. The ability to grow on various nutrient media and form colonies on the surface of the agar with a diameter of 0.1 - 0.3 mm (mycoplasmas) and 0.01 - 0.03 mm (ureaplasmas) with a convex center growing into the agar and a delicate, often lacy periphery. Typical colonies look like a fried egg.
7. The growth of mycoplasmas in the medium is suppressed by specific immune sera, which is detected in the reaction of neutralization or growth inhibition, or in the reaction of inhibition of metabolism.
Mycoplasma membranes similar to the membranes of eukaryotic cells, they are asymmetrical, the outer layer is thicker than the inner one.
The mycoplasma membrane is a mobile system consisting of 2 protein layers (external and internal) immersed in an internal lipid layer. The outer layer of the membrane is more fluid than the inner one. Proteins in the membrane make up about 40%. Peripheral proteins are easily washed out from the membrane when the pH and ionic strength of the solution changes, while integral hydrophobic proteins can only be isolated when treated with detergents. Membranes include carbohydrate-containing compounds: glycoproteins, polysaccharides and lipopolysaccharides. Lipids account for about 40%, of which 60% are neutral lipids. The main component of the latter is cholesterol. It is a necessary component of the cultivation environment
The absence of a number of enzyme systems would put mycoplasmas at an extremely disadvantageous position when competing with other microorganisms. However, it should be borne in mind that mycoplasma cells are very closely related to the host cells, and the absence of some enzyme systems is compensated by the presence of enzymes and mechanisms by which mycoplasmas extract the necessary substances from the cells of higher organisms.
The basis of modern ideas about the phylogeny of prokaryotes is data on the primary structure of ribosomal RNA, in particular 16S and 5S RNA, which in evolution are the most conservative biological macromolecules. Based on this analysis, it is assumed that the ancestors of mycoplasmas separated from the clostridial branch of Gram-positive bacteria, which already had a low level of G+C pairs in DNA. It has been suggested that in the process of the origin of mycoplasmas from Gram-positive bacteria, the main role belonged to L-transformation, i.e. L-phores were the primary and main step in the further evolution of mycoplasmas.
Currently, more than 100 species of mycoplasmas are known (the number of detected species is constantly growing). Mycoplasmas are isolated from plants, mollusks, insects, fish, birds and mammals. Humans are the natural host of at least 14 mycoplasma species: M. buccale, M. faucium, M. fermentans, M. genitalium, M. Lipophilum, M. orale, M. hominis, M. salivanura, M. penetrans, M. pirum , M. pneumoniae, M. spermatofilium, U. urealyticum, Acholeplasma laidlavii. The biological properties of these types of mycoplasmas are shown in Table 1. Less commonly, M. primatum and other species are isolated from humans, the natural hosts of which are considered to be various animals.
Table 1.
Biological properties of mycoplasmas isolated from humans.
| Kinds | Metabolism of arginine or glucose | Interaction with red blood cells | Tetrazole reduction | Formation of films and stains on the surface of the medium | Phosphatase activity |
|
| Hemadsorption | Hemolysis and hemagglutination |
|||||
| M. pneumoniae | Glucose | + | + | + | - | - |
| M. hominis | Arginine | - | - | - | - | - |
| M. genitalium | Glucose | + | - | Weak in aerobic conventional + anaerobic | - | - |
| M. fermentans | Arginine, glucose | - | - | - | + | + |
| M. salivarium | Arginine | + (erythr. sheep, human) | + | - | + | - |
| M. orale | Arginine | - | - | - | - | - |
| M. buccale | Arginine | - | - | - | - | - |
| M. facium | Arginine | - | - | - | + | - |
| M. lipophilum | Arginine | - | - | - | + | - |
| M. arthritidis | Arginine | - | - | - | - | + |
| M. penetrans | Arginine, glucose | + | Weak b-hemolysis | - | - | + |
| M. pirum | Arginine, glucose | - | - | Weak in aerobic and anaerobic. conventional | - | - |
| M. primatum | Arginine | - | - | - | - | - |
| U. urealyticum | Urea | + | + (erythr. rabbit and guinea pig) | - | - | - |
| Acholeplasma laidlavii | glucose | + | + | - | - | - |
Penetration and interaction with the cell. Mycoplasmas enter the body by airborne droplets or contact routes, including genital routes, overcome the mucous layers covering the epithelium and reach the cells of epithelial tissues, probably through chemotaxis.
Some types of mycoplasmas have microvilli and special terminal structures containing actin-like protein, with the help of which mycoplasmas actively move and attach to the cells of the infected organism. Mycoplasmas can be adsorbed on almost any eukaryotic cells, multiply on their surface and in the intercellular spaces in the immediate vicinity of cell membranes. The contact between the membranes of mycoplasmas and the membranes of the host cells is so close that there is reason to speak in some cases about the fusion of the contacting membranes. The ability for such fusion is considered as one of the pathogenicity factors of mycoplasmas. The process of attachment of mycoplasmas to host cells apparently consists of two phases. The first, the phase of nonspecific interaction, occurs as a result of Brownian motion of mycoplasmas and random collisions with host cells. The next phase, ligand-receptor interaction, has been studied in some detail in some types of mycoplasmas. It is known that the main adhesin of M. pneumoniae is the P1 protein with a molecular weight of 160 kDa, although other proteins with lower molecular weights also participate in binding. Some types of mycoplasmas interact with the host cell by the type of lipid-lipid adhesion, and the possibility of exchange of individual fragments of contacting membranes has been shown. It has also been established that hydrophobic bonds are involved in the adhesion of mycoplasmas. The nature of the receptors in the membrane of host target cells is also being actively studied. Thus, it has been shown that in the membrane of erythrocytes these are sialoglycoproteins. The binding of mycoplasmas to receptors of a glycoprotein nature can lead to disruption of the normal physiological functions of these receptors, cellular contacts, cellular cooperation and interaction between cells during growth, changes in membrane architecture and ion transport through the membrane.
As a result of the interaction of mycoplasmas and cells, change in the antigenic profile of interacting membranes and as a consequence, induction of various autoimmune reactions. Adsorption of mycoplasmas on lymphocytes can lead to nonspecific polyclonal activation of T and B cells and the subsequent development of autoimmune reactions, or to suppression of lymphocyte proliferation and, consequently, an immunosuppressive effect.
Strong binding of mycoplasmas to the cell ensures their resistance to the mechanical movement of the cilia of ciliated epithelial cells, and the preferential location of adsorbed mycoplasmas in the invaginates of the host cell protects them from the action of antibodies, which contributes to their long-term persistence.
Apparently, there is no other group of prokaryotes whose pathogenetic role information would be so contradictory and confusing. Most human mycoplasma species appear to be commensals in healthy humans. Other species (M. pneumoniae, M. hominis, U. urealyticum, M. fermentans, M. penetrans) have potential pathogenicity.
Due to their evolutionary advancement, mycoplasmas should probably be considered as the next generation of bacterial pathogens, and new approaches must be developed to properly and fully understand their potential pathogenetic role.
Antibiotic sensitivity. Due to the absence of a cell wall, mycoplasmas are resistant to all drugs whose action is associated with the biosynthetic processes of cell wall proteins and are sensitive to inhibitors of the synthesis of membrane and intracytoplasmic proteins. They are sensitive to tetracycline antibiotics, macrolides, lincosamines, aminoglycosides and quinolones. Data on the sensitivity of three types of mycoplasmas to antibiotics in vitro are given in Table. 2.
All three types of mycoplasmas are highly sensitive to tetracyclines; the most effective are doxycycline, vibromycin and minocycline. All mycoplasmas are sensitive To the newest quinolones.
Table 2.
Relative in vitro susceptibility of M. hominis. U. urealyticum and M. pneumoniae to antibiotics,
inhibiting the growth of mycoplasmas (compiled based on summary data)
| Antibiotics, mcg/ml | M. hominis | U. urealyticum | M. pneumoniae |
| Tetracyclines |
|||
| Tetracycline | 0,05 - 0,2 | 0,05 - 6,0 | 0,006 - 0,8 |
| Oxytegracycline | 1,6-4 | 6,5-62 | 0,3-6,3 |
| Chlortetracycline | 1-1,2 | - | 1,6-6,3 |
| Metacycline | 0,2-8 | - | 0,4-3,1 |
| Doxycycline | 0,1-0,4 | 0,01-0,5 | 0,2 |
| Minocycline | 0,2-0,8 | 0,5-62 | - |
| Macrolides, lincosamines, streptogrammins | |||
| Erythromycin | 461 | 0,4-3 | 0,01 |
| Oleandomycin | 512 | 5,9 | 0,05 |
| Spiromycin | 46,9 | 41,9 | 0,33 |
| Josamycin | 0,1 | 0,45 | 0,02 |
| Rosaramycin | 0,05 | 0,04 | 0,01 |
| Lincomycin | 0,65 | 73 | 4,9 |
| Midecamycin | 0,6 | 0,6 | 0,02 |
| Pristinamycin | 0,1-0,5 | 0,1-1,0 | 0,03 - 0,05 |
| Virginomycin | 0,8 | 1,3 | 0.15 |
| Clindamycin | 0,03 | 2,62 | 1,5 |
| Chloramphenicol (chloramphenicol) | 0,2-1,6 | 0,5-6,2 | 0,8 - 6,4 |
| Aminoglycosides | |||
| Streptomycin | 0,4 - 5,0 | 0,4 - 12,5 | 1,1-1,2 |
| Kanamycin | 1,6-12,5 | 1,6-50 | 3,1-12,5 |
| Gentamicin | 0,8-12,5 | 0,4-6,2 | 0,4-0,8 |
| Quinolones | |||
| Ciprofloxacin | 1,0 | 0,5-16 | - |
| Sparfloxacin | 0,02 - 0,03 | - | - |
| Gripafloxacin | 0,05 | 0,1 -2 | - |
| VAU 12- 8039 | 0,05 | 0,05-0,5 | - |
Features of mycoplasma infections
Infections caused by mycoplasmas have the following characteristic features:1. According to clinical and morphological characteristics, mycoplasma infections are similar to diseases caused by other microorganisms: chlamydia, viruses, fungi, as well as chemicals, i.e. similar to other polyetiological diseases; they do not have their own clinical manifestations, which greatly complicates diagnosis and indicates the need to use laboratory diagnostic methods and obtain epidemiological data.
2. Mycoplasma infections can be acute, but more often have a chronic recurrent course
3. The development of mycoplasmosis is largely determined by the host's sensitivity to infection. Data on the genetic determination of sensitivity to mycoplasmas were obtained by modeling infection in congenic mice. It is logical to assume that the human population is also heterogeneous in this regard. The first publications on this topic have already appeared.
4. The nature of the pathological process depends on the entrance gate of the infection. Thus, M. hominis can cause pharyngitis and diseases of the urogenital tract in humans. Several cases of pneumonia caused by M. hominis have been described in the literature. With intrauterine mycoplasmosis of the fetus, the infection develops in the upper respiratory tract, lungs, urogenital tract, and central nervous system.
5. Mycoplasma infections are often accompanied by various immunopathological reactions, which complicate and largely determine the course of the infection. Patients with defects in the immune system are especially susceptible to mycoplasma infections. Immunosuppressive chemotherapy for organ and tissue transplantation or for tumor processes increases the risk of infections associated with mycoplasmas - representatives of the normal human microflora, as well as those acquired through contact with infected animals.
6. Mycoplasmas can cause local infection and not penetrate into the underlying tissue. However, tissue tropism is easily overcome; dissimination of the pathogen in tissues and organs is often observed, which leads to generalization of the process.
7. Mycoplasma infections are characterized by long-term persistence of the pathogen in the infected organism. One of the reasons is the wide variability of membrane proteins, which is largely associated with the presence of their multiple gene copies in the genome and the possibility of homologous recombinations between them. This increases the coding capacity of their small genome, the genetic diversity of mycoplasmas and, consequently, their ability to evade host immune surveillance. Other reasons for the long-term persistence of mycoplasmas in an infected organism are summarized in Table 3.
