Taxonomy, pneumococcus, influenza lab methods

MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION KAZAN STATE MEDICAL UNIVERSITY DEPARTMENT OF MICROBIOLOGY

PNEUMOCOCCIS

Kazan 1999

UDC 576.851.21(07)

Published by decision of the Central Coordinating and Methodological Council of Kazan State Medical University.

Compiled by:

(Head of the Department of Microbiology, Doctor of Medical Sciences, Professor O.K. Pozdeev, assistant of the Department of Microbiology, Ph.D., E.R. Fedorova.

Reviewers:

Head of the Department of Epidemiology, Kazan State Medical University, Doctor of Medical Sciences, Associate Professor M.Sh. Shafeev, Head of the Department of Epidemiology of the Kazan State Medical Academy, Doctor of Medical Sciences, Professor V.E. Grigoriev.

Pneumococci /O.K. Pozdeev, E.R. Fedorov-Kazan: KSMU, 1999. - 14 s.

By copying, distributing, publishing the text or part of it on your resources, you assume responsibility in accordance with applicable law.

Exclusively for the use of the work for personal purposes (Article 18, Article 26 of the Law of the Russian Federation "On Copyright and Related Rights"). Commercial reproduction is prohibited.

Kazan State Medical University, 1999

Pneumococcus (Streptococcus pneumoniae) was first identified by Pasteur (1881) while working on a rabies vaccine and initially considered it the causative agent of rabies. The etiological role of the microorganism in the development of pneumonia in humans was proved by Frenkel and Weichselbaum (1884). Bacteria colonize human and animal organisms and are included in the group of so-called "oral" streptococci. They are the main causative agents of pneumonia, they can also cause pleurisy, meningitis, creeping corneal ulcers, purulent inflammation of the middle ear, septic conditions and other diseases. In the IX edition of the Burgey Bacteria Key (1994), pneumococci are included in the 17th group "Gram-positive cocci".

Epidemiology of lesions. Pneumococcus is one of the main causative agents of bacterial pneumonia registered outside hospitals (2-4 cases per 1000 people); At least 500,000 cases of pneumococcal pneumonia are observed annually in the world (the real value is much higher). Children and the elderly are most susceptible to infection. The reservoir of infection is patients and carriers (20-50% of preschool children and 20-25% of adults); the main route of transmission is contact; during outbreaks also airborne. The peak incidence occurs in the cold season. In the vast majority of cases, clinical forms of infection develop when the body's resistance is impaired (including due to cold stress), as well as against the background of other pathologies (sickle cell anemia, Hodgkin's disease, HIV infection, myeloma, diabetes mellitus, conditions after splenectomy, etc.) or with alcoholism. Variants 1, 2, and 3 play the greatest epidemiological significance in pathology in adults; in children - 1, 2, 3 and 14 options. Virulence of serovars in descending order - 3, 1, 2, 5, 7 and 8 options. White mice are sensitive to pneumococci (when infected, they die from septicemia within a day), calves, lambs, piglets, dogs and monkeys.

Morphology. Pneumococci are immobile, they do not form spores, they have a slightly elongated shape, resembling the contours of a candle flame. In smears from clinical material, they are arranged in pairs, each of which is surrounded by a thick capsule. In smears from culture media, they can be located in short chains and be more rounded. On simple media, they form a thin capsule; its development is stimulated by the introduction of blood, serum or ascitic fluid. Capsule formation is most pronounced in type III bacteria. When arranged in chains, the capsule may be common.

cultural properties. Pneumococci aerobes or facultative anaerobes; during cultivation, capnophilic conditions (5-10% CO2) are preferred. Chemoorganogrophy and grow well on blood or serum media, supplemented by the addition of 0.1% glucose. They can grow in the temperature range of 28-42 °C, the optimum is 37 °C. Optimum pH -7.6-7.8. On dense media, they form delicate translucent, well-defined colonies with a diameter of about 1 mm. Sometimes they can be flat with a central indentation; like other streptococci, colonies never merge

between themselves. On blood agar, they form small translucent colonies of a greenish-gray color. The center of the colonies is darker, the periphery is lighter. Under the colony and along its periphery, a zone of a-hemolysis is visible in the form of a greenish discolored zone (due to the transition of hemoglobin to methemoglobin). Colonies of type III pneumococcus often have a mucous consistency, their size is up to 2 mm. They are unclear, I can merge with each other. They form S- and R-forms of colonies. During the transition from the S- to the R-form, they lose the ability to synthesize a capsule. On liquid media with serum and 0.2% glucose give a uniform turbidity and a small flocculent precipitate. With prolonged cultivation, the sediment increases.

Sustainability. Pneumococci belong to the group of unstable microorganisms. In dry sputum, they persist for up to two months. Can be stored for a long time at low temperatures; at a temperature of 60 ° C, they die within 3-5 minutes. A 3% solution of carbolic acid kills them in 1-2 minutes. Optochin (at a concentration of 1:100,000) and bile are detrimental to pneumococci, which is used to identify bacteria.

Pneumococci differ from other microorganisms in a number of properties (Table 1).

Table 1 Biochemical properties of pneumococci

Test substrate	Result	Test substrate	Result
Growth at 100°C
		Raffinose
Medium with 6.5% Nad
a-hemolysis
B-hemolysis		Trehalose
		Phosphatase
hippurate		β-galactosidase
Glycerol
Designations: "+" - 90% or more strains are positive; (+) - 80-89% of strains are positive; d - 21-79% of strains are positive; (-) - 11-20% of strains are positive; “- - 90% or more of the strains are negative.

Antigenic structure. Several types of antigens have been found in pneumococci: a polysaccharide, 0-somatic antigen located in the cell wall; polysaccharide capsular K-antigens and M-protein. The polysaccharide somatic antigen is similar to the C-substance of other streptococci. The relationship determines the similarity of the chemical structure of ributeichoic acids associated with choline phosphate. Capsular antigens also have a polysaccharide nature, consisting of monosaccharides repeated in various combinations: D-glucose, D-galactose and L-rhamnose. According to the structure of capsular antigens, pneumococci are divided into 84 serovars. It should be remembered that capsular antigens cross-react with antisera to group A and B streptococcal antigens, as well as with sera to Klebsiella and Escherichia antigens. During the transition from S to R-form, capsular antigens are lost. For pneumococcal serotyping, group sera are produced, indicated by Latin letters (A, B, C, D, etc.) and serovariant, indicated by Roman numerals. Agglutinating serum III is not included in serum mixtures. It is released separately and cannot be bred. In humans, pneumococci of I, II and III serotypes are most often isolated. They are the most virulent for humans, so the agglutination reaction is initially set using antisera for these variants. If the result is negative, the agglutination reaction is put with a mixture of sera A, B, C, etc. (until a positive result is obtained), and then with separate antisera. For faster identification of serovars, the Neufeld reaction (immune swelling of the capsule) is used. The method is based on the ability of pneumococcal capsules to increase in volume in the presence of homologous antiserum, which is recorded by light-optical microscopy. Capsular polysaccharides have sensitizing properties, manifested in the development of a delayed-type hypersensitivity reaction, detected using skin tests.

pathogenicity factors. The main factor is the capsule, which protects bacteria from the microbicidal potential of phagocytes and leads them away from the action of opsonins. Non-encapsulated strains are practically avirulent and are rare. Most of the pool of anti-pneumococcal ATs are AT to Ag capsules. An important function of the capsule and M-protein is also to provide adhesion to the mucosa. Substance-C, which interacts specifically with C-reactive protein, is essential. The consequence of such a response is the activation of the complementary cascade and the release of mediators of the acute phase of inflammation; their accumulation in the lung tissue stimulates the migration of polymorphonuclear phagocytes. The formation of powerful inflammatory infiltrates is accompanied by a violation of the homeostasis of the lung tissue and its hepatization. Pneumococci produce endotoxin, a- and b-hemolysins (pneumolysins), leukocidin. a-pneumolysin is a thermolabile protein capable of neutralizing 0-streptolysin,

erythrogenic substance, neuraminidase. Pneumococci also synthesize a number of enzymes that contribute to the pathogenesis of lesions - muramidase, hyaluronidase (promotes the spread of microorganisms in tissues), peptidase (cleaves IgA).

The pathogenesis of lesions. The biotope of pneumococci is the upper respiratory tract. The pathogenesis of most pneumonias involves aspiration of saliva containing S. pneumoniae and entry of bacteria into the lower airways. Violation of protective draining mechanisms - cough shock and mucociliary clearance is essential. In adults, lobar lesions of the lungs are more often observed, in children and the elderly, peribronchial or focal lesions dominate. The formation of powerful inflammatory infiltrates is accompanied by a violation of the homeostasis of the lung tissue and its hepatization. Infections with the most virulent serovar 3 may be accompanied by the formation of cavities in the lung parenchyma. From the primary focus, the pathogen can penetrate into the pleural cavity and pericardium or disseminate hematogenously and cause meningitis, endocarditis and articular lesions.

Clinical manifestations. Classic pneumococcal pneumonia begins suddenly; note the rise in body temperature, productive cough and chest pain. In debilitated individuals and the elderly, the disease develops slowly, with a slight fever, impaired consciousness, and signs of pulmonary heart failure. Streptococcal meningitis is recorded in all age groups; they are characterized by a violent onset with a rise in body temperature, stiffness of the neck muscles, headache, nausea and vomiting. Meningeal vascular lesions are often accompanied by loss of consciousness; among children and in old age, mortality can reach 80%. Hematogenous pneumococcal lesions, as well as sinusitis, mastoiditis, otitis media, endocarditis, and peritonitis, are quite common in immunocompromised individuals (eg, HIV-infected) or patients with splenectomy. After the illness, unstable immunity develops, which is type-specific in nature and due to the appearance of antibodies against the typical capsular polysaccharide.

Treatment. The basis of therapy for pneumococcal infection is antibiotics - penicillin, tetracycline, levomycetin, vancomycin, rifampicin, ceftriaxone.

Prevention. Nonspecific prevention of pneumococcal infections is aimed at identifying patients and carriers with their subsequent treatment. For specific prevention of infection, children over two years of age, adults at risk, as well as healthy individuals during the outbreak of the disease are vaccinated with the polyvalent polysaccharide vaccine Pneumovex 23. The drug contains 23 pneumococcal capsular polysaccharide antigens (1, 2, 3, 4, 5, 6B 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 1-9F, 19A, 20, 22F , 23F, 33F). Antigens

pneumococci were obtained from 90% of strains isolated from the blood of patients with invasive pneumococcal infection in the USA and corresponding to the strains encountered in Russia. Immunization is carried out twice with an interval of 5-8 years.

After vaccination, artificial, active, type-specific immunity is created.

Laboratory diagnostics. The "gold standard" is the isolation of the pathogen. It should be remembered that the material must be examined quickly, because. bacteria are prone to rapid autolysis due to the activity of intracellular enzymes. The material for the study is sputum, pleural effusion and other exudates, cerebrospinal fluid, blood, mucus from the nose and throat, discharge from eye ulcers, discharge from the ear, urine, pieces of organs (in case of death of the patient). A signal response to pneumococcal infection can be issued if neutrophils and gram-positive lanceolate diplococci are detected in sputum smears (at least 10 per field of view). Otherwise, resort to isolation of the pathogen.

The first stage of the study. Pathological material is subjected to preliminary bacterioscopy (except for blood). Sputum is placed in a sterile Petri dish, washed, a purulent-mucous lump is captured with a loop, rubbed on a glass slide, dried and stained according to Gram. In the smear, Gram-positive lanceolate or oval-shaped cocci surrounded by a capsule are found (capsule formation is observed only in pneumococci isolated from sick and infected animals). Identification of pneumococcal capsules can be carried out using the Burri-Gins method. Inoculation of pathological material is carried out on 5-10% blood or serum agar and enrichment medium (8-10% serum broth). If pneumococcal sepsis is suspected, 5-10 ml of the patient's blood is inoculated into 45-90 ml of serum broth. The cerebrospinal fluid, if it is clear, is centrifuged and a few drops from the sediment are inoculated onto nutrient media. Semi-liquid serum agar is used as the enrichment medium. The cultures are incubated at 37°C for 24 hours. The best method for isolating a pure culture of pneumococci is to infect white mice with pathological material. Sputum, washed in a Petri dish with sterile saline, is ground in a sterile mortar with a sterile pestle or broken glass with the addition of saline in a ratio of 1:2-1:5. The suspension is settled, the supernatant in the amount of 0.5-1 ml is administered to white mice intraperitoneally. In the presence of pneumococci in the material, mice die within 72 hours. In smears from organs and blood, typical pneumococci are found. Organs and blood are also cultured on serum broth and on Petri dishes with blood or serum agar.

The second stage of the study. Study the nature of growth on nutrient media. On blood agar, pneumococcal colonies are small, round, with smooth

edges, tender, surrounded by a zone of greening of the environment (which is very reminiscent of the growth of green streptococci). On serum agar, the colonies are tender, translucent and transparent. With bacterioscopy of smears stained by Gram. find Gram-positive diplococci without capsules. After bacterioscopy, colonies suspected of pneumococci are subcultured on slant serum or blood agar or serum broth. Microscopy of smears from the enrichment medium, along with various microflora, can reveal gram-positive cocci arranged in pairs or short chains. The material from the enrichment medium is transferred to solid nutrient media. Crops are incubated at 37°C for 24 hours.

The third stage of the study. On blood agar slant, pneumococci form a delicate, thin, translucent coating. On whey broth, pneumococci cause turbidity and light sediment. In smears from solid culture media, pneumococci can have a different appearance. Along with diplococci of an elongated shape with pointed outer ends resembling a candle flame, there are cells of the correct oval and round shape. In broth culture, pneumococci are often arranged in chains. Based on the morphological and cultural properties of pneumococci, it is difficult to distinguish from viridescent streptococci, therefore, a set of special tests has been proposed for their differentiation:

Solubility in bile (deoxycholate test);

The ability to decompose inulin;

Sensitivity to optochin;

Agglutination reaction with specific anti-pneumococcal sera;

Ability to degrade glucose, maltose, sucrose, lactose, mannitol, sorbitol and salicin.

The most accessible methods that differentiate pneumococci from other streptococci are a test with optochin (inhibits their growth); from green streptococci they are distinguished by the ability to ferment inulin, as well as sensitivity to bile.

Deoxycholate test. After a preliminary bacterioscopy, 10 drops of isolated pure culture (preferably broth) are added to a test tube with 5 drops of sterile bovine bile. The control is a culture introduced into a test tube with 5 drops of saline. After 30-60 minutes of incubation at 37 °C, complete lysis of the culture is observed in the form of clearing in the test tube with bile, the mixture remains cloudy in the control tube. It should be remembered that avirulent cultures of pneumococcus are resistant to bile.

Bile resistance can also be tested by inoculation in 10% bile broth. The test material is added to the medium, while the broth becomes cloudy. After a 24-hour incubation at 37°C, the presence of pneumococci will be indicated by clearing of the broth as a result of bacterial lysis.

You can also use discs soaked in a 20% bile solution. The discs are placed on the grown culture in the dish and incubated for 1-2 hours at 37°C. In the presence of pneumococci, colonies lyse around the disk at a distance of 1-2 mm.

Inulin test. The culture of pneumococcus is seeded on the medium with inulin. To do this, 200 ml of sterile distilled water, 18 ml of litmus tincture and 3 g of inulin are added to 100 ml of bovine serum heated at 56 ° C for 30 minutes, sterilized with flowing steam for 30 minutes. The cultures are incubated at 37°C for 24 hours. Pneumococcus decomposes inulin, causing the medium to turn red. The green streptococcus does not cause reddening of the medium.

Test with optochin. The test pneumococcal culture is inoculated into serum broth with optochin at a dilution of 1:100,000 or 1:200,000. Pneumococcus does not grow on such an environment. It is also possible to determine sensitivity to optochin by inoculation on 10% blood agar containing optochin at a dilution of 1:50,000. The control is culture on blood agar. Pneumococci do not grow on the medium with Optochin; growth of pneumococci is observed on the control medium. You can use disks impregnated with 6 μg of optochin, which are applied after inoculation to the surface of the medium. In pneumococci, a growth inhibition zone of at least 18 mm in diameter forms around the disc.

Virulence test. The daily culture of pneumococcus grown on whey broth is diluted with 1% sterile peptone water (pH - 7.6) or slightly alkaline broth to 1:10. The diluted culture is administered intraperitoneally to white mice weighing 16-20 g in a volume of 0.5 ml and observed for 72 hours. From the organs of the dead mouse, inoculations are made on nutrient media and smears-imprints are microscopically examined. Highly virulent cultures include pneumococci, which cause the death of mice after the introduction of the culture at a dilution of 1:10. Avirulent cultures do not cause death in mice.

Serotyping of pneumococci. The 18-hour culture is tested in the Sabin microagglutination test. 4 drops of pneumococcal culture are applied to a glass slide. To 1 drop add a drop of type 1 antipneumococcal serum, to the 2nd - type II serum, to the 3rd - serum - 111, to the 4th - a drop of normal serum. The mixtures on the glass are mixed with a loop and examined under a magnifying glass or under a microscope at low magnification. In a positive case, agglutination is observed in one of the first three drops. The type of pneumococcus is determined in the agglutination reaction with specific agglutinating sera of the first three fixed types. Cultures that do not agglutinate with these types of sera are assigned to the X-group. The reaction is set as follows. Pour the 18-hour broth culture in 0.5 ml into test tubes. Then add in an equal volume of serum, diluted with saline in a ratio of 1:5. Controls are 2 test tubes, one of which contains the test culture mixed with

normal rabbit serum, and the other - only the test culture. The contents of the test tubes are thoroughly shaken and placed for 2 hours in a thermostat at a temperature of 37 °C, after which a preliminary calculation of the reaction is carried out. The final results are noted after an additional incubation at room temperature for 20 hours. Agglutination is scored four plus if the contents of the tubes are completely clear and the agglutination culture is a dense film that does not break when shaken; by three pluses if, with complete clarification of the contents of the tube, the agglutinating culture is easily broken into pieces; two pluses - if enlightenment does not occur, particles of an agglutinated culture are clearly visible to the naked eye in the cloudy contents of the tube; with agglutination by one plus in a test tube, a fine-grained mixture of glued pneumococci is found. With a negative reaction visible to the eye, agglutination is not observed;

the contents of the test tubes after shaking is a uniform turbidity.

Typing of X-group pneumococci is carried out using group

sera containing a mixture of typical agglutinating sera taken

in equal volumes. The following group sera are prepared by

mixing equal volumes of undiluted standard diagnostic

sera (Lund, I960):

A -1, II, IV, V, XVIII serovars;

B - VI, VIII, XIX serovars;

C - VII, XX. XXIV, XXXI, XL serovars;

D - IX, XI, XVI, XXXVI. XXXVII serovars;

E - X, XXI. XXXIII, XXXIX serovars;

F-XII. XVII. XXII, XXXVII, XXXII, XLI serovars;

G - XIII, XXV. XXIX, XXXIV, XXXV, XXXVIII, XLII, XLVII serovars;

J-XLIII. XLIV, XLV, XLVI serovars.

Type III agglutinating serum is used per se (without mixing with other typical sera) due to the difficulty of obtaining it in a sufficiently high titer. Typing is carried out in two steps: first with the help of group sera, and then with individual sera of the group with which a positive reaction was obtained. Pneumococcal serotyping is used primarily for epidemiological studies of the results of specific serotherapy and seroprophylaxis.

Microagglutination of pneumococci by the Sabin method can be obtained by mixing anti-pneumococcal sera with exudate from the abdominal cavity of a mouse infected with the patient's sputum. Already four hours after infection, a pure culture of pneumococci is found in the exudate, giving positive Sabin agglutination.

Accelerated methods for detecting and typing pneumococci. 1. Neufeld method or the phenomenon of pneumococcal capsule swelling. One lump of freshly isolated sputum of the patient is applied to three

coverslips, to each of them add a drop of undiluted specific anti-pneumococcal serum (1, II, III types) and a drop of Loeffler's blue. The drops are thoroughly mixed, covered with a glass slide with a hole smeared around the edges with petroleum jelly. After two minutes, the hanging drops are examined under a microscope with an immersion system. In a positive case, a sharp increase in pneumococcal capsules is seen. With a negative result, the capsules are hardly cherished. The swelling reaction is specific and does not give a positive result with other capsular bacteria. I do not use it for the study of sputum from patients treated with sulfonamides and antibiotics, tk. in this case, capsular pneumococci can be isolated.

2. Method of precipitation. 5-10 ml of sputum is boiled in a water bath until a dense clot is obtained. The clot is triturated and a small amount of saline is added, boiled again for several minutes to extract the specific polysaccharide from pneumococci. The suspension is centrifuged, with the resulting clear liquid and specific typical sera in precipitation tubes, a ring precipitation reaction is performed. The appearance of a ring at the interface between liquids indicates a positive result.

3. Determination of pneumococcal capsules according to Burri. A drop of the test material and a drop of ink are applied to the end of the glass slide. The mixture is stirred and a smear is made, dried in air and, without fixing, microscopically. The background of the drug is dark smoky, microbial bodies and their capsules are not stained. The preparation made according to Burri can be fixed with Nikiforov's mixture, washed with water, stained with Tsilya fuchsin, diluted 1:3 for 3-5 minutes. On the dark background of the smear, unstained capsules stand out, inside of which there are bacteria of a bright crimson color (Gins method).

scarlet fever cause various serotypes of beta-hemolytic streptococci with M-antigen and producing erythrogenin (toxigenic streptococci of serogroup A) - (Streptococcus pyogenes). In the absence of antitoxic immunity, scarlet fever occurs, in the presence of angina.

Clinical picture

 Intoxication - fever, general malaise, headaches.

 Scarlatina rash - finely punctate, with moderate pressure with a glass spatula, the spots are more clearly visible. When pressed harder, the rash gives way to a golden-yellowish skin tone. It appears on the 1-3rd day of illness and is localized mainly on the cheeks, in the groin, on the sides of the body. The skin of the nasolabial triangle remains pale and rash-free. The rash usually lasts 3-7 days, then fades away, leaving no pigmentation. Characterized by a thickening of the rash on the folds of the limbs - axillary, elbow, popliteal areas.

 Scarlet tongue - on the 2-4th day of illness, the patient's tongue becomes pronounced grainy, bright red, the so-called "crimson" tongue.

 Angina is a constant symptom of scarlet fever. It can be more severe than the usual sore throat.

 Peeling of the skin - occurs after the disappearance of the rash (14 days after the onset of the disease): in the area of ​​the palms and feet it is large-lamellar, starting from the fingertips; on the trunk, neck, auricles scaly peeling.

Pneumococci, taxonomy. Properties. Serological groups. Distinctive features from other streptococci. Caused diseases. Principles and methods of laboratory diagnostics.

Morphology and biological properties. Pneumococci (Streptococcus pneumoniae) are paired cocci of an oval, slightly elongated lanceolate shape, resembling a candle flame. They can also be located in short chains, resembling streptococci. They are non-motile, do not form spores, and are Gram-positive.
They are grown on media with the addition of protein: blood, serum, with ascitic fluid. On blood agar, pneumococcal colonies are small, resembling dewdrops, transparent in transmitted light, with a depressed center, surrounded by a zone of incomplete hemolysis, a greenish tint, similar to the colonies of viridescent streptococcus. On liquid media, they give a gentle turbidity, sometimes forming a precipitate. They are quite active biochemically: they decompose glucose, lactose, maltose, inulin and other carbohydrates with the formation of acid, do not liquefy gelatin, do not form indole. The splitting of inulin is a differential diagnostic feature that helps to distinguish pneumococci from streptococci, which do not decompose inulin. An important distinguishing feature is the ability of pneumococci to dissolve in bile, while streptococci are well preserved in it.

Pathogenesis and clinic. Pneumococci are the causative agents of lobar pneumonia in humans. They can also cause creeping corneal ulcers, upper respiratory catarrhs, meningitis, endocarditis, joint damage, and other diseases.

After the disease, immunity is low-tensioned, short-term, type-specific.
Microbiological diagnostics. The material for the study is sputum, blood, throat swab, cerebrospinal fluid. Due to the fact that pneumococcus dies quickly, pathological material must be delivered to the laboratory as soon as possible for research.

Meningococcus. Taxonomy, properties. Antigenic structure of meningococci, classification. Pathogenesis of meningococcal infection, clinical manifestations. Principles and methods of microbiological diagnostics. Differentiation of the causative agent of meningococcal infection and other meningococci. specific prophylaxis.

N. meningitidis (meningococci).

Meningococcus is the causative agent of meningococcal infection - a severe anthroponosis with airborne droplet transmission of the pathogen. The main source is carriers. The natural reservoir is the human nasopharynx. Morphological, cultural and biochemical properties are similar to gonococcus. Differences - they ferment not only glucose, but also maltose, produce hemolysin. They have a capsule that is larger and has a different structure than that of the gonococcus.

antigenic composition. They have four main antigenic systems.

1. Capsular group-specific polysaccharide antigens. Serogroup A strains most commonly cause epidemic outbreaks.

2. Protein antigens of the outer membrane. According to these antigens, meningococci of serogroups B and C are divided into classes and serotypes.

3. Genus- and species-specific antigens.

4. Lipopolysaccharide antigens (8 types). They have a high toxicity, cause a pyrogenic effect.

pathogenicity factors. Adhesion factors and colonization - pili and outer membrane proteins. Invasiveness factors - hyaluronidase and other produced enzymes (neuraminidase, proteases, fibrinolysin). Of great importance are capsular polysaccharide antigens that protect microorganisms from phagocytosis.

Immunity resistant, antimicrobial.

Laboratory diagnostics based on bacterioscopy, isolation of the culture and its biochemical identification, serological diagnostic methods. The inoculation of the material is carried out on solid and semi-liquid nutrient media containing blood, ascitic fluid, and blood serum.

Oxidase positive cultures are considered to belong to the genus Neisseria. Meningococcus is characterized by the fermentation of glucose and maltose. Belonging to the serogroup is determined in the agglutination test (RA).

Gonococcus. Taxonomy, properties. Pathogenesis of gonococcal infection, features of immunity. Principles and methods of laboratory diagnostics of acute and chronic gonorrhea, blennorrhea. RSK Borde-Zhangu, purpose, mechanism, response accounting. Prevention of blennorrhea in newborns. Prevention and treatment of gonorrhea. specific therapy.

N.gonorrheae (gonococcus).

Gonococcus is the causative agent of gonorrhea, a sexually transmitted disease with inflammatory manifestations in the urinary tract. The substrate for colonization is the epithelium of the urethra, rectum, conjunctiva of the eye, pharynx, cervix, fallopian tubes and ovary.

Diplococci stain well with methylene blue and other aniline dyes, pleomorphic (polymorphism). Very whimsical to the conditions of cultivation and nutrient media. Of the carbohydrates, only glucose is fermented.

Antigenic structure very variable - characterized by phase variations (disappearance of antigenic determinants) and antigenic variations (changes in antigenic determinants).

pathogenicity factors. The main factors are drank, with the help of which gonococci carry out adhesion and colonization of epithelial cells of the mucous membrane of the urinary tract, and lipopolysaccharide(endotoxin, released during the destruction of gonococci). Gonococci synthesize IgAI, a protease that cleaves IgA.

Laboratory diagnostics. Bacterioscopic diagnosis includes Gram stain and methylene blue. Typical signs of gonococcus are gram-negative staining, bean-shaped diplococci, intracellular localization.

Sowing is carried out on special media (KDS-MPA from rabbit meat or bovine heart with serum, ascites-agar, blood agar).

Causative agents of gaseous anaerobic infection. Taxonomy. Properties. characteristics of toxins. Pathogenesis, clinical forms. Principles and methods of laboratory diagnostics, drugs for specific prevention and treatment.

Gas gangrene is an anaerobic polyclostridial (i.e. caused by various types of clostridia) wound (traumatic) infection. Of primary importance is C.perfringens, less often C.novyi, as well as other types of clostridia in persistent associations with each other, aerobic pyogenic cocci and putrefactive anaerobic bacteria.

C.perfringens is a normal inhabitant of the intestines of humans and animals, it enters the soil with feces. It is the causative agent of wound infection - it causes disease when the pathogen enters the wound under anaerobic conditions. It is highly invasive and toxigenic. Invasiveness is associated with the production of hyaluronidase and other enzymes that have a destructive effect on muscle and connective tissues. Main pathogenicity factor - exotoxin, which has hemo-, necro-, neuro-, leukotoxic and lethal effects. In accordance with the antigenic specificity of exotoxins, they are isolated serotypes pathogen. Along with gas gangrene, C. perfringens causes food poisoning (they are based on the action of enterotoxins and necrotoxins).

Features of pathogenesis. In contrast to purulent diseases caused by aerobes, anaerobic infection is not dominated by inflammation, but necrosis, edema, gas formation in tissues, poisoning with toxins and tissue decay products.

Immunity- predominantly antitoxic.

Laboratory diagnostics includes bacterioscopy of wound discharge, isolation and identification of the pathogen, detection and identification of toxin in bioassays using a neutralization reaction with specific antitoxic antibodies.

Prevention and treatment. The prevention of gas gangrene is based on timely and correct surgical treatment of wounds. In case of severe wounds, antitoxic serums are administered against the main types of clostridium, 10 thousand IU each, for medicinal purposes - 50 thousand IU each.

Clostridia tetanus. Taxonomy. Properties, characteristics of toxins. The pathogenesis of the disease. Descending tetanus. Clinic. Principles and methods of laboratory diagnostics. The purpose of bacteriological research, preparations for specific prevention and treatment.

Tetanus is an acute wound infection characterized by lesions neurotoxin motor cells of the spinal cord and brain, which manifests itself in the form of spasms of the striated muscles. People and farm animals get sick. Soil, especially contaminated with human and animal feces, is a constant source of tetanus infection.

Pathogen - C.tetani - a large spore-forming gram-positive bacillus. Spores are located terminally (a type of drumstick), mobile due to flagella - peritrichous. Mandatory anaerobic. The spores are very resistant.

antigenic properties. The causative agent has O- and H- antigens.

pathogenicity factors. The main factor is the strongest exotoxin. Its two main fractions are distinguished - tetanospasmin (neurotoxin) and tetanolysin (hemolysin). Neurotoxin in the central nervous system penetrates into the area of ​​myoneural synapses, is transmitted from neuron to neuron in the area of ​​synapses, accumulates in the motor areas of the spinal and brain, blocks synaptic transmission. Death occurs from paralysis of the respiratory center, asphyxia (damage to the muscles of the larynx, diaphragm, intercostal muscles) or paralysis of the heart.

Laboratory diagnostics. Microbiological diagnostics includes bacterioscopy of raw materials, culture for isolation of the pathogen and its identification, detection of tetanus toxin.

Isolation of the pathogen is carried out according to the standard scheme for anaerobes, using various dense and liquid (Kitt-Tarozzi medium) media, identification is based on morphological, cultural, biochemical and toxigenic properties.

The simplest and most effective method of microbiological diagnostics is a bioassay on white mice. One group is infected with the test material, the second (control) - after mixing the samples with antitoxic tetanus serum. In the presence of tetanus toxin, the experimental group of mice dies, while the control group remains alive.

Treatment and emergency prevention. Donor tetanus immunoglobulin (antitoxin), antitoxic serum (350 IU/kg), antibiotics (penicillins, cephalosporins) are used. To create vaccine immunity, tetanus toxoid is used, more often as part of the DTP vaccine (tetanus toxoids, diphtheria and killed whooping cough).

Clostridia botulinum. Taxonomy. Properties. Characteristics of toxins, difference from exotoxins of pathogens of other foodborne infections. Principles and methods of laboratory diagnostics. Drugs for specific prevention and treatment.

Botulism is a severe food poisoning associated with the use of products contaminated with C.botulinum, and is characterized by a specific lesion of the central nervous system. It got its name from lat. botulus - sausage.

Exciter properties. Large polymorphic gram-positive rods, motile, have peritrichous flagella. Spores are oval, located subterminally (tennis racket). Eight types of toxins are formed, differing in antigenic specificity, and, accordingly, 8 types of pathogen are isolated. Among the most important characteristics is the presence or absence of proteolytic properties (casein hydrolysis, production of hydrogen sulfide).

The toxin has a neurotoxic effect. The toxin enters the body with food, although it can probably accumulate when the pathogen multiplies in the tissues of the body. The toxin is thermolabile, although boiling for up to 20 minutes is necessary for complete inactivation. The toxin is rapidly absorbed in the gastrointestinal tract, penetrates into the blood, selectively acts on the nuclei of the medulla oblongata and ganglion cells of the spinal cord. Neuroparalytic phenomena develop - swallowing disorders, aphonia, dysphagia, ophthalmo-plegic syndrome (strabismus, double vision, eyelid drooping), paralysis and paresis of the pharyngeal and laryngeal muscles, respiratory and cardiac arrest.

Laboratory diagnostics. The principles are common to clostridia.

Treatment and prevention. The basis is the early use of antitoxic sera (polyvalent or, when the type is established, homologous). Prevention is based on the sanitary and hygienic regime in the processing of food products. Home-made canned mushrooms and other products stored under anaerobic conditions are especially dangerous.

11. Pseudomonas aeruginosa. Taxonomy. Properties. Caused diseases.
Role in nosocomial infections. Principles and methods of laboratory diagnostics.

The genus pseudomonas, P. aeruginosa (Pseudomonas aeruginosa) is one of the main causative agents of local and systemic purulent-inflammatory processes in medical hospitals.

The pathogen is ubiquitous (water, soil, plants, animals), occurs normally in humans (most often in the intestines, on the skin and mucous membranes). Morphology- Gram-negative straight or slightly curved rod, movable, located in smears singly, in pairs or in short chains. Synthesizes mucus (capsular substance), especially more virulent mucoid strains.

cultural properties. It is an aerobe and has a set of enzymes corresponding to the type of respiration (cytochromes, cytochrome oxidase, dehydrases). On liquid media it forms a grayish-silver film. On dense media, the phenomenon of iridescent lysis is often observed. By the end of the day due to pigment synthesis pyocyanin a blue-green color of the culture appears.

biochemical properties. Pseudomonas aeruginosa is characterized by low saccharolytic activity (oxidizes only glucose), high proteolytic activity, and the formation of a beta-hemolysis zone on blood agar. Synthesizes trimethylamine, which gives crops a pleasant smell of jasmine. Produces the production of bacteriocins - pyocins.

Antigenic and pathogenic properties. The main antigens of Pseudomonas aeruginosa are a group-specific somatic O-antigen and a type-specific flagellar H-antigen. O-antigenic complex - an aggregate of LPS with proteins and lipids of the cell wall, has the properties of endotoxin, is one of the main factors of pathogenicity. Pseudomonas aeruginosa has a large set of pathogenicity factors - endotoxin (LPS, similar to other gram-negative bacteria), a number of exotoxins - cytotoxin, exoenzyme S, hemolysins, exotoxin A (the most important, resembles diphtheria exotoxin), enzymes (collagenase, neuraminidase, proteases).

Laboratory diagnostics. P.aeruginisa received its name for the bluish - green staining of the detachable wounds and dressings. The main diagnostic method is bacteriological. Important is the detection of the pigment pyocyanin. Treatment and specific prevention. There is no specific prevention. With food poisoning and intestinal dysbacteriosis caused by Pseudomonas aeruginosa, a complex intesti - bacteriophage, which includes pseudomonas phage, is effective. Of the antibacterial drugs, aminoglycosides, cephalosporins and quinolones are more often used.

Conditionally pathogenic gram-negative bacteria - causative agents of purulent-inflammatory processes (Proteus, Klebsiella, miraculous rod, etc.), taxonomy. General characteristics of enterobacteria. Principles and methods of laboratory diagnostics.

Genus Klebsiella.

The genus Klebsiella belongs to the Enterobacteriaceae family. A feature of the representatives of the genus is the ability to form a capsule. The main species is K. Pneumoniae. Cause opportunistic lesions - nosocomial pneumonia, urinary tract infections, diarrhea in newborns. Klebsiella cause mastitis, septicemia and pneumonia in animals, are constantly found on the skin and mucous membranes of humans and animals. Klebsiella - straight, motionless sticks of various sizes. facultative anaerobes. Oxidase - negative, catalase - positive.

pathogenicity factors. These include a polysaccharide capsule (K-antigen), endotoxin, fimbriae, siderophore system (binds ferrous ions and reduces their content in tissues), thermolabile and thermostable exotoxins.

Clinical manifestations. K.pneumoniae (subsp. pneumoniae) is characterized by hospital bronchitis and bronchopneumonia, lobar pneumonia, urinary tract infections, lesions of the meninges, joints, spine, eyes, as well as bacteremia and septicopyemia. The subspecies ozaenae causes a special form of chronic atrophic rhinitis - lake.

Laboratory diagnostics. The main method is bacteriological. Treatment. One of the features of Klebsiella is their multidrug resistance and the development of lesions against the background of a decrease in the body's resistance. Antibiotics are used for generalized and sluggish chronic forms of Klebsiellesis, usually in combination with drugs that stimulate the immune system.

Genus Proteus.

The genus Proteus belongs to the Enterobacteriaceae family. The genus was named in honor of the son of Poseidon Proteus, capable of changing his appearance. Representatives of the genus are able to change the external manifestations of growth on dense nutrient media, and are also distinguished by the greatest pleomorphism (morphology variability) compared to other Enterobacteria.

Proteins break down tyrosine, restore nitrates, oxidase is negative, catalase is positive. They live in the intestines of many species of vertebrates and invertebrates, soil, sewage, and decaying organic residues. Can cause urinary tract infections in humans, as well as septic lesions in patients with burns and after surgery. Quite often they also cause food poisoning. P.vulgaris and P.mirabilis have the most common role in pathology.

cultural properties. Proteas grow on simple media over a wide range of temperatures. The optimum pH is 7.2-7.4, the temperature is from +35 to 37 degrees Celsius. Proteus colonies in the O-form are rounded, semi-transparent and convex, H-forms give continuous growth. The growth of proteas is accompanied by a putrid odor. The phenomenon of swarming is characteristic, H-forms give on MPA a characteristic creeping growth in the form of a bluish-smoky delicate veil. When sowing according to the Shushkevich method in the condensation moisture of freshly cut MPA, the culture gradually rises in the form of a veil up the surface of the agar. Diffuse turbidity of the medium with a thick white sediment at the bottom is noted on the BCH.

pathogenicity factors. These include LPS of the cell wall, the ability to “swarm”, fimbria, proteases and urease, hemolysins and hemagglutinins.

Laboratory diagnostics. The main method is bacteriological. Differential diagnostic media (Ploskirev), enrichment media and MPA are used according to the Shushkevich method. Treatment. In case of intestinal dysbacteriosis associated with proteas (colitis), it is possible to use proteus phage and preparations containing it (intestifag, coliproteus bacteriophage).

"Wonderful stick" (Serratia marcescens), type of bacteria from among the pigment microorganisms. Gram-negative motile (peritrichous) non-spore-bearing rods. By type of exchange - facultative anaerobe. On the surface of the agar forms smooth or granular dark and bright red colonies with a metallic sheen. Lives in soil, water, food. Developing on bread (at high humidity), in milk, it colors them red; such products are not allowed to be sold. Conditionally pathogenic for animals and humans; may cause suppuration.

13. Escherichia. Taxonomy. Diseases caused by Escherichia coli. Pathogenic variants of diarrheagenic Escherichia. Antigenic structure, classification. Features of microbiological diagnostics. Differentiation of diarrheagenic Escherichia from conditionally pathogenic.

Escherichia are the most common aerobic intestinal bacteria that, under certain conditions, can cause a wide group of human diseases, both intestinal (diarrhea) and extraintestinal (bacteremia, urinary tract infections, etc.) localization. The main species - E. coli (E. coli) - the most common causative agent of infectious diseases caused by enterobacteria. This pathogen is an indicator of faecal contamination, especially water.

cultural properties. On liquid media, E. coli gives diffuse turbidity, on dense media it forms S- and R-forms of colonies. On the Endo medium for Escherichia, lactose-fermenting Escherichia coli form intensely red colonies with a metallic sheen, non-fermenting - pale pink or colorless colonies with a darker center, on Ploskirev's medium - red with a yellowish tint, on Levin's medium - dark blue with a metallic sheen .

biochemical properties. Escherichia coli in most cases ferments carbohydrates (glucose, lactose, mannitol, arabinose, galactose, etc.) with the formation of acid and gas, forms indole, but does not form hydrogen sulfide, and does not liquefy gelatin.

The main pathogenicity factors of diarrheal E.coli.

1. Factors of adhesion, colonization and invasion associated with pili, fimbrial structures, outer membrane proteins. They are encoded by plasmid genes and promote colonization of the lower small intestine.

2. Exotoxins: cytotonins (stimulate hypersecretion of fluid by intestinal cells, disrupt water-salt metabolism and contribute to the development of diarrhea) and enterocytotoxins (act on cells of the intestinal wall and capillary endothelium).

3. Endotoxin (lipopolysaccharide).

Depending on the presence of various pathogenicity factors, diarrheagenic Escherichia coli are divided into five main types: enterotoxigenic, enteroinvasive, enteropathogenic, enterohemorrhagic, enteroadhesive.

4. Pathogenic E. coli is characterized by the production of bacteriocins (colicins).

Enterotoxigenic E. coli have a high molecular weight thermolabile toxin, similar in action to cholera, cause cholera-like diarrhea (gastroenteritis in young children, traveler's diarrhea, etc.).

Enteroinvasive Escherichia coli able to penetrate and multiply in the cells of the intestinal epithelium. They cause profuse diarrhea with an admixture of blood and a large number of leukocytes (an indicator of an invasive process) in the stools. Clinically resembles dysentery. The strains have some similarities with Shigella (non-motile, do not ferment lactose, have high enteroinvasive properties).

Enteropathogenic E.coli- the main causative agents of diarrhea in children. At the heart of the lesions is the adhesion of bacteria to the intestinal epithelium with damage to the microvilli. Characterized by watery diarrhea and severe dehydration.

Enterohemorrhagic Escherichia coli cause diarrhea mixed with blood (hemorrhagic colitis), hemolytic-uremic syndrome (hemolytic anemia in combination with renal failure). The most common serotype of enterohemorrhagic Escherichia coli is O157:H7.

Enteroadhesive E.coli do not form cytotoxins, are poorly studied.

Laboratory diagnostics. The main approach is the isolation of a pure culture on differential diagnostic media and its identification by antigenic properties. They put RA with a set of polyvalent OK (to O- and K-antigens) sera.

Among pathogenic streptococci, S.pneumoniae (pneumococcus) occupies a special place. It plays a very important role in human infectious pathology. This species is one of the main causative agents of lobar pneumonia. According to far from complete data, there are more than 500,000 cases of pneumonia each year in the world caused by pneumococci, especially in children and the elderly. In addition to inflammation of the lungs, this microbe causes meningitis, endocarditis, peritonitis, otitis media, rhinitis, sinusitis, sepsis, creeping corneal ulcer and a number of other diseases. For laboratory diagnostics, bacterioscopic, bacteriological and biological methods are used. Material for the study of sputum, pus, blood, cerebrospinal fluid, mucus from the oropharynx and nasopharynx, discharge from the maxillary sinus, eyes and ears. It is important to immediately send the material to the laboratory and analyze it very quickly, since pneumococci are susceptible to autolysis.

Bacterioscopic examination

Bacterioscopic examination of the material (except for blood) is reduced to the manufacture of two smears. One of them is stained by Gram, the second - by Burri-Gins, which makes it possible to identify the capsule. Pneumococci are located in the form of lanceolate diplococci, surrounded by a common capsule. If 10 or more typical diplococci are detected in the field of view, it is highly likely that S.pneumoniae is present. However, primary microscopy does not give the right to make a final diagnosis, since smears may contain capsular non-pathogenic diplococci - representatives of the normal microflora. Therefore, it is necessary to carry out sowing of clinical material and isolate a pure culture.

Bacteriological research

In case of sepsis, 10 ml of blood is inoculated at the patient's bedside into a vial containing 100 ml of serum or sugar broth, incubated for 18-20 hours at 37 ° C, then sown on blood agar, a pure culture is isolated and identified. With meningitis, the cerebrospinal fluid is centrifuged and the sediment is cultured on blood agar. On it, pneumococci grow in the form of small round colonies surrounded by a green zone; a characteristic depression is visible in the center of the colony. Sowing sputum or pus on nutrient media is not advisable, since the presence of saprophytic microflora inhibits the growth of S.pneumoniae. It is better to introduce the test material into the abdominal cavity of white mice. Bioassay is a fast, reliable and accurate method for isolating a pure pneumococcal culture. White mice are very sensitive to these bacteria and within 10-12 hours after infection, pneumococci penetrate into the blood and parenchymal organs, causing sepsis. Culture of blood from the heart or pieces of internal organs during the autopsy of animals allows you to isolate a pure culture of the pathogen. To identify pneumococci, their properties are used. Unlike other types of streptococci, S.pneumoniae does not grow on a medium with optochin, inulin is fermented and is very sensitive to the action of bile (deoxycholate test). Rapid lysis of pneumococci under the action of bile can be detected if 0.5 ml of bile is added to 1 ml of broth culture. After 15-20 minutes of stay in the thermostat, complete lysis of bacterial cells occurs. To determine the serovars of pneumococci (now there are 85 of them), an agglutination reaction on glass with typical sera or the phenomenon of "capsule swelling" is used. In the presence of homologous serum, the pneumococcal capsule swells strongly. Even better, serotyping is done with commercial latex agglutination or coagglutination reagents that display capsular antigens. Among streptococci, the genus Enterococcus is also important, the most significant species of which are E.faecalis, E.faecium and E.durans. They are quite widespread in nature. Their main ecological niche is the intestines of humans and animals, but they are also found in the normal microflora of the skin of the perineum, genitourinary organs, oropharynx and nasopharynx. They can cause suppuration of wounds, bacteremia, damage to the urogenital system, especially in patients with long-term functioning catheters, food poisoning, intestinal dysbacteriosis, less often endocarditis. In smears from the test material, enterococci are arranged in pairs, short chains or in the form of clusters, gram-positive. Bacteriological diagnosis of enterococcal infections is carried out without any difficulties, since these bacteria grow well on simple media. Agar dif-3 is selective for them (up to 600 ml of 3% MPA add 400 ml of 40% bile). After 24 hours of incubation, the colonies that have grown have a size of 0.4-1.0 mm of a grayish color. On blood agar, incomplete or complete hemolysis occurs around the colonies. Unlike viridescent streptococci, enterococci can grow on MPA with 6.5% NaCl, reducing milk with methylene blue at 37°C after 4-6 hours. Identification of isolated cultures is carried out according to morphological, cultural and biochemical characteristics.

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The causative agent of lobar pneumonia (pneumonia) is pneumococcus - Diplococcus pneumoniae, first discovered by Pasteur in the saliva of a man who died of rabies (1881).
Morphology and tinctorial properties. Pneumococci (Fig. 67 and 68 on the insert) are paired cocci that have an elongated shape like a lancet. Therefore, they are otherwise called lanceolate diplococci. Forming short chains, pneumococci become similar to streptococci, and therefore II. F. Gamaleya called them Streptococcus lanceolatus. The cell size ranges from 0.5X0.75 to 1X1.5 microns. They do not have spores or flagella. A distinctive feature of pneumococcus is the formation of a capsule, which is well expressed in pathological materials (sputum, blood, etc.). When cultivated on nutrient media, the capsule is lost. Pneumococci easily perceive aniline dyes and stain positively according to Gram.
Cultural and biochemical properties.

Rice. 68. Pneumococci in sputum smear.

Pneumococci are aerobes and facultative anaerobes. The temperature optimum is about 37°. Grow on media containing animal protein (blood or serum agar, ascitagar).
On the surface of the agar, after 24 hours, small colonies are formed, resembling streptococcal, but smaller and more transparent.
On slant agar, with abundant inoculation, a very delicate transparent coating is obtained, consisting of the smallest, non-merging colonies, on broth - a slight turbidity and a small flaky precipitate.
Freshly isolated strains do not grow on gelatin. Old laboratory strains of pneumococci can produce small whitish colonies already at 18-22°C. Gelatin is not liquefied.
They grow well on milk, curdling it with the formation of acid.
On blood agar, a zone of incomplete hemolysis with a greenish-brown staining of the medium is formed around the colonies.

Rice. 67. Pneumococci in pure culture from broth.

Pneumococci decompose sucrose, raffinose and lactose. The most important feature is the decomposition of inulin. Most streptococci do not have this property. Virulent pneumococci are soluble in bile.
Antigenic structure and serological types of pneumococci. The cytoplasm of pneumococci contains a protein antigen common to all pneumococci. This antigen determines their species specificity. The capsule contains specific polysaccharide antigens (hapten), which differ in their chemical composition in different pneumococci (typical antigens). Based on these typical antigens, all pneumococci are divided into three main groups (I, II, III) and a fourth combined group (X-group) using the agglutination and precipitation reaction. The X-group includes more than 70 types.
resistance. On artificial nutrient media, pneumococci quickly die (4-7 days). Under a layer of vaseline oil in liquid and semi-liquid media containing protein, they remain viable for 3-12 months.
Pneumococci tolerate drying well: in dry sputum in diffused light, they persist for up to 2 months. When heated to 52-55 °, they die in 10 minutes, at 60 ° - even faster. In a solution of carbolic acid (3%), pneumococci die in 1-2 minutes.
Pneumococci are especially sensitive to optochin. Under the influence of the latter, they die at a concentration of 1: 1,000,000.
Toxin formation and pathogenicity for animals. Pneumococcal venom is an endotoxin. Of laboratory animals, white mice and rabbits are more sensitive to pneumococcus. Parenteral administration of virulent pneumococci after 24-48 hours causes the death of animals with sepsis. At autopsy at the injection site, fibrinous exudate is detected; the spleen is enlarged and hyperemic.
Pathogenesis and diseases in humans. The entrance gate of infection is usually the mucous membrane of the pharynx. The introduction of pneumococci into the body and their penetration into the tissue of the lung can, apparently, occur both through the lymphatic and circulatory system, and directly through the branching of the bronchi. The most common disease is croupous pneumonia, which is characterized by a sudden onset, high fever, sometimes with chills, pain in the side when breathing, headache, sometimes loss of consciousness, delirium, strong arousal. In the future, there is a cough with a characteristic rusty-red sputum. In the lungs, there is a process that captures more often one, less often - two or three lobes.
Sources of infection are a sick person and a bacteriocarrier. Infection from the outside occurs both aerogenically - by droplets from the carrier, and through dust infection. Pneumococci can persist in dried sputum for a long time (about 2 months) and enter the air with dust.
When examining healthy people, pathogenic pneumococci are often found in the nasopharynx, so the possibility of autoinfection is not excluded, and factors that weaken the body's resistance, such as hypothermia, play a significant role.
In addition to croupous pneumonia, pneumococci cause inflammation of the middle ear, meninges (meningitis), as well as the mucous membrane of the nose and sinuses, sore throat, creeping ulcer of the cornea and inflammation of the lacrimal sac.
Immunity. Transferred pneumonia does not confer immunity. The disease may recur more than once. This is due to the presence of many types of pneumococci and the fact that past pneumonia increases the body's sensitivity to pneumococci.
The serum of recovered patients contains antibodies (agglutinins, etc.).
By the time of the crisis in pneumonia, the concentration of antibodies in the blood reaches a significant titer, and phagocytosis increases dramatically (I. Ya. Chistovich). Based on these data, immunity in pneumonia should be considered primarily as phagocytic, in which antibodies (bacteriotropins) play an important role.
Microbiological diagnostics. Materials for research in pneumococcal diseases are sputum, blood and pus taken from various lesions, less often cerebrospinal fluid.
Pathological material (excluding blood) is examined bacterioscopically, bacteriologically and by infecting white mice. The latter method has to be resorted to because the source material, especially sputum, usually contains abundant extraneous microflora, which, when the material is directly sown on nutrient media, makes it difficult to isolate pneumococcus.
Smears from sputum, pus, etc. are Gram-stained. Under the microscope, lanceolate diplococci, Gram-stained positively surrounded by a capsule, are found.
Cultures are isolated on blood agar or ascig agar. After 24-48 hours of growth at 37°, characteristic colonies appear if pneumococcus is present. Colonies are plated on slant serum or ascites agar and the isolated culture is checked for solubility in bile and for the ability to degrade inulin.
Infection of a white mouse is the surest way to isolate a culture of pneumococcus. Material from a patient or a corpse (sputum, pus, a piece of an organ, etc.) is placed in a sterile cup, then ground in a sterile mortar, with 1-2 ml of sterile broth and 0.5 ml of this suspension is injected intraperitoneally to a white mouse. After the death of the mouse, which occurs within 12-48 hours, blood cultures are taken from the heart, and in almost all cases a pure culture of pneumococcus is obtained.
If sepsis is suspected, 10-20 ml of blood is inoculated into ascitic or serum broth. After enrichment from the broth, inoculations are made on blood agar, and the isolated pure culture is identified by morphological and biochemical characteristics.
specific therapy and chemotherapy. Currently, sulfanilamide preparations and antibiotics (penicillin, biomycin, tetracycline, etc.) are used with great success for the treatment of lobar pneumonia.

The genus Streptococcus includes: Streptococcus pyogenes (hemolytic) and Streptococcus pneumoniae (pneumococcus). Streptococci were first discovered by Billroth (1874), L. Pasteur (1879). They were studied by E. Rosenbach (1884).

Streptococcus pyogenes (hemolytic)

Morphology. Streptococci are cocci that have a spherical shape. The diameter of each coccus is on average 0.6-1 μm, however, they are characterized by polymorphism: there are small and large cocci, strictly spherical and oval. Streptococci are arranged in a chain, which is the result of their division in the same plane. Chain lengths vary. On a dense nutrient medium, the chains are usually short; on liquid ones, they are long. Streptococci are immobile, do not have spores (see Fig. 4). Freshly isolated cultures sometimes form a capsule. On ultrathin sections, a microcapsule is visible, under it there is a three-layer cell wall and a three-layer cytoplasmic membrane. Gram-positive.

cultivation. Streptococci are facultative anaerobes. Grow at a temperature of 37 ° C and pH 7.6-7.8. The optimal media for their cultivation are media containing blood or blood serum. On dense nutrient media, streptococcal colonies are small, flat, cloudy, grayish in color. On blood agar, some varieties of streptococci form hemolysis. β-hemolytic streptococci form a clear zone of hemolysis, α-hemolytic streptococci form a small greenish zone (the result of the transition of hemoglobin to methemoglobin). There are streptococci that do not give hemolysis.

On sugar broth, streptococci grow with the formation of parietal and near-bottom fine-grained sediment, while the broth remains transparent.

Enzymatic properties. Streptococci have saccharolytic properties. They break down glucose, lactose, sucrose, mannitol (not always) and maltose to form acid. Their proteolytic properties are poorly expressed. They coagulate milk, gelatin does not liquefy.

toxin formation. Streptococci form a number of exotoxins: 1) streptolysins - destroy red blood cells (O-streptolysin has a cardiotoxic effect); 2) leukocidin - destroys leukocytes (formed by highly virulent strains); 3) erythrogenic (scarlet fever) toxin - causes the clinical picture of scarlet fever - intoxication, vascular reactions, rash, etc. The synthesis of erythrogenic toxin is determined by the prophage; 4) cytotoxins - have the ability to cause glomerulonephritis.

Streptococci have various antigens. The cytoplasm of the cell contains an antigen of a specific nucleoprotein nature - the same for all streptococci. Protein type antigens are located on the surface of the cell wall. A polysaccharide group antigen was found in the cell wall of streptococci.

According to the composition of the polysaccharide group-specific antigen fraction, all streptococci are divided into groups, denoted by capital Latin letters A, B, C, D, etc. up to S. In addition to groups, streptococci are divided into serological types, which are indicated by Arabic numerals.

Group A includes 70 types. This group includes most streptococci that cause various diseases in humans. Group B includes mainly opportunistic human streptococci. Group C includes streptococci pathogenic to humans and animals. Group D consists of streptococci that are not pathogenic to humans, but this group includes enterococci, which are inhabitants of the intestinal tract of humans and animals. Getting into other organs, they cause inflammatory processes: cholecystitis, pyelitis, etc. Thus, they can be attributed to conditionally pathogenic microbes.

The belonging of the isolated cultures to one of the serological groups is determined using a precipitation reaction with group sera. To determine serological types, an agglutination reaction with type-specific sera is used.

Streptococci are fairly stable in the environment. At a temperature of 60 ° C, they die after 30 minutes.

In dried pus and sputum, they persist for months. The usual concentrations of disinfectants destroy them in 15-20 minutes. Enterococci are much more resistant, disinfectant solutions kill them only after 50-60 minutes.

Animal susceptibility. Cattle, horses, dogs, and birds are susceptible to pathogenic streptococci. From laboratory animals rabbits and white mice are sensitive. However, streptococci pathogenic for humans are not always pathogenic for experimental animals.

Sources of infection. People (sick and carriers), less often animals or infected products.

Transmission routes. Airborne and airborne dust, sometimes food, contact-household is possible.

Diseases can occur as a result of exogenous infection, as well as endogenously - with the activation of opportunistic streptococci that live on the mucous membranes of the pharynx, nasopharynx, and vagina. A decrease in the body's resistance (cooling, starvation, overwork, etc.) can lead to autoinfections.

Of great importance in the pathogenesis of streptococcal infections is preliminary sensitization - as a result of a previously transferred disease of streptococcal etiology.

When penetrating into the bloodstream, streptococci cause a severe septic process.

Diseases in humans more often cause β-hemolytic streptococci of serological group A. They produce pathogenicity enzymes: hyaluronidase, fibrinolysin (streptokinase), deoxyribonuclease, etc. In addition, a capsule, M-protein, which have antiphagocytic properties, are found in streptococci.

Streptococci cause various acute and chronic infections in humans, both with the formation of pus and non-suppurative, differing in clinical picture and pathogenesis. Suppurative - phlegmon, abscesses, wound infections, non-suppurative - acute infections of the upper respiratory tract, erysipelas, scarlet fever, rheumatism, etc.

Streptococci often cause secondary infections in influenza, measles, whooping cough and other diseases and often complicate wound infections.

Immunity. By nature, immunity is antitoxic and antibacterial. Postinfectious antimicrobial immunity is weak. This is due to the weak immunogenicity of streptococci and a large number of serovars that do not give cross-immunity. In addition, with streptococcal diseases, an allergization of the body is observed, which explains the tendency to relapse.

Prevention. It comes down to sanitary and hygienic measures, strengthening the overall resistance of the body. Specific prophylaxis has not been developed.

Treatment. Apply antibiotics. More often, penicillin is used, to which streptococci have not acquired resistance, as well as erythromycin and tetracycline.

The value of streptococcus in the etiology of rheumatic heart disease. The pathogenesis of rheumatic heart disease is not well understood. But a number of facts speak in favor of the role of streptococcus in the development of this disease:

1. In patients with rheumatic heart disease, B-hemolytic streptococcus is sown from the pharynx.

2. Rheumatism often occurs after suffering a sore throat, tonsillitis, pharyngitis, sensitizing the body.

3. Antistreptolysin, antistreptohyaluronidase - antibodies to streptococcal enzymes, toxins are found in the blood serum of patients.

4. Indirect confirmation of the role of streptococcus is the successful treatment with penicillin.

Recently, L-forms of streptococcus have been given importance in the occurrence of chronic forms of rheumatic heart disease.

Prevention of exacerbations of rheumatic heart disease is reduced to the prevention of streptococcal diseases (for example, in spring and autumn, a prophylactic course of penicillin administration is carried out). Treatment is reduced to the use of antibacterial drugs - penicillin.

The value of streptococcus in the etiology of scarlet fever. G. N. Gabrichevsky (1902) was the first to suggest that hemolytic streptococcus is the causative agent of scarlet fever. But since the streptococci isolated in other diseases did not differ from the causative agents of scarlet fever, this opinion was not shared by everyone. It is now established that scarlet fever is caused by group A streptococci that produce erythrogenic toxin.

In those who have been ill, immunity arises - persistent, antitoxic. Its tension is determined by setting the Dick reaction - intradermal injection of erythrogenic toxin. In those who are not sick around the injection site, hyperemia and edema occur, which is characterized as a positive reaction (lack of antitoxin in the blood serum). In those who have been ill, such a reaction is absent, since the antitoxin formed in them neutralizes the erythrogenic toxin.

Prevention. Isolation, hospitalization. Contact, weakened children are given gamma globulin. Specific prophylaxis has not been developed.

Treatment. Use penicillin, tetracycline. In severe cases, antitoxic serum is administered.

The purpose of the study: detection of streptococcus and determination of its serovar.

Research material

1. Mucus from the throat (tonsillitis, scarlet fever).

2. Scraping from the affected area of ​​the skin (erysipelas, streptoderma).

3. Pus (abscess).

4. Urine (nephritis).

5. Blood (suspected sepsis; endocarditis).

Basic research methods

1. Bacteriological.

2. Microscopic.

Research progress

Second day of research

Take the cups out of the thermostat and inspect. In the presence of suspicious colonies, smears are made from a part of them, stained according to Gram and microscopically. If streptococci are found in the smear, part of the remaining colony is subcultured into test tubes on agar with serum to isolate a pure culture and on broth with blood in test tubes. By the end of the day, a 5-6-hour culture from broth or agar is subcultured onto Marten's broth with 0.25% glucose to determine the serological group in the Lensfield precipitation reaction. Test tubes and vials are placed in a thermostat and left until the next day.

Third day of research

The cultures are removed from the oven, the purity of the culture is checked on the agar slant, smears are made, Gram stained and microscoped. In the presence of a pure culture of streptococcus, they are sown on Hiss media (lactose, glucose, maltose, sucrose and mannitol), milk, gelatin, 40% bile and put in a thermostat.

Look through Martin's broth. In the presence of specific growth, a Lensfield precipitation test is performed to determine the serological group.

Setting up the precipitation reaction according to Lensfield. The daily culture grown on Martin's broth is poured into several centrifuge tubes, centrifuged for 10-15 minutes (3000 rpm).

The supernatant is poured into a jar with a disinfectant solution, and the precipitate is poured into a sterile isotonic sodium chloride solution and centrifuged again. To the precipitate collected from all centrifuge tubes, add 0.4 ml of 0.2% hydrochloric acid. Then the tube is placed in a water bath and boiled for 15 minutes, shaking occasionally. After boiling, the resulting suspension is again centrifuged. The antigen is then extracted into the supernatant, which is poured into a clean tube and neutralized with 0.2% sodium hydroxide solution to pH 7.0-7.2. Bromothymol blue (0.01 ml of a 0.04% solution) is added as an indicator. With this reaction, the color changes from straw yellow to blue.

Then, 0.5 ml of antistreptococcal group sera, which are prepared by immunizing rabbits, are poured into 5 precipitation tubes (see Chapter 19). Serum A is introduced into the 1st tube, serum B into the 2nd, serum C into the 3rd, serum D into the 4th, isotonic sodium chloride solution (control) into the 5th. After that, with a Pasteur pipette, the resulting extract (antigen) is carefully layered along the wall into all test tubes.

With a positive reaction in a test tube with homologous serum, a thin milky-white ring is formed at the border of the extract with serum (Fig. 38).

Fourth day of research

The results are recorded (Table 25).

Currently, deoxyribonuclease is being determined, as well as antistreptohyaluronidase, antistreptolysin-O.

test questions

1. What are the main methods of laboratory research for the detection of streptococci do you know?

2. What is the Lensfield precipitation reaction for?

3. Why should the antigen be transparent during this reaction? Describe the technique for staging this reaction.

Get antistreptococcal serum A, B, C, D and isotonic sodium chloride solution from the teacher. Set the precipitation reaction, show the results to the teacher and draw.

Nutrient media

agar with blood(see chapter 7).

Serum Agar(see chapter 7).

Hiss media(dry).

Meat peptone gelatin (MPG). To 100 ml of MPB add 10-15 g of finely chopped gelatin. Gelatin should swell when slowly heated in a water bath (at a temperature of 40-50 ° C). A 10% solution of sodium carbonate (baking soda) is added to the melted gelatin and the pH is adjusted to 7.0. It is then immediately filtered through a pleated filter. Filtration is slow. To speed up the process, filtration can be done in a hot autoclave. The filtered medium is poured into test tubes of 6-8 ml and sterilized. Sterilization is carried out either fractionally at a temperature of 100 ° C for 3 days in a row, or simultaneously at 110 ° C for 20 minutes in an autoclave. Cooling of the medium is carried out in test tubes placed vertically.

Milk preparation. Fresh milk is brought to a boil, put in a cool place for a day, freed from cream, boiled again. Leave for a day and remove the top layer. Skimmed milk is filtered through a layer of cotton wool, then alkalized with 10% sodium carbonate solution to pH 7.2 and poured into test tubes of 5-6 ml.

Bouillon Martin. An equal amount of peptone Marten (minced meat from pork stomachs exposed to hydrochloric acid) is added to the meat water. The resulting mixture is boiled for 10 minutes, alkalized with 10% sodium hydroxide solution to pH 8.0, 0.5 sodium acetate is added, boiled again and poured into sterile dishes. 0.25% glucose is added to Martin's broth.

Wednesday Kitt - Tarozzi(see chapter 34).

Streptococcus pneumoniae (pneumococcus)

Pneumococci were first described by R. Koch (1871).

Morphology. Pneumococci are diplococci in which the sides of the cells facing each other are flattened and the opposite sides are elongated, so they have a lanceolate shape resembling a candle flame (see Fig. 4). The size of pneumococci is 0.75-0.5 × 0.5-1 μm, they are arranged in pairs. In liquid nutrient media, they often form short chains, resembling streptococci. Prevmococci are immobile, do not have spores, form a capsule in the body that surrounds both cocci. The capsule contains a heat-resistant substance antiphagin (which protects pneumococcus from phagocytosis and the action of antibodies). When growing on artificial nutrient media, pneumococci lose their capsule. Pneumococci are gram positive. Gram-negative bacteria are found in old cultures.

cultivation. Pneumococci are facultative anaerobes. Grow at a temperature of 36-37 ° C and a pH of 7.2-7.4. They are demanding on media, since they cannot synthesize many amino acids, therefore they grow only on media with the addition of native protein (blood or serum). On agar with serum form small, delicate, fairly transparent colonies. On agar with blood, moist greenish-gray colonies grow, surrounded by a green zone, which is the result of the conversion of hemoglobin to methemoglobin. Pneumococci grow well in broth with the addition of 0.2% glucose and in broth with whey. Growth in liquid media is characterized by diffuse turbidity and dusty sediment at the bottom.

Enzymatic properties. Pneumococci have a fairly pronounced saccharolytic activity. They break down: lactose, glucose, sucrose, maltose, inulin with the formation of acid. Do not ferment mannitol. Their proteolytic properties are poorly expressed: they coagulate milk, do not liquefy gelatin, and do not form indole. Pneumococci dissolve in bile. Cleavage of inulin and dissolution in bile is an important diagnostic feature that distinguishes Streptococcus pneumoniae from Streptococcus pyogenes.

pathogenicity factors. Pneumococci produce hyaluronidase, fibrinolysin, etc.

toxin formation. Pneumococci produce endotoxin, hemolysin, leukocidin. The virulence of pneumococci is also associated with the presence of antiphagin in the capsule.

Antigenic structure and classification. In the cytoplasm of pneumococci there is a protein antigen common to the entire group, and in the capsule there is a polysaccharide antigen. According to the polysaccharide antigen, all pneumococci are divided into 84 serovars. Serovars I, II, III are the most common pathogens for humans.

Environmental resistance. Pneumococci belong to the group of unstable microorganisms. A temperature of 60 ° C destroys them in 3-5 minutes. They are quite resistant to low temperatures and drying. In dried sputum, they remain viable for up to 2 months. On a nutrient medium, they remain no more than 5-6 days. Therefore, when cultivating, it is necessary to do reseeding every 2-3 days. Conventional solutions of disinfectants: 3% phenol, sublimate at a dilution of 1:1000 destroy them in a few minutes.

Pneumococci are especially sensitive to optochin, which kills them at a dilution of 1:100,000.

Animal susceptibility. Humans are the natural host of pneumococci. However, pneumococci can cause illness in calves, lambs, piglets, dogs, and monkeys. Of the experimental animals, white mice are highly sensitive to pneumococcus.

Sources of infection. A sick person and a bacteriocarrier.

Transmission routes. Airborne, may be airborne.

entrance gate. The mucous membrane of the upper respiratory tract, eyes and ear.

Diseases in humans. Pneumococci can cause purulent-inflammatory diseases of different localization. Specific for pneumococci are:

1) lobar pneumonia;

2) creeping ulcer of the cornea;

The most common disease is croupous pneumonia, which affects one, less often two or three lobes of the lung. The disease is acute, accompanied by high fever, cough. It usually ends critically.

Immunity. After the illness, unstable immunity remains, since pneumonia is characterized by relapses.

Prevention. It comes down to sanitary and preventive measures. Specific prophylaxis has not been developed.

Treatment. Antibiotics are used - penicillin, tetracycline, etc.

test questions

1. Morphology of pneumococci. Cultivation and enzymatic properties.

2. What factors determine the pathogenicity of pneumococci and what protects pneumococci from phagocytosis?

3. What are the main gates of pneumococcal infection. What diseases are caused by pneumococci?

Microbiological research

The purpose of the study: detection of pneumococcus.

Research material

1. Phlegm (pneumonia).

2. Mucus from the pharynx (tonsillitis).

3. Discharge from the ulcer (creeping ulcer of the cornea).

4. Discharge from the ear (otitis media).

5. Pus (abscess).

6. Pleural punctate (pleurisy).

7. Blood (suspected sepsis).

1 (It is better to take morning sputum (with specific pneumonia, sputum has a rusty color).)

Basic research methods

1. Microscopic.

2. Microbiological.

3. Biological.

Research progress

biological sample. A little (3-5 ml of sputum) is emulsified in a sterile broth, 0.5 ml of this mixture is injected intraperitoneally to a white mouse. After 6-8 hours, the mice show signs of the disease. At this time, pneumococcus can already be detected in the exudate of the abdominal cavity. The exudate is taken with a sterile syringe. Smears are made from it, stained according to Gram and microscoped. To isolate a pure culture, the exudate is inoculated onto agar with serum. If the mouse dies or becomes ill, blood is cultured from the heart on serum agar to isolate a pure culture. Crops are placed in a thermostat.

An accelerated method for determining the type of pneumococcus(reaction of microagglutination). 4 drops of exudate from the abdominal cavity of an infected mouse are applied to a glass slide. Type I agglutinating serum is added to the first drop, type II serum to the second, type III to the third, and isotonic sodium chloride solution (control) to the fourth.

Type I and II sera are pre-diluted in a ratio of 1:10, and type III serum - 1:5. All drops are stirred, dried, fixed and stained with diluted magenta. With a positive result in one of the drops, microbial aggregation (agglutination) is noted.

Second day of research

The cultures are removed from the thermostat, examined, and smears are made from suspicious colonies. In the presence of gram-positive lanceolate diplococci in smears, 2-3 colonies are isolated on a slant of agar with serum to obtain a pure culture. Crops are placed in a thermostat. Smears are made from the broth, Gram-stained, and microscoped.

Third day of research

Crops are removed from the thermostat. Check the purity of the culture - make smears, Gram stain and microscope. If Gram-positive lanceolate diplococci are present in the isolated culture, the isolated culture is identified by inoculation:

1) on the Hiss media (lactose, glucose, sucrose, maltose), sowing is carried out in the usual way - by injection into the medium;

2) on the medium with inulin;

3) on the medium with optochin;

4) put a sample with bile.

Inulin test. The studied culture is seeded on a nutrient medium containing inulin and litmus tincture, and placed in a thermostat. After 18-24 hours, the crops are removed from the thermostat. In the presence of pneumococci, the medium turns red (streptococci do not change the consistency and color of the medium).

Determination of sensitivity to optochin. The isolated culture is seeded on 10% blood agar containing optochin 1:50,000. Pneumococci, unlike streptococci, do not grow on media containing optochin.

Bile test. 1 ml of the studied broth culture is poured into agglutination tubes. A drop of rabbit bile is added to one of them, the second test tube serves as a control. Both test tubes are placed in a thermostat. After 18-24 hours, lysis of pneumococci occurs, which is expressed in the clearing of a cloudy broth. In the control, the suspension remains cloudy.

A sample with bile can be placed on a dense nutrient medium. To do this, a grain of dry bile is applied to a colony of pneumococci grown in agar and serum plates - the colony dissolves - disappears.

Fourth day of research

The results are recorded (Table 26).

Note. to - the breakdown of carbohydrates with the formation of acid.

Currently, serological research methods (RSK and RIGA) are widely used to determine streptococcal antibodies. Determination of the group and serovar of the isolated culture is carried out using fluorescent antibodies.

Determination of pneumococcal virulence. Daily broth culture of pneumococcus is diluted with 1% peptone water from 10 -2 to 10 -8 , 0.5 ml of each dilution is administered to two white mice. The culture that caused the death of mice at a dilution of 10 -7 is assessed as virulent, at a dilution of 10 -4 -10 -6 it is considered moderately virulent. The culture that did not cause the death of mice is avirulent.

test questions

1. What methods of isolating a pure culture of pneumococci do you know?

2. Which animal is most susceptible to pneumococcus?

3. What reactions are put with the exudate of an infected mouse and for what purpose?

4. From which representatives of pyogenic cocci should pneumococcus be differentiated and by what test?

5. How to determine the virulence of pneumococci?

Exercise

Draw up a sputum examination scheme, indicating its stages by day.

Nutrient media

Serum Agar(see chapter 7).

Whey broth(see chapter 7).

agar with blood(see chapter 7).

Hiss media(dry).

Inulin test medium. To 200 ml of distilled water add 10 ml of inactivated bovine serum, 18 ml of litmus tincture and 3 g of inulin. Sterilize with flowing steam at 100°C for 3 consecutive days. Bile broth (see chapter 7).