Actinomycetes. Cunning actinomycetes Actinomycetes morphology
Morphology and physiology
Thin straight or slightly curved sticks and threads with true branching. Short rods often with club-shaped ends, resembling corynebacteria. Branched rods with branching threads at the ends are typical. Gram-positive, immobile, non-spore-forming, non-acid-resistant. Unlike fungi, they do not form conidia. Facultative anaerobes, require additional supply of CO2 Chemoorganotrophs with a fermentative type of metabolism. When carbohydrates are fermented, they form acid. On solid media, microcolonies characteristic of individual species are formed after 24 hours, and macrocolonies form after 7-14 days. They ferment glucose to form acid and have weak proteolytic activity.Antigens
The cell walls contain non-identical species-specific antigens, which makes it possible to divide them into 5 serogroups. A. israelii belongs to serogroup D, A. naeslun-dii belongs to serogroup A, A. bovis belongs to serogroup D. Each serogroup is divided into serovars.Pathogenicity and pathogenesis
Virulence factors and toxins of actinomycetes found in the pathology of humans and animals have not been sufficiently studied. The pathogen is localized on epithelial cells the mucous membrane of the oral cavity and other cavities and organs, as well as skin cells where inflammatory foci are formed - actinomyomas - in the case of immunodeficiency conditions. Druses of actinomycetes are found in actinomycomas. From the primary focus, the pathogen spreads in the body either by contact or lymphogenous route. There is no bacteremia. Secondary foci of infection can form in different organs (lungs, liver, etc.). The primary disease is often accompanied by a secondary one bacterial infection.Immunity
Post-infectious immunity is characterized by weak tension. Antimicrobial antibodies are detected in the blood serum, which do not have protective properties. The formation of HRT is noted.Ecology and epidemiology
Actinomycosis is an anthroponotic infection. However, actinomycetes that affect many animals (cattle and small cattle, pigs, etc.), in particular A.bovis, according to some authors, can cause disease in humans. The disease is believed to occur endogenously, since A. israelii is found in the oral cavity healthy people. Exogenous infection also occurs when people are infected by aerosol from external environment. It has been shown that actinomycetes not only retain their viability, but also reproduce in environment.Actinomycosis
Actinomycosis is a chronic granulomatous purulent inflammation of various tissues and organs caused by actinomycetes. It is accompanied by tissue infiltration, abscesses, fistulas, and the formation of dense grains (drusen). The main pathogens are Actinomyces israelii and A. bovis. The material for laboratory diagnosis is manure, sputum, urine, feces, cerebrospinal fluid, punctures and biopsies of affected tissues. The best results are obtained by microscopic and bacteriological research methods.Bacterioscopy
Bacterioscopy is performed to identify drusen. To do this, in a thin layer of liquid or rarefied material in a Petri dish, purulent pieces or grains are selected under a magnifying glass, transferred to a glass slide in a drop of 10-20% NaOH solution, slightly heated, covered with a coverslip and examined under a microscope (x40). Drusen look like continuous-like accumulations of mycelium, from which hyphae with thickenings at the ends extend radially to the periphery (like the rays of the sun). Stained preparations of drusen are also examined microscopically. For this purpose, grains or purulent pieces are washed with water, placed on a glass slide, covered with a second glass, gently pressed, separated, obtaining two uniform smears. They are stained with Gram and Ziehl-Neelsen. Immersion microscopy reveals mycelial cells, rod-shaped and coccoid formations, as well as spores. According to Gram, spores are colored dark purple, and drusen are pink. When stained according to Ziehl-Neelsen, the mycelium is blue and the spores are pink. If drusen do not appear, ordinary smears are prepared from the material being examined and stained with Gram. In this case, under a microscope, bundles of mycelium or individual filaments of purple color are detected. Identification of drusen or thin non-septate mycelium and spores confirms the diagnosis of actinomycosis.Bacteriological research
Pathological material from patients is usually contaminated with foreign microflora. To get rid of it, the material is centrifuged in a solution of penicillin and streptomycin, then washed from antibiotics with a 0.85% NaCl solution. To get rid of the accompanying flora, you can add a 50% glycerol solution to a test tube and keep it for 2-3 days at 37 ° C. The material treated in this way is sown on Sabouraud’s medium, blood or serum agar. Crops are incubated at 37°C for 3-5 days.A. israelii forms colorless, pasty, bumpy colonies on serum agar. Aerial mycelium is poorly formed. In smears from colonies, rods, spherical and flask-like elements are visible. Old colonies become fluffy, as if dusted with flour.A. bovis grows under anaerobic conditions, forming colorless paste-like colonies, which are early covered with white aerial mycelium. The colonies are firmly adhered to the agar and are not removed with a loop. In smears from colonies, non-septate mycelium can be seen, disintegrating into rod-shaped and cocci-shaped formations. Identification of isolated cultures is carried out according to a number of characteristics. Both types do not soften milk, do not liquefy gelatin, and do not reduce nitrates.Serological diagnosis
Serological diagnosis is rarely performed. Complement fixation and indirect hemagglutination reactions are used. The antigen is actinolysate. But both reactions can give positive results in other diseases (lung cancer, severe purulent processes). Immunological tests are of slightly greater diagnostic importance: the leukocyte migration inhibition reaction and the allergy test with actinolysate, as well as quantitation immunocompetent cells. Intradermal test with visceral actinomycosis it is often negative.13. Morphology and ultrastructure of actinomycetes. Pathogenic representatives. Actinomycetes are producers of antibiotics.
Actinomycetes – radiant mushrooms, belonging to the genus Actinomyces.
They are prokaryotes and are located under plants.
Morphology : actinomycete cells usually have the form of long and branching filaments, reminiscent in some cases of the mycelium of unicellular fungi, but rod-shaped and coccoid forms are also found. Mycelium threads are 100-600 µm long and 0.2-1.2 µm thick.
Ultrastructure : cell wall, cytoplasmic membrane that limits the cytoplasm, which contains the nucleoid, ribosomes, and intracellular inclusions. Mesosomes of actinomycetes are derivatives of the cytoplasmic membrane. The peptidoglycan of some actinomycetes contains arabinose, galactose, and other sugars that are absent in bacteria.
Actinomycetes reproduce by spores, transverse division, and budding.
Pathogenic representatives actinomycetes cause actinomycosis and nocardiosis in humans. These are Actinomyces israelli, Nocardia asteroides and others. The causative agents of actinomycosis outside the body, on a nutrient medium, are long branching threads, in places breaking up into fragments. In the human body, pathogenic actinomycetes form drusen - intertwined threads in the center with separate threads extending in the form of rays along the periphery. Hence the name: actinomycetes - radiant fungi. The ends of the threads immersed in the fabric are thickened, slimy and have a different chemical composition, and, like a bacterial capsule, protect the microbe from phagocytosis.
Actinomycetes – producers of antibiotics: Many actinomycetes, whose habitat is soil, produce antibiotics that are widely used in medical practice.
Actinomycetesgram +; detected by simple staining methods or Gram staining.
Actinomycetes are unicellular microorganisms that belong to prokaryotes. Their cells have the same structure as bacteria: a cell wall containing peptidoglycan, a cytoplasmic membrane; the cytoplasm contains the nucleoid, ribosomes, mesosomes, and intracellular inclusions. Therefore, pathogenic actinomycetes are sensitive to antibacterial drugs. At the same time, they have a form of branching intertwining threads similar to mushrooms, and some actinomycetes belonging to the Strentomycetes family reproduce by spores. Other families of actinomycetes reproduce by fragmentation, that is, the disintegration of filaments into separate fragments.
Actinomycetes are widespread in the environment, especially in soil, and participate in the cycle of substances in nature. Among actinomycetes there are producers of antibiotics, vitamins, and hormones. Most antibiotics currently used are produced by actinomycetes. These are streptomycin, tetracycline and others.
Pathogenic representatives of actinomycetes cause actinomycosis and nocardiosis in humans. These are Actinomyces israelli, Nocardia asteroides and others. The causative agents of actinomycosis outside the body, on a nutrient medium, are long branching threads, in places breaking up into fragments. In the human body, pathogenic actinomycetes form drusen - intertwined threads in the center with separate threads extending in the form of rays along the periphery. Hence the name: actinomycetes - radiant fungi. The ends of the threads, immersed in the tissue, are thickened, slimed and have a different chemical composition, and, like a bacterial capsule, protect the microbe from phagocytosis.
Pathogenic representatives: Treponema pallidum - causes syphilis, Treponema pertenue - the causative agent of yaws, Borrelia recurrentis - relapsing fever, Borrelia burgdorferi - Lyme disease, Leptospira interrogans - leptospirosis.
S., unlike other bacteria, does not perceive aniline dyes well. They are detected in preparations of “crushed” or “hanging” drops in a dark-field or phase-contrast microscope, as well as by Romanovsky-Giemsa staining with a mixture of methylene blue, eosin and azure: a working dye solution is applied to the smear (2 drops of dye per 1 ml of distilled water) for 10-20 minutes; then the preparation is washed with water and dried in air. Borrelia, unlike Treponema and Leptospira, are easily stained with aniline dyes.
Spirochetes are prokaryotes. They have characteristics common to both bacteria and protozoan microorganisms. These are single-celled microbes, shaped like long, thin, spirally curved cells, capable of active movement. Under unfavorable conditions, some of them can turn into cysts.
Electron microscope studies made it possible to establish the structure of spirochete cells. These are cytoplasmic cylinders surrounded by a cytoplasmic membrane and a cell wall containing peptidoglycan. The cytoplasm contains the nucleoid, ribosomes, mesosomes, and inclusions. Under the cytoplasmic membrane there are fibrils that provide various movements of spirochetes - translational, rotational, flexion.
Saprophytic spirochetes are present in the environment. Several non-pathogenic species are permanent inhabitants of the human body. Species pathogenic to humans belong to three genera: Treponema, Borrelia, Leptospira. They differ in the shape and arrangement of the curls. Treponemas consist of 8-12 curls of equal size, the position of which does not change during movement. Borrelia form 5-8 curls, changing with movement like the movement of a snake. Leptospires consist of 40-50 very small permanent whorls, the ends are curved in the form of hooks and have thickenings. When moving, the ends of the leptospires bend in different directions, and a shape is formed in the form of the Russian letter C or the Latin S. Spirochetes, with the exception of Borrelia, do not perceive aniline dyes well, so they are stained using the Romanovsky-Giemsa method. It is best to observe live spirochetes in a dark field of view.
Pathogenic representatives of spirochetes: Treponema pallidum - causes syphilis, Borrelia recurrentis - relapsing fever, Borrelia burgdorferi - Lyme disease, Leptospira interrogans - leptospirosis.
Systematic position of actinomycetes
ACTINOMYCETES
Methods for studying the morphology of Leptospires.
1. Dark-field microcopy. Leptospires are poorly stained with aniline dyes, so they are usually studied in a living state by microscopy of native preparations in a dark field of view (Fig. 46).
However, during microscopy of the material under study (blood, urine, cerebrospinal fluid, crushed organs of corpses), sometimes it is not possible to see leptospira in a dark field. Therefore, it is better to examine the supernatant liquid of centrifugates of the material being studied.
2. Leptospires are poorly stained with aniline dyes; when stained according to Romanovsky-Giemsa, they are pink.
The systematic position of spirochetes is presented in Table. 10.
Table 10
Actinomycetes- Gram+, polymorphic: most often they form filaments with a diameter of 0.2–2 µm and a length of up to 600 µm, sometimes thin, straight or slightly curved rods 0.2–1x2.5 µm are found, as well as coccoid forms (Fig. 47, 48) . The ultrastructure of actinomycetes does not differ from true bacteria, however, sugars not found in other prokaryotes (galactose, arabinose, xylose), mycolic acids and large amounts of fatty acids are found in the composition of the CS peptidoglycan.
Rice. 47. Pure culture of actinomycetes, Gram stain
Rice. 48. Microscopy of pus in cervicofacial actinomycosis, Gram stain
The filamentous cells of actinomycetes, like those of unicellular fungi, are not separated by septa (septa) and are called hyphae. A collection of hyphae is called mycelium. The mycelium develops from a small bud, which gradually extends into a rod, and then into a short thread with lateral branches. At the ends of the aerial mycelium, actinomycetes form conidia or spore carriers (straight, wavy or spiral).
Exospores (oval, round, cylindrical, with a smooth surface or spines) serve for the reproduction of actinomycetes; they are not heat-resistant, but can withstand drying. Some actinomycetes have a capsule.
Actinomycetes reproduce asexually (by exospores, transverse fission, budding and fragmentation of the mycelium into rod-shaped or coccoid forms).
Previously, actinomycetes were mistakenly considered fungi, which was reflected in the name (from the Greek aktis - ray and myces - mushroom). Actinomycetes are bacteria because:
– have nucleoid , and not the true core characteristic of mushrooms;
– sensitive to bacteriophages and antibiotics , and not to antifungal drugs;
– grow well in a slightly alkaline environment , and mushrooms - in sour.
Representatives of actinomycetes and their role in pathology. Representatives of the genus Actinomyces- free-living microorganisms that live in humus-rich soil, on plants, and in water. When plowing the soil, the mycelium threads are damaged and released essential oil(1,10-dimethyl-9-decalol), which causes a “spring” smell.
Some UP actinomycetes, being symbionts of humans and animals, are present in oral cavity, dental plaque, lacunae of the tonsils, in the salivary glands, on the mucous membrane of the gastrointestinal tract, found in stones of the biliary and urinary tracts.
Infection develops at the site of introduction inflammatory focus and is formed granuloma - a very dense and almost painless infiltrate consisting of macrophages and actinomycetes. Granuloma grows into surrounding tissues, resulting in The skin is involved in the process: it becomes cyanotic-purple. When the granuloma suppurates, its center undergoes necrotic decay, and when the abscess is opened, a fluctuation first appears, then long-term non-healing fistulas are formed. Pus is released from the fistulas, which contains Druze - white or yellow granules, 1–2 mm in diameter. Then fibrosis (scarring) of the granuloma occurs. A secondary, predominantly staphylococcal, infection plays a role in the formation of suppuration.
Actinomycosis is characterized by damage to various organs and tissues.
The main clinical forms of actinomycosis:
1) cervical-maxillofacial occurs most often (usually the field of tooth extraction), has a relatively favorable course compared to other forms. The actinomycosis process can involve the cheeks, lips, tongue, tonsils, trachea, orbits, and larynx.
2) thoracic(organs of the chest cavity). It begins gradually: weakness, low-grade fever, cough, initially dry, then with mucopurulent sputum, often mixed with blood (the sputum has an earthy smell). Then bronchitis develops. The course is severe, without treatment the outcome is fatal.
3) abdominal; Primary lesions are localized in the ileocecal region and in the appendix region, this often mimics appendicitis or intestinal obstruction, then other parts of the colon are affected. Spreading, the infiltrate captures other organs: liver, kidneys, spine, and can reach abdominal wall. Without etiotropic treatment, mortality reaches 50%. Secondary lesions in abdominal actinomycosis can spread to genitourinary organs.
4) actinomycosis of bones and joints occurs as a result of the transfer of actinomycotic infiltrate from neighboring organs or is a consequence of hematogenous introduction of the fungus. Often the process is preceded by trauma. Osteomyelitis of the bones of the leg, pelvis, spine, and joint damage have been described. Despite pronounced bone changes, patients retain the ability to move; if the joints are damaged, the function is not seriously impaired.
5) actinomycosis of the central nervous system.
6) in education subgingival calculus Another type of actinomycetes is involved - A. viscosus. Thanks to the micellar cell structure A. viscosus act as a matrix in which other microorganisms and dietary fiber residues are retained, which subsequently undergo calcification. A. viscosus also contributes to the occurrence of tooth root caries.
No. 22 Actinomycetes. Taxonomy. Characteristic. Microbiological diagnostics.
Treatment.
Morphology. Genus Actynomyces Branching bacteria. They do not contain chitin in the cell wall; the wall has the structure of gram-positive bacteria. The mycelium looks like thin straight rods, forming threads. Feature actinomycetes - the ability to form well-developed mycelium. Rod-shaped forms, often with thickened ends, are arranged singly, in pairs, in V- and Y-shapes in the smear. All morphological forms are capable of true branching, especially in a thioglycollate semi-liquid medium. Gram staining is poor, often forming granular or clear-shaped forms; non-acid-resistant. Typical view - Actinomycesbovis.
Cultural properties. Obligate and facultative anaerobes. They grow slowly, crops should be cultivated for 7 days. Temperature optimum for growth is 37C. Some strains give?-?-hemolysis on media with blood. Some species form filamentous microcolonies resembling mycelium; on the 7th day they form S-shaped colonies, sometimes colored yellow/red. A.odontolyticus On blood agar it forms red colonies with a zone of hemolysis.
Biochemical activity. Chemoorganotrophs. Carbohydrates are fermented to form acid without gas; fermentation products are acetic, formic, lactic and succinic acids. The presence of catalase and the ability to reduce nitrates into nitrites do not form indole.
Antigenic structure. ELISA distinguishes 6 groups: A, B, C, D, E and F.
Antimicrobial sensitivity.
Sensitive to penicillins, tetracycline, erythromycin, but resistant to antimycotics. Sensitive to the effects of commonly used antiseptics and disinfectants.
Epidemiology. The source of infection is soil. The transmission mechanism is contact, and the transmission route is wound. They colonize the oral mucosa of humans and mammals.
Pathogenesis. Causes opportunistic infection.
Clinic. Actinomycosis is a chronic opportunistic infection of humans and animals caused by anaerobic and facultative anaerobic actinomycetes, which is characterized by granulomatous inflammation.
Microbiological diagnostics
: Material for research - sputum, cerebrospinal fluid, pus from fistulas, tissue biopsy.
Used for diagnosis bacterioscopic, bacteriological, serological And allergologicalmethods.
Bacteroscopically: by detecting drusen of actinomycetes in the test material, which look like small yellowish or grayish-white grains with a greenish tint. According to Gram, spores are colored dark purple, mycelium is purple, and drusen are pink.
To suppress the growth of accompanying microflora, pus and sputum are centrifuged in a solution of penicillin and streptomycin before inoculation. Inoculated on nutrient media (sugar agar) and cultivated under aerobic and anaerobic conditions. The ability of isolated cultures to curdle and peptonize milk is determined - a characteristic characteristic of actinomycetes. Isolation of anaerobic species confirms the diagnosis of actinomycosis.
For serodiagnosis, RSC with actinolysate is performed. The reaction is not specific enough, since positive results can be observed with lung cancer and severe suppurative processes. The use of extracellular proteins of actinomycetes as an antigen instead of actinolysate increases the sensitivity of RSC. The same AG can also be used to stage RIGA.
An allergy test is carried out with actinolysate. Diagnostic value have positive tests.
Treatment. The use of penicillin, tetracycline, erythromycin, clindamycin.
Prevention. Specific prevention - no. Nonspecific - increased immune status.
Actinomycetes are branching bacteria. They do not contain chitin or cellulose in their cell wall, unlike fungi, they have the structure of gram-positive bacteria. The mycelium is primitive. Thin straight or slightly curved rods measuring 0.2-1.0×2.5 microns, often form threads up to 10-50 microns long.
Capable of forming a well-developed mycelium, in some species it is long and sparsely branched, in others it is short and strongly branched, the mycelium hyphae are not septate. Rod-shaped forms, often with angular ends, are located singly or in pairs in the smear. V- and Y-shaped or in the form of a palisade. All morphological forms are capable of true branching, especially in thioglycolate semi-liquid medium. Gram stains poorly, often form granular or clear-shaped forms, do not form conidia, and are not acid-fast. The type species is Actinomyces hovis.
Properties of actinomycetes
Obligate and facultative anaerobes, capnophiles. They grow slowly, crops should be cultivated for 7-14 days. Temperature optimum for growth is 37 C. Some strains produce hemolysis on media with blood. Some species form filamentous microcolonies resembling mycelium, and on days 7-14 they form crumbly S-shaped colonies, sometimes colored yellow or red. Actinomyces israelii tends to form long branching mycelium, which over time disintegrates into polymorphic coccoid, tube-shaped and other elements. It grows poorly on simple nutrient media; it grows better on protein media containing whey; forms transparent, colorless, pasty, usually smooth colonies, tightly merging with the medium. The aerial mycelium is scanty and does not form pigments; on some media, for example, on blood agar, it can form white lumpy colonies; on blood agar, A. odontoiyticm forms red colonies with a hemolysis zone.
Biochemical activity of actinomycetes
Actinomycetes are chemoorganotrophs. Carbohydrates are fermented with the formation of acid without gas; fermentation products are acetic, formic, lactic and succinic acids (but not iropionic acid). The presence of catalase and the ability to reduce nitrates to nitrites vary among different types, indole does not form. Species differentiation is based on differences in the ability to ferment carbohydrates and in several other biochemical tests.
In ELISA, serogroups A, B, C, D, E, F are distinguished.
Ecological niche of actinomycetes
The main habitat is soil. They are constantly found in water, air, on various objects, the surfaces of plants, animals and humans. They colonize the mucous membrane of the oral cavity and mammals.
Resistance of actinomycetes in the environment
When exposed to air they die instantly.
Sensitivity to antimicrobial drugs. Sensitive to penicillins, tetracycline, erythromycin and clindamycin. Sensitive to the effects of commonly used antiseptics and disinfectants.
Pathogenesis
Actinomycetes cause opportunistic infections.
Epidemiology of actinomycosis
The source of infection is soil. A multiplicity of mechanisms, routes and factors of transmission is characteristic, although the purest mechanism of transmission is contact, and the transmission route is wounded. Susceptibility to actins and cets, as to all opportunistic microbes, is low in individuals with normal status and increased in immunodeficient hosts.
Symptoms of actinomycosis
Actinomycosis is a chronic opportunistic infection of humans and animals caused by anaerobic and facultative anaerobic actinomycetes, which is characterized by granulomatous inflammation with polymorphic symptoms.
The disease actinomycosis is manifested by the formation of a granuloma, which undergoes necrotic disintegration with the formation of pus, emerging through fistulas onto the surface of the skin and mucous membranes of varying consistency, yellowish-white in color, sometimes mixed with blood, often containing drusen. At the same time, granuloma fibrosis is noted. Depending on the location, cervicofacial, thoracic, abdominal, genitourinary, musculoskeletal, musculocutaneous, septic and other forms of the disease are distinguished.
Immunity has not been studied enough.
Laboratory diagnosis of actinomycosis
The materials for the study are sputum, cerebrospinal fluid, pus from fistulas, punctates of unopened foci of softening, scrapings from granulations, tissue obtained by biopsy.
To diagnose actinomycosis, bacterioscopic, bacteriological, serological and allergological methods are used.
Usually the diagnosis is made bacterioscopically by the detection of drusen of actinomycetes in the test material, which have the appearance of small yellowish or grayish-white grains with a greenish tint. Under low magnification, formations of a circular shape with a structureless center and the periphery of a radial structure are visible; under high magnification, tangles with pigmented grains are visible in the center; along the periphery, hyphae extend radially from this ball of mycelium in the form of rays with flask-shaped thickenings at the ends. According to Gram, spores are colored dark purple, mycelium - purple. According to Ziehl-Nelson, mycelium is colored blue and spores are colored red.
The final diagnosis is made based on the isolation of the pathogen. To suppress the growth of accompanying microflora, pus and sputum are centrifuged in a solution of penicillin and streptomycin before inoculation, then washed with an isotonic NaCI solution to remove antibiotics. They are inoculated on nutrient media (sugar agar, Sabouraud's medium, etc.) and cultivated under aerobic and anaerobic conditions. Pure culture is isolated and identified according to the generally accepted scheme. The ability of isolated cultures to curdle and peptonize milk is determined - a characteristic characteristic of actinomycetes. Isolation of anaerobic species confirms the diagnosis of actinomycosis.
For serodiagnosis of actinomycosis, RSK with actinolysate is performed. The reaction is not specific enough, since positive results can be observed with mild and severe suppurative processes. The use of extracellular proteins of actinomycetes as an antigen instead of actinolysate increases the sensitivity of CSC. The same antigen can also be used to stage RNGA.
An allergy test is carried out with actinolysate. Only positive and strongly positive tests have diagnostic value. With visceral actin and goat allergy tests are often negative.
Treatment of actinomycosis
Satisfactory results can be achieved using penicillin, tetracycline, erythromycin, clindamycin.
Prevention of actinomycosis
Specific actinomycosis has not been developed. Nonspecific prevention is aimed at immune status.