Taxonomy, pneumococcus, laboratory methods for influenza

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

PNEUMOCOCCUS

KAZAN 1999

UDC 576.851.21(07)

Published by decision of the Central Coordination and Methodological Council of the 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, Candidate of Medical Sciences, 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, 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 accept responsibility in accordance with current legislation.

Exclusively for 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 isolated by Pasteur (1881) while working on an anti-rabies vaccine and was initially considered the causative agent of rabies. The etiological role of the microorganism in the development of pneumonia in humans was proven by Frenkel and Weichselbaum (1884). The bacteria colonize human and animal organisms and belong to the group of so-called “oral” streptococci. They are the main causative agents of pneumonia, and can also cause pleurisy, meningitis, creeping corneal ulcers, purulent inflammation of the middle ear, septic conditions and other diseases. In the IX edition of Bergey's guide to bacteria (1994), pneumococci are included in group 17, “Gram-positive cocci.”

Epidemiology of lesions. Pneumococcus is one of the main causative agents of bacterial pneumonia recorded outside hospitals (2-4 cases per 1000 people); Every year, at least 500,000 cases of pneumococcal pneumonia are observed 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 transmission route is contact; during outbreaks it is 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. Options 1, 2 and 3 play the greatest epidemiological significance in pathology in adults; for children - 1, 2, 3 and 14 options. The virulence of serovars in descending order is variants 3, 1, 2, 5, 7 and 8. White mice (if infected, they die from septicemia within 24 hours), calves, lambs, piglets, dogs and monkeys are sensitive to pneumococci.

Morphology. Pneumococci are immobile, they do not form spores, and have a slightly elongated shape, reminiscent of the contours of a candle flame. In smears of clinical material, they are arranged in pairs, each of which is surrounded by a thick capsule. In smears from culture media, they may 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 are aerobes or facultative anaerobes; When cultivating, capnophilic conditions (5-10% CO2) are preferred. They are chemoorganogrophs and grow well on blood or serum media supplemented with 0.1% glucose. They can grow in the temperature range of 28-42 °C, optimum - 37 °C. Optimum pH -7.6-7.8. On dense media they form delicate, translucent, clearly defined colonies with a diameter of about 1 mm. Sometimes they can be flat with a central depression; like other streptococci, colonies never fuse

between themselves. On blood agar they form small translucent greenish-gray colonies. 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 (which is due to the transition of hemoglobin to methemoglobin). Colonies of type III pneumococcus often have a mucous consistency and are up to 2 mm in size. They are a bit cloudy and can merge with each other. They form S- and R-forms of colonies. When transitioning from the S- to the R-form, they lose the ability to synthesize the capsule. On liquid media with serum and 0.2% glucose they give uniform turbidity and a small flocculent sediment. With prolonged cultivation, the sediment increases.

Sustainability. Pneumococci belong to the group of unstable microorganisms. They persist in dry sputum for up to two months. Able to 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 have a detrimental effect on 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
Wednesday 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: 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 in the chemical structure of ribitteichoic acids associated with choline phosphate. Capsule antigens also have a polysaccharide nature, consisting of monosaccharides repeated in various combinations: D-glucose, D-galactose and L-rhamnose. Based on the structure of capsular antigens, pneumococci are divided into 84 serovars. It should be remembered that capsular antigens cross-react with antisera to antigens of group A and B streptococci, as well as with sera to Klebsiella and Escherichia antigens. During the transition from S to R form, capsular antigens are lost. For serotyping of pneumococci, group sera are produced, designated by Latin letters (A, B, C, D, etc.) and serovariant sera, designated by Roman numerals. Agglutinating serum III is not included in serum mixtures. It is released separately and cannot be bred. In humans, pneumococci of serovariants I, II and III are most often isolated. They are the most virulent for humans, so the agglutination test is initially performed using antisera to these variants. If the result is negative, the agglutination reaction is performed 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 capsule swelling) 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 diverts them from the action of opsonins. Non-encapsulated strains are practically avirulent and are rare. The majority of the pool of antipneumococcal ATs consists of ATs to Ag capsules. An important function of the capsule and M-protein is also to ensure adhesion to the mucosa. Substance C is essential, as it specifically interacts with C-reactive protein. The consequence of such a reaction 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 disruption of the homeostasis of the lung tissue and its hepatization. Pneumococci produce endotoxin, a- and beta-hemolysins (pneumolysins), and leukocidin. α-pneumolysin is a thermolabile protein capable of neutralizing O-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 (breaks down IgA).

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 penetration of bacteria into the lower airways. Violation of protective drainage mechanisms - cough impulse and mucociliary clearance - is essential. In adults, lobar lung lesions are more often observed; in children and the elderly, peribronchial or focal lesions predominate. The formation of powerful inflammatory infiltrates is accompanied by a disruption 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 the pleural cavity and pericardium or disseminate hematogenously and cause meningitis, endocarditis and joint lesions

Clinical manifestations. Classic pneumococcal pneumonia begins suddenly; They note a rise in body temperature, a productive cough and chest pain. In weakened persons and the elderly, the disease develops slowly, with slight fever, impaired consciousness and signs of pulmonary heart failure. Streptococcal meningitis is registered 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. Lesions of the vessels of the meninges are often accompanied by loss of consciousness; among children and the elderly, mortality can reach 80%. Quite often, hematogenous pneumococcal lesions, as well as sinusitis, mastoiditis, otitis media, endocarditis and peritonitis, are noted in people with immunodeficiencies (for example, HIV-infected) or patients with splenectomy. After an illness, unstable immunity develops, which is type-specific and is caused by the appearance of antibodies against the typical capsular polysaccharide.

Treatment. The basis of treatment for pneumococcal infection is antibiotics - penicillin, tetracycline, chloramphenicol, 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 a disease outbreak are vaccinated with the polyvalent polysaccharide vaccine "Pneumovex 23". The drug contains 23 capsular polysaccharide antigens of pneumococci (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 the strains isolated from the blood of patients with invasive pneumococcal infection in the USA and corresponding to the strains found 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 pathogen isolation. 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 pharynx, discharge from eye ulcerations, discharge from the ear, urine, pieces of organs (in case of death of the patient). A signal response to pneumococcal infection can be issued when neutrophils and gram-positive lanceolate diplococci (at least 10 per field of view) are detected in sputum smears. Otherwise, they resort to isolating the pathogen.

The first stage of the study. Pathological material is subjected to preliminary bacterioscopy (except blood). The sputum is placed in a sterile Petri dish, washed, a purulent mucous lump is captured with a loop, ground on a glass slide, dried and stained with a Gram stain. The smear reveals gram-positive lancet-shaped or oval-shaped cocci surrounded by a capsule (capsule formation is observed only in pneumococci isolated from sick and infected animals). Detection 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 on enrichment medium (8-10% whey broth). If sepsis of a pneumococcal nature is suspected, 5-10 ml of the patient's blood is inoculated into 45-90 ml of whey broth. The cerebrospinal fluid, if it is clear, is centrifuged and a few drops from the sediment are inoculated onto nutrient media. Semi-solid whey agar is used as an enrichment medium. The crops 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 while adding saline in a ratio of 1:2-1:5. The suspension is settled, the supernatant liquid in an amount of 0.5-1 ml is administered intraperitoneally to white mice. If pneumococci are present in the material, mice die within 72 hours. Typical pneumococci are found in smears from organs and blood. Organs and blood are also cultured on whey broth and on Petri dishes with blood or serum agar.

Second stage of the study. The growth pattern on nutrient media is studied. On blood agar, colonies of pneumococci are small, round, with smooth

edges, tender, surrounded by a zone of greening of the medium (which is very reminiscent of the growth of viridans streptococci). On serum agar, the colonies are delicate, translucent and transparent. During bacterioscopy of Gram-stained smears. Gram-positive diplococci without capsules are detected. After bacterioscopy, colonies suspected of pneumococci are subcultured onto slanted serum or blood agar or into whey broth. When microscopying smears from the enrichment medium, along with various microflora, gram-positive cocci can be detected, arranged in pairs or short chains. The material from the enrichment medium is transferred to solid nutrient media. The crops are incubated at 37°C for 24 hours.

The third stage of the study. On slants of blood agar, pneumococci form a delicate, thin, translucent coating. In whey broth, pneumococci cause turbidity and a slight sediment. In smears from solid culture media, pneumococci can have different appearances. Along with diplococci of elongated shape with pointed outer ends, reminiscent of a candle flame, there are cells of regular 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 viridans streptococci, therefore a set of special tests has been proposed for their differentiation:

Solubility in bile (deoxycholate test);

Ability to decompose inulin;

Sensitivity to optochin;

Agglutination reaction with specific antipneumococcal sera;

The ability to decompose glucose, maltose, sucrose, lactose, mannitol, sorbitol and salicin.

The most accessible methods for differentiating pneumococci from other streptococci are a test with optochin (inhibits their growth); They are distinguished from viridans streptococci by their ability to ferment inulin, as well as sensitivity to bile.

Deoxycholate test. After 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 added to a test tube with 5 drops of saline solution. 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; in the control tube the mixture remains cloudy. It should be remembered that avirulent pneumococcal cultures are resistant to bile.

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

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

Test for inulin. The pneumococcal culture is inoculated on a medium with inulin. To do this, add 200 ml of sterile distilled water, 18 ml of litmus tincture and 3 g of inulin to 100 ml of bovine serum heated at 56 °C for 30 minutes, and sterilize with running steam for 30 minutes. The crops are incubated at 37 °C for 24 hours. Pneumococcus decomposes inulin, causing the medium to turn red. Viridans streptococcus does not cause redness of the environment.

Test with optochin. The test pneumococcal culture is inoculated on whey broth with optochin at a dilution of 1:100,000 or 1:200,000. Pneumococcus does not grow on such a medium. You can also determine sensitivity to optochin by plating on 10% blood agar containing optochin at a dilution of 1:50,000. The control is to inoculate the 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 soaked in 6 μg of optochin, which are applied to the surface of the medium after inoculation. In pneumococci, a growth inhibition zone of at least 18 mm in diameter forms around the disc.

Virulence test. A daily culture of pneumococcus grown in whey broth is diluted with 1% sterile peptone water (pH - 7.6) or slightly alkaline broth up 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. The organs of a dead mouse are inoculated onto nutrient media and the fingerprint smears are examined microscopically. Highly virulent cultures include pneumococci, which cause the death of mice after the introduction of a 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 reaction. 4 drops of pneumococcal culture are applied to a glass slide. To 1 drop add a drop of antipneumococcal serum type 1, to the 2nd - type II serum, to the 3rd - serum - 111, to the 4th - a drop of normal serum. The mixtures on glass are mixed with a loop and examined under a magnifying glass or microscope at low magnification. In a positive case, agglutination is observed in one of the first three drops. The type of pneumococcus is determined by an agglutination reaction with specific agglutinating sera of the first three fixed types. Cultures that are not agglutinated by these types of sera are classified as X-group. The reaction is set up as follows. Pour 0.5 ml of 18-hour broth culture into test tubes. Then an equal volume of serum is added, diluted with saline in a ratio of 1:5. The 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 tubes are thoroughly shaken and placed in a thermostat at 37 °C for 2 hours, after which a preliminary calculation of the reaction is carried out. Final results are noted after additional storage at room temperature for 20 hours. Agglutination is assessed as four pluses if the contents of the tubes are completely cleared and the agglutination culture is a dense film that does not break when shaken; three pluses if, when the contents of the tube are completely cleared, the agglutinating culture easily breaks into parts; two pluses - if clearing does not occur, particles of the agglutinated culture are clearly visible to the naked eye in the turbid contents of the test tube; with agglutination for one plus, a fine-grained mixture of glued pneumococci is found in the test tube. In case of a negative reaction visible to the eye, agglutination is not observed;

The contents of the test tubes after shaking are uniformly cloudy.

Typing of X-group pneumococci is carried out using group

sera containing a mixture of typical agglutinating sera taken

in equal volumes. Prepare the following group sera by

mixing equal volumes of undiluted standard diagnostic

serums (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 standard sera) due to the difficulty of obtaining it in a sufficiently high titer. Typing is carried out in two stages: first with the help of group sera, and then with individual sera of the group with which a positive reaction was obtained. Serotyping of pneumococci is used primarily for epidemiological studies of the results of specific serotherapy and seroprophylaxis.

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

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

coverslips, to each of them add a drop of undiluted specific antipneumococcal serum (types 1, II, III) and a drop of Loeffler's blue. The drops are thoroughly mixed and covered with a glass slide with a well smeared around the edges with Vaseline. 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 visible. If the result is negative, the capsules are hardly treasured. The swelling reaction is specific and does not give a positive result with other capsular bacteria. I do not use it for examining sputum from patients treated with sulfonamides and antibiotics, because in this case, non-capsular pneumococci can be isolated.

2. Precipitation method. 5-10 ml of sputum is boiled in a water bath until a dense clot is obtained. The clot is ground and a small amount of saline is added and boiled again for several minutes to extract the specific polysaccharide from pneumococci. The suspension is centrifuged, and a ring precipitation reaction is performed with the resulting clear liquid and specific standard sera in precipitation tubes. The appearance of a ring at the interface between the 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 slide. The mixture is mixed and a smear is made, dried in air and, without fixing, examined under a microscope. The background of the preparation is dark smoky; microbial bodies and their capsules are not stained. The preparation prepared according to Burri can be fixed with Nikiforov’s mixture, rinsed with water, and stained with Ziel fuchsin diluted 1:3 for 3-5 minutes. Against the dark background of the smear, unpainted capsules stand out, inside of which there are bacteria of a bright crimson color (Hins method).

Scarlet fever cause various serotypes of beta-hemolytic streptococci that have M-antigen and produce erythrogenin (toxigenic streptococci of serogroup A) - (Streptococcus pyogenes). In the absence of antitoxic immunity, scarlet fever occurs, and in the presence of sore throat.

Clinical picture

 Intoxication - fever, general malaise, headaches.

 Scarlet fever rash is pinpoint, with moderate pressure with a glass spatula the spots are more clearly visible. When pressed harder, the rash gives way to a golden-yellowish tint to the skin. It appears on days 1-3 of illness and is localized mainly on the cheeks, groin, and sides of the body. The skin of the nasolabial triangle remains pale and free of rash. The rash usually lasts 3-7 days, then fades away, leaving no pigmentation. The rash thickens on the bends of the limbs - axillary, elbow, popliteal areas.

 Scarlet tongue - on the 2-4th day of illness, the patient’s tongue becomes distinctly granular, bright red in color, the so-called “raspberry” tongue.

 Sore throat is a constant symptom of scarlet fever. It may be more severe than ordinary sore throat.

 Peeling of the skin - occurs after the rash disappears (14 days from the onset of the disease): in the area of ​​​​the palms and feet it is large-plate, starting from the tips of the fingers; There is pityriasis-like peeling on the body, neck, and ears.

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 oval, slightly elongated, lanceolate-shaped cocci arranged in pairs, resembling a candle flame. They can also be located in short chains, resembling streptococci. Motile, do not form spores, gram-positive.
They are grown on media with added protein: blood, serum, and ascitic fluid. On blood agar, colonies of pneumococci are small, resembling dewdrops, transparent in transmitted light, with a depressed center, surrounded by a zone of incomplete hemolysis, greenish in color, similar to colonies of viridans streptococcus. In liquid media they produce a slight cloudiness, sometimes forming a precipitate. Biochemically they are quite active: they decompose glucose, lactose, maltose, inulin and other carbohydrates to form acid, do not liquefy gelatin, and do not form indole. The breakdown of inulin is a differential diagnostic feature that helps distinguish pneumococci from streptococci, which do not degrade 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, catarrh of the upper respiratory tract, meningitis, endocarditis, joint damage and other diseases.

After an illness, immunity is low-strained, short-term, and type-specific.
Microbiological diagnostics. The materials for the study are sputum, blood, throat swab, and cerebrospinal fluid. Due to the fact that pneumococcus dies quickly, pathological material must be delivered to the laboratory for examination as soon as possible.

Meningococci. 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 prevention.

N.meningitidis (meningococci).

Meningococcus is the causative agent of meningococcal infection - strict anthroponosis with airborne transmission of the pathogen. The main source is media. 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, and produce hemolysin. They have a capsule that is larger in size and has a different structure than that of gonococcus.

Antigenic composition. They have four main antigenic systems.

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

2. Protein antigens of the outer membrane. Based on 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 high toxicity and cause pyrogenic effects.

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

Immunity durable, antimicrobial.

Laboratory diagnostics based on bacterioscopy, culture isolation and its biochemical identification, serological diagnostic methods. The material is inoculated 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 fermentation of glucose and maltose. Belonging to a serogroup is determined by an agglutination test (RA).

Gonococci. Taxonomy, properties. Pathogenesis of gonococcal infection, characteristics of immunity. Principles and methods of laboratory diagnosis of acute and chronic gonorrhea, blenorrhea. RSK Bordet-Gengou, purpose, mechanism, reaction accounting. Prevention of blenorrhea 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 genitourinary tract. The substrate for colonization is the epithelium of the urethra, rectum, conjunctiva of the eye, pharynx, cervix, fallopian tubes and ovary.

Diplococci, easily stained with methylene blue and other aniline dyes, are pleomorphic (polymorphism). They are very picky about cultivation conditions 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 genitourinary tract, and lipopolysaccharide(endotoxin released when gonococci are destroyed). Gonococci synthesize IgAI protease, which breaks down IgA.

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

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

Causative agents of gas 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. The main 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 a causative agent of wound infection - it causes disease when the pathogen enters wounds under anaerobic conditions. It is highly invasive and toxicogenic. Invasiveness is associated with the production of hyaluronidase and other enzymes that have a destructive effect on muscle and connective tissue. 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 foodborne toxic infections (they are based on the action of enterotoxins and necrotoxins).

Features of pathogenesis. Unlike purulent diseases caused by aerobes, with anaerobic infection it is not inflammation that predominates, but necrosis, edema, gas formation in tissues, poisoning with toxins and tissue breakdown products.

Immunity- predominantly antitoxic.

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

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

Clostridia tetanus. Taxonomy. Properties, characteristics of toxins. Pathogenesis of the disease. Descending tetanus. Clinic. Principles and methods of laboratory diagnostics. The purpose of bacteriological research, drugs 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 convulsions of the striated muscles. People and farm animals get sick. Soil, especially contaminated with human and animal excrement, is a constant source of tetanus infection.

The causative agent is C.tetani - a large spore-forming gram-positive rod. The spores are located terminally (type of drumstick), and are mobile due to flagella - peritrichs. Obligatory anaerobe. The spores are very resistant.

Antigenic properties. The pathogen has O- and H-antigens.

Pathogenicity factors. The main factor is the strongest exotoxin. There are two main fractions of it: tetanospasmin (neurotoxin) and tetanolysin (hemolysin). The neurotoxin enters the central nervous system into the areas of myoneural synapses, is transmitted from neuron to neuron in the area of ​​synapses, accumulates in the motor areas of the spinal cord and brain, and 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 starting materials, culture to isolate the pathogen and its identification, and detection of tetanus toxin.

Isolation of the pathogen is carried out according to the standard scheme for anaerobes, using various solid and liquid (Kitt-Tarozzi medium) media, identification 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, most often as part of DTP vaccines (tetanus toxoids, diphtheria and killed pertussis bacilli).

Clostridium botulism. Taxonomy. Properties. Characteristics of toxins, difference from exotoxins of pathogens of other food infections. Principles and methods of laboratory diagnostics. Drugs for specific prevention and treatment.

Botulism is a severe food poisoning associated with the consumption of products contaminated with C.botulinum and characterized by specific damage to the central nervous system. It got its name from Lat. botulus - sausage.

Properties of the pathogen. Large polymorphic gram-positive rods, motile, have peritrichous flagella. The spores are oval and located subterminally (tennis racket). They produce eight types of toxins, differing in antigenic specificity, and accordingly, 8 types of pathogens are distinguished. Among the most important characteristics is the presence or absence of proteolytic properties (hydrolysis of casein, 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 heat labile, although boiling for up to 20 minutes is necessary for complete inactivation. The toxin is quickly absorbed in the gastrointestinal tract, penetrates the blood, selectively acts on the nuclei of the medulla oblongata and ganglion cells of the spinal cord. Neuro-paralytic phenomena develop - swallowing disorders, aphonia, dysphagia, ophthalmo-plegic syndrome (strabismus, double vision, drooping eyelids), paralysis and paresis of the pharyngeal and laryngeal muscles, cessation of breathing and cardiac activity.

Laboratory diagnostics. The principles are common to clostridia.

Treatment and prevention. It is based on the early use of antitoxic serums (polyvalent or, when the type is established, homologous). Prevention is based on a sanitary and hygienic regime when processing food products. Homemade 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 distributed everywhere (water, soil, plants, animals), and is found normally in humans (most often in the intestines, on the skin and mucous membranes). Morphology- gram-negative straight or slightly curved rod, mobile, located singly, in pairs or in short chains in smears. Synthesizes mucus (capsular substance), especially the 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 solid media the phenomenon of rainbow lysis is often observed. By the end of the day due to the synthesis of pigment pyocyanin A blue-green color appears in the culture.

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 jasmine scent. Produces the production of bacteriocins - pyocins.

Antigenic and pathogenic properties. The main antigens of Pseudomonas aeruginosa are group-specific somatic O-antigen and type-specific flagellar H-antigen. O-antigenic complex - an aggregate of LPS with proteins and lipids of the cell wall, has endotoxin properties and is one of the main pathogenicity factors. 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, reminiscent of diphtheria exotoxin), enzymes (collagenase, neuraminidase, proteases).

Laboratory diagnostics. P.aeruginisa received its name for the bluish-green coloring of wound discharge and dressing material. The main diagnostic method is bacteriological. The detection of the pyocyanin pigment is important. Treatment and specific prevention. There is no specific prevention. For food toxic infections and intestinal dysbiosis caused by Pseudomonas aeruginosa, a complex intesti-bacteriophage, which includes a pseudomonas phage, is effective. Among the antibacterial drugs, aminoglycosides, cephalosporins and quinolones are most often used.

Opportunistic gram-negative bacteria - causative agents of purulent-inflammatory processes (Proteus, Klebsiella, miraculous bacillus, 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. They cause opportunistic lesions - hospital-acquired pneumonia, urinary tract infections, diarrhea in newborns. Klebsiella causes mastitis, septicemia and pneumonia in animals and is constantly found on the skin and mucous membranes of humans and animals. Klebsiella are straight, motionless rods 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), heat-labile and heat-stable 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 - ozen.

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 klebsiella, usually in combination with drugs that stimulate the immune system.

Genus Proteus.

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

Proteas break down tyrosine, reduce nitrates, oxidase is negative, catalase is positive. They live in the intestines of many species of vertebrate and invertebrate animals, soil, wastewater, and decaying organic matter. Can cause urinary tract infections in humans, as well as septic lesions in burn patients and after surgery. Quite often they also cause food poisoning. P.vulgaris and P.mirabilis play a role most often in pathology.

Cultural properties. Proteas grow on simple media over a wide temperature range. Optimal pH is 7.2-7.4, temperature is from +35 to 37 degrees Celsius. Colonies of proteas in the O-form are round, semi-digital and convex, while the H-forms give continuous growth. The growth of proteas is accompanied by a putrid odor. The swarming phenomenon is characteristic; H-forms give characteristic creeping growth on the MPA 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. On the MPB, diffuse turbidity of the medium with a thick white sediment at the bottom is noted.

Pathogenicity factors. These include cell wall LPS, the ability to “swarm”, fimbriae, proteases and urease, hemolysins and hemagglutinins.

Laboratory diagnostics. The main method is bacteriological. Differential diagnostic media (Ploskirev), enrichment media and MPA according to the Shushkevich method are used. Treatment. For intestinal dysbacteriosis associated with Proteas (colitis), you can use Proteus phage and drugs that contain it (intestiphage, coliproteus bacteriophage).

"Wonderful stick" (Serratia marcescens), a type of bacteria from among pigment microorganisms. Gram-negative motile (peritrichous) non-spore-bearing rods. By type of metabolism - facultative anaerobe. On the surface of the agar it forms smooth or granular dark and bright red colonies with a metallic sheen. Lives in soil, water, and food. Developing on bread (at high humidity) and in milk, it turns them red; such products are not allowed for sale. 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 opportunistic ones.

Escherichia is 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 is E. coli (Escherichia coli) - the most common causative agent of infectious diseases caused by enterobacteria. This pathogen is an indicator of fecal contamination, especially in water.

Cultural properties. On liquid media, E. coli produces diffuse turbidity; on solid media, it forms S- and R-form colonies. On Endo, the main medium for Escherichia, lactose-fermenting E. coli form intensely red colonies with a metallic sheen; non-fermenting ones form pale pink or colorless colonies with a darker center; on Ploskirev’s medium they are red with a yellowish tint; on Levin’s medium they are dark blue with a metallic sheen. .

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

The main pathogenicity factors of diarrheagenic E.coli.

1. Factors of adhesion, colonization and invasion associated with pili, fimbrial structures, and 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 promote the development of diarrhea) and enterocytotoxins (act on the cells of the intestinal wall and capillary endothelium).

3. Endotoxin (lipopolysaccharide).

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

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

Enterotoxigenic E.coli have a high-molecular heat-labile toxin, similar in action to cholera, causing cholera-like diarrhea (gastroenteritis in young children, travelers' diarrhea, etc.).

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

Enteropathogenic E.coli- the main causative agents of diarrhea in children. The lesions are based on the adhesion of bacteria to the intestinal epithelium with damage to 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, poorly studied.

Laboratory diagnostics. The main approach is the isolation of a pure culture on differential diagnostic media and its identification by antigenic properties. RA is diagnosed 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, every year in the world there are more than 500 thousand pneumonia caused by pneumococci, especially often in children and the elderly. In addition to pneumonia, this microbe causes meningitis, endocarditis, peritonitis, otitis media, rhinitis, sinusitis, sepsis, creeping corneal ulcers and a number of other diseases. To carry out laboratory diagnostics, bacterioscopic, bacteriological and biological methods are used. The material for examination is sputum, pus, blood, cerebrospinal fluid, mucus from the mouth and nasopharynx, discharge from the maxillary sinus, eyes and ears. It is important to immediately send the material to the laboratory and examine it very quickly, since pneumococci are susceptible to autolysis.

Bacterioscopic examination

Bacterioscopic examination of material (except blood) comes down to making two smears. One of them is stained with Gram, the second with 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 to conclude 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 normal microflora. Therefore, it is necessary to inoculate clinical material and isolate a pure culture.

Bacteriological research

For sepsis at the patient's bedside, inoculate 10 ml of blood into a vial containing 100 ml of whey or sugar broth, incubate for 18-20 hours at 37 ° C, then inoculate on blood agar, isolate and identify a pure culture. For meningitis, the cerebrospinal fluid is centrifuged and the sediment is inoculated onto 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. It is not advisable to culture sputum or pus on nutrient media, since the presence of saprophytic microflora suppresses the growth of S. pneumoniae. It is better to introduce the test material into the abdominal cavity of white mice. A bioassay is a fast, reliable and accurate method for isolating a pure culture of pneumococci. White mice are very sensitive to these bacteria and within 10-12 hours after infection, pneumococci penetrate the blood and parenchymal organs, causing sepsis. Cultivation of blood from the heart or pieces of internal organs during autopsy of animals allows one 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 by bile can be detected if 0.5 ml of bile is added to 1 ml of broth culture. After 15-20 minutes in the thermostat, complete lysis of bacterial cells occurs. To determine pneumococcal serovars (there are currently 85 of them), the glass agglutination reaction with standard sera or the phenomenon of “capsule swelling” is used. In the presence of homologous serum, the pneumococcal capsule swells greatly. It is even better to perform serotyping using commercial reagents in latex agglutination or coaglutination reactions, through which capsular antigens are revealed. Among streptococci, the Enterococcus genus 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 as part of the normal microflora of the skin of the perineum, genitourinary organs, oro- and nasopharynx. They can cause suppuration of wounds, bacteremia, damage to the urogenital system, especially in patients with long-functioning catheters, foodborne toxic infections, dysbacteriosis of the intestinal tract, and, less commonly, endocarditis. In smears from the test material, enterococci are located 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 and are grayish in color. On blood agar, incomplete or complete hemolysis occurs around the colonies. Unlike viridans 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 crops 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 person who died of rabies (1881).
Morphology and tinctorial properties. Pneumococci (Fig. 67 and 68 in the inset) are paired cocci with an elongated lancet-like shape. Therefore, they are otherwise called lanceolate diplococci. Forming short chains, pneumococci become similar to streptococci, and therefore II. F. Gamaleya named them Streptococcus lanceolatus. The cell size ranges from 0.5X0.75 to 1X1.5 μm. They do not have spores or flagella. A distinctive feature of pneumococcus is the formation of a capsule, which can be clearly expressed in pathological materials (sputum, blood, etc.). When cultivated on nutrient media, the capsule is lost. Pneumococci easily accept aniline dyes and stain positively on the Gram.
Cultural and biochemical properties.

Rice. 68. Pneumococci in sputum smear.

Pneumococci are aerobes and facultative anaerobes. Temperature optimum is about 37°. They grow on media containing animal protein (blood or serum agar, ascitagar).
After 24 hours, small colonies are formed on the surface of the agar, reminiscent of streptococcal colonies, but smaller and more transparent.
On slanted agar, with abundant inoculation, a very delicate transparent coating is obtained, consisting of tiny, non-merging colonies; on broth, there is slight turbidity and a small flaky sediment.
Freshly isolated strains do not grow on gelatin. Old laboratory strains of pneumococci can produce small whitish colonies already at 18-22°. Gelatin is not liquefied.
They grow well in milk, curdling it to form acid.
On blood agar, a zone of incomplete hemolysis with a greenish-brown coloration of the medium forms around the colonies.

Rice. 67. Pneumococci in pure culture from broth.

Pneumococci degrade sucrose, raffinose and lactose. The most important feature is the decomposition of inulin. Most streptococci do not have this property. Virulent pneumococci are bile soluble.
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 among different pneumococci (type antigens). Based on these typical antigens, using the agglutination and precipitation reaction, all pneumococci are divided into three main groups (I, II, III) and a fourth group (X-group). The X-group includes more than 70 types.
Resistance. On artificial nutrient media, pneumococci die quickly (4-7 days). Under a layer of petroleum jelly in liquid and semi-liquid media containing protein, they remain viable for 3-12 months.
Pneumococci tolerate drying well: they persist in dry sputum in diffuse light for up to 2 months. When heated to 52-55° they die in 10 minutes, at 60° they die even faster. In a solution of carbolic acid (3%), pneumococci die within 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. Among 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 symptoms of sepsis. Upon autopsy, fibrinous exudate is found at the injection site; the spleen is enlarged and hyperemic.
Pathogenesis and diseases in humans. The entry point for infection is usually the mucous membrane of the pharynx. The introduction of pneumococci into the body and their penetration into the lung tissue can apparently occur both through the lymphatic and circulatory system, and directly through the branches of the bronchi. The most common disease is lobar 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, and severe agitation. Subsequently, a cough appears with characteristic rusty-red sputum. In the lungs, a process is observed that often involves one, less often two or three lobes.
The sources of infection are the sick person and the bacteria carrier. Infection from the outside occurs both aerogenously - 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 cannot be excluded, and factors that weaken the body's resistance, such as hypothermia, play a significant role.
In addition to lobar pneumonia, pneumococci cause inflammation of the middle ear, meninges (meningitis), as well as the mucous membrane of the nose and air sinuses, tonsillitis, creeping corneal ulcers and inflammation of the lacrimal sac.
Immunity. Having pneumonia does not provide immunity. The disease can recur several times. This is explained by the presence of many types of pneumococci and the fact that past pneumonia increases the body's sensitivity to pneumococci.
The serum of those who have recovered contains antibodies (agglutinins, etc.).
By the time of the crisis with pneumonia, the concentration of antibodies in the blood reaches a significant titer, and phagocytosis increases sharply (I. Ya. Chistovich). Based on these data, immunity in pneumonia should be considered primarily as phagocytic, in which antibodies (bacteriotropins) play a major role.
Microbiological diagnostics. The materials for research in pneumococcal diseases are sputum, blood and pus taken from various lesions, and 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 foreign microflora, which, when directly inoculating the material on nutrient media, makes it difficult to isolate pneumococcus.
Smears from sputum, pus, etc. are Gram stained. Under a microscope, lanceolate diplococci surrounded by a capsule are found, Gram stained positively.
To isolate cultures, inoculate them on blood agar or ascig agar. After 24-48 hours of growth at 37°C, in the presence of pneumococcus, characteristic colonies appear. Colonies are sown on slants of whey or ascites agar, and the isolated culture is tested for solubility in bile and the ability to decompose inulin.
Infecting a white mouse is the most reliable way to isolate a pneumococcal culture. Material from a patient or corpse (sputum, pus, a piece of 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 into 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, the broth is inoculated onto blood agar and the isolated pure culture is identified by morphological and biochemical characteristics.
Specific therapy and chemotherapy. Currently, sulfonamide drugs and antibiotics (penicillin, biomycin, tetracycline, etc.) are used with great success to treat lobar pneumonia.

The genus Streptococcus includes: Streptococcus pyogenes (hemolytic) and Streptococcus pneumoniae (pneumococcus). Streptococci were first discovered by Billroth (1874) and 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 microns, but 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. The length of the chains is different. On a solid nutrient medium the chains are usually short, on a liquid medium they are long. Streptococci are immobile and do not have spores (see Fig. 4). Freshly isolated cultures sometimes form a capsule. In ultrathin sections, a microcapsule is visible, under which there is a three-layer cell wall and a three-layer cytoplasmic membrane. Gram positive.

Cultivation. Streptococci are facultative anaerobes. They grow at a temperature of 37° C and a pH of 7.6-7.8. The optimal media for their growth are media containing blood or blood serum. On solid nutrient media, colonies of streptococci are small, flat, cloudy, and grayish in color. Some species of streptococci form hemolysis on blood agar. 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 produce hemolysis.

In sugar broth, streptococci grow with the formation of near-wall and 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 weakly expressed. They curdle milk, but do not liquefy gelatin.

Toxin formation. Streptococci produce 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 - determines 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.

Various antigens have been found in streptococci. The cytoplasm of the cell contains a specific antigen of 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.

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

Group A includes 70 types. This group includes most streptococci that cause various diseases in humans. Group B includes mainly streptococci that are opportunistic for humans. Group C includes streptococci pathogenic for humans and animals. Group D consists of streptococci that are non-pathogenic to humans, but this group also 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 classified as opportunistic 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 quite stable in the environment. At a temperature of 60°C they die after 30 minutes.

They remain in dried pus and sputum for months. Normal 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 sensitive to pathogenic streptococci. Among laboratory animals, rabbits and white mice are sensitive. However, streptococci that are pathogenic for humans are not always pathogenic for experimental animals.

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

Transmission routes. Airborne and airborne dust, sometimes foodborne, possibly household contact.

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, fasting, overwork, etc.) can lead to autoinfections.

Pre-sensitization is of great importance in the pathogenesis of streptococcal infections - as a consequence of a previously suffered disease of streptococcal etiology.

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

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

Streptococci cause various acute and chronic infections in humans, both with the formation of pus and non-suppurative ones, 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. The nature of immunity is antitoxic and antibacterial. Post-infectious antimicrobial immunity is low-strength. This is explained by the weak immunogenicity of streptococci and a large number of serovars that do not provide cross-immunity. In addition, with streptococcal diseases, 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 prevention has not been developed.

Treatment. Antibiotics are used. Penicillin, to which streptococci have not become resistant, is often used, as well as erythromycin and tetracycline.

The importance of streptococcus in the etiology of rheumatic carditis. The pathogenesis of rheumatic carditis has not been studied enough. But a number of facts speak in favor of the role of streptococcus in the development of this disease:

1. In patients with rheumatic carditis, B-hemolytic streptococcus is cultured from the throat.

2. Rheumatism often occurs after suffering from tonsillitis, tonsillitis, pharyngitis, which sensitize the body.

3. In the blood serum of patients, antistreptolysin and antistreptohyaluronidase are detected - antibodies to streptococcal enzymes and toxins.

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 carditis.

Prevention of exacerbations of rheumatic carditis comes down to the prevention of streptococcal diseases (for example, in the spring and autumn, a preventive course of penicillin is administered). Treatment comes down to the use of antibacterial drugs - penicillin.

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

Those who have recovered from the disease develop immunity - stable, antitoxic. Its tension is determined by staging the Dick reaction - intradermal injection of an erythrogenic toxin. In those who are not sick, hyperemia and swelling occur around the injection site, which is characterized as a positive reaction (absence of antitoxin in the blood serum). In those who have recovered from the disease, such a reaction is absent, since the antitoxin they have formed neutralizes the erythrogenic toxin.

Prevention. Isolation, hospitalization. Gamma globulin is administered to contact, weakened children. Specific prevention has not been developed.

Treatment. Penicillin and tetracycline are used. In severe cases, antitoxic serum is administered.

Purpose of the study: identification of streptococcus and determination of its serovar.

Material for research

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

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

3. Pus (abscess).

4. Urine (nephritis).

5. Blood (suspicion of sepsis; endocarditis).

Basic research methods

1. Bacteriological.

2. Microscopic.

Progress of the study

Second day of the study

Remove the cups from the thermostat and inspect. If there are suspicious colonies, smears are made from some of them, stained with Gram and microscopically examined. If streptococci are detected 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 into Martin broth with 0.25% glucose to determine the serological group in the Lensfield precipitation reaction. Test tubes and bottles are placed in a thermostat and left until the next day.

Third day of the study

The crops are removed from the thermostat, the purity of the culture is checked on a slanted agar, smears are made, stained with Gram and microscopically examined. If there is a pure culture of streptococcus, inoculate on Hiss media (lactose, glucose, maltose, sucrose and mannitol), milk, gelatin, 40% bile and place in a thermostat.

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

Setting up the precipitation reaction according to Lensfield. A daily culture grown in Martin broth is poured into several centrifuge tubes and centrifuged for 10-15 minutes (3000 rpm).

The supernatant liquid is poured into a jar with a disinfectant solution, and the sediment is filled with a sterile isotonic sodium chloride solution and centrifuged again. 0.4 ml of 0.2% hydrochloric acid is added to the sediment collected from all centrifuge tubes. Then the test tube is placed in a water bath and boiled for 15 minutes, shaking occasionally. After boiling, the resulting suspension is centrifuged again. The antigen is extracted into the supernatant, which is poured into a clean test tube and neutralized with a 0.2% sodium hydroxide solution to a pH of 7.0-7.2. Bromothymol blue (0.01 ml of 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 are poured into 5 precipitation tubes, which are prepared by immunizing rabbits (see Chapter 19). Serum A is added to the 1st tube, serum B to the 2nd, serum C to the 3rd, serum D to the 4th, isotonic sodium chloride solution (control) to the 5th. After this, using a Pasteur pipette, carefully layer the resulting extract (antigen) into all test tubes along the wall.

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

Fourth day of research

The results are recorded (Table 25).

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

Control questions

1. What basic laboratory testing methods for identifying streptococci do you know?

2. Why use the Lensfield precipitation reaction?

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

Obtain antistreptococcal serums A, B, C, D and isotonic sodium chloride solution from the teacher. Set up the precipitation reaction, show the results to the teacher and sketch them.

Culture media

Blood agar(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 heated slowly in a water bath (at a temperature of 40-50 ° C). A 10% sodium carbonate solution (baking soda) is added to the melted gelatin and the pH is set to 7.0. Then immediately filter 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 6-8 ml tubes 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.

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

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

Wednesday Kitta - 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, reminiscent of a candle flame (see Fig. 4). The size of pneumococci is 0.75-0.5 × 0.5-1 microns, they are located in pairs. In liquid nutrient media they often form short chains, becoming similar to streptococci. Preumococci are immobile, do not have spores, and in the body form a capsule surrounding both cocci. The capsule contains a heat-resistant substance antifagin (protecting 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 older cultures.

Cultivation. Pneumococci are facultative anaerobes. They 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, so they grow only on media with the addition of native protein (blood or serum). On serum agar they form small, delicate, rather transparent colonies. On blood agar, moist colonies of greenish-gray color 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 quite pronounced saccharolytic activity. They break down: lactose, glucose, sucrose, maltose, inulin to form acid. Mannitol is not fermented. Their proteolytic properties are weakly expressed: they curdle milk, do not liquefy gelatin, and do not form indole. Pneumococci dissolve in bile. The breakdown 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, and 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. Based on the polysaccharide antigen, all pneumococci are divided into 84 serovars. Among those pathogenic for humans, serovars I, II, and III are most common.

Resistance to environmental factors. Pneumococci belong to the group of unstable microorganisms. A temperature of 60° C kills 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. They can be stored on a nutrient medium for no more than 5-6 days. Therefore, when cultivating, it is necessary to reseed every 2-3 days. Conventional solutions of disinfectants: 3% phenol, sublimate in 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. The natural host of pneumococci is humans. However, pneumococci can cause disease 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 bacteria carrier.

Transmission routes. Airborne droplets, maybe airborne dust.

Entrance gate. Mucous membrane of the upper respiratory tract, eyes and ears.

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

1) lobar pneumonia;

2) creeping corneal ulcer;

The most common disease is lobar pneumonia, which affects one, less often two or three lobes of the lung. The disease is acute, accompanied by high fever and 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 prevention has not been developed.

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

Control 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 do pneumococci cause?

Microbiological examination

Purpose of the study: identification of pneumococcus.

Material for research

1. Sputum (pneumonia).

2. Mucus from the throat (sore throat).

3. Discharge from an ulcer (creeping corneal ulcer).

4. Discharge from the ear (otitis media).

5. Pus (abscess).

6. Pleural punctate (pleurisy).

7. Blood (suspicion of sepsis).

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

Basic research methods

1. Microscopic.

2. Microbiological.

3. Biological.

Progress of the study

Biological sample. A little (3-5 ml of sputum) is emulsified in a sterile broth, 0.5 ml of this mixture is injected intraperitoneally into a white mouse. After 6-8 hours, the mouse shows signs of 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 with Gram and examined under a microscope. To isolate a pure culture, the exudate is inoculated onto serum agar. If the mouse dies or becomes ill, blood from the heart is cultured onto serum agar to isolate a pure culture. The crops are placed in a thermostat.

Accelerated method for determining the type of pneumococcus(microagglutination reaction). 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 is added to the second, type III is added to the third, and isotonic sodium chloride solution (control) is added to the fourth.

Type I and II serums 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 fuchsin. If the result is positive, microbial crowding (agglutination) is noted in one of the drops.

Second day of the study

The cultures are removed from the thermostat, examined, and smears are made from suspicious colonies. If gram-positive lanceolate diplococci are present in the smears, 2-3 colonies are isolated on a serum agar slant to obtain a pure culture. The crops are placed in a thermostat. Smears are made from the broth, stained with Gram and examined under a microscope.

Third day of the study

The crops are removed from the thermostat. They check the purity of the culture - make smears, Gram stain and microscope. If there are gram-positive lanceolate diplococci in the isolated culture, the isolated culture is identified by culturing:

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

2) on a medium with inulin;

3) on a medium with optochin;

4) perform a bile test.

Inulin test. The culture under study is sown 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 inoculated on 10% blood agar containing optochin 1:50000. Pneumococci, unlike streptococci, do not grow on media containing optochin.

Bile test. 1 ml of the test broth culture is poured into agglutination tubes. A drop of rabbit bile is added to one of them, the second 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 bile sample can be performed on a solid nutrient medium. To do this, a grain of dry bile is applied to a colony of pneumococci grown in dishes with agar and serum - the colony dissolves and disappears.

Fourth day of research

The results are recorded (Table 26).

Note. j - 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. A 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. A culture that does not cause the death of mice is avirulent.

Control questions

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

2. Which animal is most sensitive to pneumococcus?

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

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

5. How to determine the virulence of pneumococci?

Exercise

Make a diagram of the sputum examination, indicating its stages by day.

Culture media

Serum agar(see chapter 7).

Whey broth(see chapter 7).

Blood agar(see chapter 7).

Hiss media(dry).

Sample medium with inulin. 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 days in a row. Bile broth (see Chapter 7).