Microbiological diagnosis of streptococcal infections. streptococcal infections
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QUESTIONS AND ANSWERS
MICROBIOLOGY
"PRIVATE MICROBIOLOGY"
Questions and answers
Microbiology
Manual for self-preparation for the final lesson in the section
"Private microbiology"
Version 1.00
Editor-in-Chief and Editor-in-Chief esclkm ([email protected])
Questions 1-43 scored esclkm and Vano
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Used Books:
- Lecture notes;
- Computer version of the lectures of Kaskevich L.I.;
- Borisov.
- and other available literature
1. Staphylococci, general characteristics. role in human pathology. Pathogenicity factors and mechanisms of pathogenesis of staphylococcal infections. Microbiological diagnostics. Prevention and treatment. 5
2. Streptococci, classification. General characteristics. pathogenicity factors. Antigenic structure. Pathogenesis, immunity, microbiological diagnosis of streptococcal infections. 6
3. Classification of Neisseria. Meningococci, general characteristics. Meningococcal infections, mechanisms of pathogenesis, immunity, diagnostic methods, prevention. IDS. 8
4. Gonococci, general characteristics. Mechanisms of pathogenesis and immunity. Microbiological diagnosis of acute and chronic gonorrhea. 9
5. General characteristics of the Enterobacteriaceae family. 11
6. General principles of bacteriological diagnosis of acute intestinal infections (AII). Nutrient media for enterobacteria. Classification, principles of operation, application. eleven
7. Materials for research in AEI: methods of taking and nature of the material depending on the clinical form of the disease and the stage of pathogenesis. 12
8. General principles of serological diagnosis of acute intestinal infections. 13
9. Escherichia coli, general characteristics. The biological role of Escherichia coli. Diseases caused by Escherichia. 13
10. Salmonella. General characteristics. Genus representatives. Serological classification according to Kaufman-White. Molecular biological typing. 14
11. Causative agents of typhoid fever, paratyphoid A and B, general characteristics. Phage typing. Vi-antigen and its significance. 15
12. Mechanisms of pathogenesis and methods of microbiological diagnosis of typhoid fever and paratyphoid fever. 15
13. Immunity in typhoid fever. Serological diagnosis of typhoid fever and paratyphoid fever. specific prophylaxis. 16
14. Etiology of food intoxications and toxic infections of a bacterial nature. Materials and diagnostic methods. 16
15. Salmonellosis. Characteristics of pathogens and diagnostic methods. Nosocomial salmonellosis. 17
16. Causative agents of dysentery. Classification. Characteristic. Pathogenesis, immunity to dysentery. Methods of microbiological diagnostics of acute and chronic dysentery. 18
17. Klebsiella. Classification, general characteristics. Pathogenesis, immunity, methods of microbiological diagnostics of Klebsiellosis. 19
18. Pseudomonas aeruginosa, general characteristics, pathogenicity factors. role in human pathology. 19
19. Causative agents of intestinal yersiniosis, general characteristics. Pathogenesis. Methods for the diagnosis of yersiniosis. 20
20. Causative agent of diphtheria, general characteristics. Differences from non-pathogenic corynebacteria. Mechanisms of pathogenesis. Methods of microbiological and molecular biological diagnostics of diphtheria. 21
21. Diphtheria toxin and its properties. Anatoxin. Immunity in diphtheria and its nature. Determination of the intensity of antitoxic immunity. Specific immunotherapy and specific prophylaxis. 22
22. Pertussis causative agent, general characteristics. Differentiation with the causative agent of parapertussis. Pathogenesis, immunity. Microbiological diagnostics. Specific prophylaxis of whooping cough. 23
23. General characteristics of causative agents of tuberculosis. Pathogenesis, immunity, diagnostic methods and specific prevention of tuberculosis. Mycobacteriosis. 24
24. The causative agent of leprosy. Characteristics, pathogenesis, immunity of the disease. 26
25. Especially dangerous infections (SDI). Classification Basic rules for the mode of operation, taking, sending infectious material during the OOI. General principles for diagnosing ASI .. 27
26. Causative agents of cholera. Systematics. General characteristics. Differentiation of biovars. Pathogenesis, immunity, specific prevention. Methods of microbiological diagnostics. 28
27. Plague causative agent, general characteristics. plague pathogenesis. Immunity, prevention. 29
28. The causative agent of anthrax, characteristics. Pathogenesis, immunity, specific prevention of anthrax. 29
29. The causative agent of tularemia, general characteristics. Pathogenesis. Immunity. specific prophylaxis. 30
30. Causative agents of brucellosis, general characteristics. Differentiation of Brucella species. Pathogenesis. Immunity. specific prophylaxis. 31
31. Spirilla family. Campylobacter, characteristics, role in human pathology. Helicobacter. 31
32. Classification and general characteristics of anaerobes. Clostridia. Bacteroides, peptococci and other non-spore-forming anaerobes. pathogenicity factors. role in human pathology. 33
33. The causative agent of tetanus, general characteristics. pathogenesis and immunity. Specific therapy and prevention. 34
34. Causative agents of gas gangrene, general characteristics. Pathogenesis. Specific prevention of gas gangrene. 34
35. The causative agent of botulism, general characteristics. Pathogenesis. Specific therapy and prevention of botulism. Clostridial gastroenteritis. 35
36. Methods for diagnosing anaerobic infections. 36
37. Classification and general characteristics of spirochetes. 36
38. Classification of treponema and treponematoses. Characteristics of the causative agent of syphilis. Pathogenesis, immunity, diagnostic methods of syphilis. 37
39. Leptospira. General characteristics. Leptospirosis pathogenesis, immunity, specific prevention. Microbiological diagnosis of leptospirosis. 38
40. Borrelia, general characteristics. Pathogenesis, immunity in relapsing fever. Microbiological diagnostics. The causative agent of Lyme borreliosis. 38
41. Systematic position and characteristics of rickettsiae. causative agents of rickettsiosis. Pathogenesis, immunity, methods of diagnosing typhus. 39
42. Characteristics of chlamydia. Causative agents of trachoma, ornithosis, respiratory and urogenital chlamydia. Mechanisms of pathogenesis and methods of diagnosis of chlamydia. 41
43. General characteristics of mycoplasmas. role in human pathology. Methods for the diagnosis of mycoplasmosis. 42
Staphylococci, general characteristics. role in human pathology. Pathogenicity factors and mechanisms of pathogenesis of staphylococcal infections. Microbiological diagnostics. Prevention and treatment.
DOMAIN → Bacteria; TYPE → Firmicutes; CLASS → Vasilli; ORDER → Vasillalles; FAMILY → Staphylococcaceae; genus → Staphylococcus; SPECIES → Staphylococcus species;
The genus Staphylococcus has 28 species, of which 14 live on the skin and mucous membranes. Some species cause disease in humans, most often these are:
S. aureus(golden),
S. epidermidis(epidermal),
S.saprophiticus(saprophytic).
Morphology.
Spherical shape, cluster-like arrangement (Greek - staphylos - bunch). There is no dispute. Motionless. Gram-positive.
facultative anaerobes. Chemoorganotrophs. Grow on normal media, can grow in the presence of 6-10% NaCl. The colonies are pigmented.
Biochemically active. Catalase positive. Oxidase negative. Contains cytochromes.
They live on the skin and mucous membranes of humans and animals. There are various ecological options. Hospital ecovars of pathogens have special properties.
Sustainability
The most resistant bacteria that do not form spores. They tolerate drying well (up to 50 days at room temperature). UV kills in 10-12 hours, boils in seconds
Resistant to NaCl, fatty acids, acidic pH. (provides nourishment to the skin)
Nosocomial strains (especially S. aureus) are characterized by increased resistance to antibiotics, antiseptics and disinfectants.
Pathogenic factors:
1) Capsule → Inhibition of phagocytosis
2) Protein A → Interaction with the Fc fragment of antibodies, sensitization
3) Peptidoglycan → Stimulation of the production of endogenous pyrogens, leukocyte chemoattractant (formation of abscesses)
4) Teicic acids → Bind fibronectin
5) Membranotoxins, or hemolysins (alpha, beta, gamma, delta toxins), leukocidin → Toxic to many cells, including erythrocytes, leukocytes, macrophages, fibroblasts. Alpha toxin is an example of a pore-forming toxin.
6) Exfoliative toxin (A, B) → Causes the "scalded skin" syndrome, destroying cell contacts - desmosomes in the granular layer of the epidermis. Superantigen
7) Toxin of toxic shock syndrome → Neurotropic, vasotropic effects. Superantigen
8) Enterotoxins (A-E) → Action on enterocytes (food intoxication). Neurotropic effects Superantigen.
9) Plasmocoagulase → Transfer of fibrinogen to fibrin, preventing contact with phagocytes
10) Hyaluronidase → Destruction of connective tissue
11) Lipase, lecithinase → Hydrolysis of lipids, lecithin
12) Fibrinolysin → Destruction of fibrin clots
13) Deoxyribonuclease → DNA cleavage, pus liquefaction
14) Keratinoid enzymes → Inactivation of bactericidal oxygen species
15) Resistance to NaCl, fatty acids → Reproduction in sweat and sebaceous glands.
Transmission mechanisms: Contact (main), Aerosol, fecal-oral
Infection can take place both exogenously and endogenously.
Features of pathogenesis. Staphylococci are opportunistic pathogens. The development of the disease and its clinical form depend on a number of conditions: impaired immunity; damage to the covers; properties of the pathogen (a set of pathogenicity factors), its quantity, entrance gate.
The development of the pathological process is possible in any biotope.
Staphylococcal infections often develop:
1) against the background of other diseases (secondary infections), for example, after influenza or other viral infections
2) in medical institutions (nosocomial infections)
Diseases: more than 100 nosological forms. The main pathogen is S. aureus
Local suppurative processes
Diseases of the bones and joints
Infections of internal organs: pneumonia (in children and the elderly), kidney damage (pyelonephritis), cystitis (often S. epidermidis and S. saprophiticus)
· Peritonitis. After operations on the abdominal organs.
CNS lesions
· Sepsis. Septicopyemia.
· Toxic shock syndrome.
Syndrome of "scalded babies". In newborns (infection through the umbilical vein), there is a detachment of the skin with blisters, intoxication. In older children - the syndrome of "scalded skin" (erythema, blisters, intoxication).
· Food poisoning.
Prevention principles
specific
A) Staphylococcal toxoid.
b) Associated Staphylo-Protein-Pseudomonas aeruginosa vaccine ( Contains concentrated toxoids of staphylococcus aureus and Pseudomonas aeruginosa, cytoplasmic antigens of staphylococcus and chemical proteus vaccine.
Non-specific prophylaxis
1) Compliance with the sanitary and anti-epidemic regime
2) Monitoring of pathogens and their drug resistance.
3) Restrictive measures.
a) invasive procedures - carried out according to strict indications.
b) immunosuppressive drugs and methods (immunosuppressants, antibiotics, chemotherapy, radiotherapy) - also according to strict indications.
Streptococci, classification. General characteristics. pathogenicity factors. Antigenic structure. Pathogenesis, immunity, microbiological diagnosis of streptococcal infections.
DOMAIN → Bacteria; TYPE → Firmicutes; CLASS → Vasilli; ORDER → Lactobacillales;
FAMILY → Streptococcaceae; GENUS → Streptococcus; SPECIES → Streptococcus species (up to 50 species)
The main features of the genus Streptococcus:
1. Cells of spherical or oval (lanceolate) shape 0.5-2.0 microns. Arranged in a chain or in pairs.
2. Motionless, no dispute. Some species have a capsule.
3. Gram-positive. Chemoorganotrophs, demanding on nutrient media, facultative anaerobes
4. Ferment sugars to form acid, but this is not a reliable differentiator within the genus
5. Unlike staphylococci, there is no catalase activity and cytochromes.
6. Usually, erythrocytes are lysed. According to hemolytic properties: beta (complete), alpha (partial), gamma (none). Capable of forming L-shapes.
Antigenic structure of the genus Streptococcus:
Cell wall polysaccharide on the basis of which they are divided into 20 groups, denoted by Latin letters. Pathogenic species belong primarily to the A. group and less often to other groups. There are species without a group antigen.
Type-specific protein antigens (M, T, R). M-protein is possessed by pathogenic species. In total, there are over 100 serotypes, most of which belong to group A streptococci. The M-protein is located superficially in the form of filamentous formations braiding the cell - fimbriae.
Capsular streptococci have capsular antigens of various chemical nature and specificity.
There are cross-reactive antigens
Group A streptococci are part of the nasopharyngeal microflora and are not normally found on the skin. The most pathogenic for humans are hemolytic streptococci of group A, belonging to the species S.pyogenes
Group A streptococci cause infections at any age and are most common in children between 5 and 15 years of age.
Group A pathogenicity factors
1) Capsule (hyaluronic acid) → Antiphagocytic activity
2) M-protein (fimbriae) → Antiphagocytic activity, destroys complement (C3b), superantigen
3) M-like proteins → Bind IgG, IgM, alpha2-macroglobulin
4) F-protein → Microbe attachment to epithelial cells
5) Pyrogenic exotoxins (erythrogenins A, B, C) → Pyrogenic effect, increased HRT, immunosuppressive effect on B-lymphocytes, rash, superantigen
6) Streptolysins: S (oxygen stable) and
O (oxygen sensitive) → Destroy white blood cells, platelets, red blood cells. Stimulate the release of lysosomal enzymes.
7) Hyaluronidase → facilitates invasion by disintegrating connective tissue
8) Streptokinase (fibrinolysin) → Destroys blood clots (thrombi), promotes the spread of microbes in tissues
9) DNase → Demolymerizes extracellular DNA in pus
10) C5a-peptidase → Destroys the C5a component of complement, chemoattractant
The pathogenesis of infections caused by S.pyogenes:
It most commonly causes a localized infection of the upper respiratory tract or skin, but can infect any organ.
Most frequent suppurative processes: abscesses, phlegmon, tonsillitis, meningitis, pharyngitis, sinusitis, frontal sinusitis. lymphadenitis, cystitis, pyelitis, etc.
Local inflammation leads to leukocytolysis in the peripheral blood, followed by tissue infiltration with leukocytes and local pus formation.
Non-suppurative processes caused by S.pyogenes:
Erysipelas,
streptoderma,
Impetigo,
Scarlet fever,
Rheumatoid infection (rheumatic fever),
Glomerulonephritis,
toxic shock,
Sepsis etc.
Treatment of streptococcal infections:It is carried out primarily with antibiotics: cephalosporins, macrolides, lincosamides
Prevention of streptococcal infections:
General sanitary and hygienic measures, prevention and treatment of acute local streptococcal infections are important. To prevent relapse (rheumatic fever) - antibiotic prophylaxis.
An obstacle to the creation of vaccines is a large number of serotypes, which, taking into account the type-specificity of immunity, makes their production hardly realistic. In the future, the synthesis of M-protein polypeptides and the hybridoma route for its production.
Associated drugs are produced abroad for the immunotherapy of infections caused by opportunistic microbes - from 4 to 19 types. These vaccines include S.pyogenes and S.pneumoniae.
Immunoprophylaxis of pneumococcal infections - a vaccine from polysaccharides of 12-14 serovariants, which often cause diseases.
A vaccine against caries is being developed.
streptococci(Streptococcus) - causative agents of a large number of infections in humans and animals, they cause erysipelas, sepsis and purulent infections, scarlet fever, tonsillitis. There are non-pathogenic varieties that live in the human mouth and intestines. Anaerobic strains of streptococci have a low degree of activity, and they are usually found in the human oral cavity and digestive tract. In some cases, they cause chronic inflammatory processes and are the causative agents of wound infections. Significantly more important in the pathogenesis of human streptococcal infections are facultative anaerobes, which are divided according to the nature of hemolysis on agar with blood into the following types:
- beta-hemolytic streptococci;
- alpha-hemolytic streptococci;
- gamma-hemolytic streptococci that do not cause visible hemolysis on solid nutrient media with blood.
The greatest pathogenicity possess beta-hemolytic streptococci, which are the causative agents of most streptococcal infections in humans. The pathogenicity of alpha-hemolytic streptococci is less pronounced. They are found in the pharyngeal mucus of healthy people, but in some cases in chroniosepsis, subacute septic endocarditis, oral infections. Gamma-hemolytic streptococci are saprophytes of the upper respiratory tract and the human intestinal tract. In some cases, they cause subacute septic endocarditis, urinary tract infections, wound infections.
Morphology of streptococci
these are immobile spherical or oval cocci with a diameter of 0.8-1 microns, forming chains of various lengths and positively staining according to Gram. Some strains form a capsule. The length of the chains is related to the growing conditions. In a liquid nutrient medium, they are longer; on dense media, they are often arranged in the form
short chains and bundles. Cocci may be ovoid before division. The division occurs perpendicular to the chain. Each coccus is divisible by 2.
Biology of streptococci
cultural properties: on agar with blood, streptococcus forms small (1-2 mm in diameter) translucent rods, grayish or colorless, which are well removed by a loop. The size of the hemolysis zone varies in different strains: group A forms a hemolysis zone slightly larger than the diameter of the colony, group B gives a large hemolysis zone. Type A streptococci form a greenish or greenish-brown hemolysis zone, cloudy or transparent, varying in size and color intensity. In some cases, the colony itself acquires a greenish coloration. In liquid nutrient media, streptococci are characterized by benthic, often rising along the walls, growth. When shaken, a granular or flaky suspension. Common growing media: meat-peptone agar with the addition of rabbit or sheep blood, semi-liquid agar with serum.
Good growth and toxin formation can be achieved on "combination broth" or on media containing casein hydrolysate and yeast extract. Hemolytic streptococci metabolize glucose with the formation of lactic and other acids, which is a factor limiting the growth of microbes in a nutrient medium. Resistance to physical and chemical factors.
Group A hemolytic streptococci can persist for a long time on objects, in dust in a dried state. However, these cultures, while maintaining viability, lose their virulence.
Group A streptococcus is highly sensitive to penicillin, which has a bactericidal effect on it. Sulfanilamide acts on streptococcus A bacteriostatically.
Modern classification of streptococci
based on their serological differences. There are 17 known serological groups: A, IN,
C, D, E, F, etc. The division into groups is based on the presence of a specific polysaccharide (substance C) in representatives of different groups. Streptococci of group A are pathogenic for humans. Streptococci of different groups differ not only in their ability to cause diseases in humans and animals and in their natural habitat, but also in biochemical and cultural characteristics.
In addition to serological differences, when differentiating strains, the following indications are taken into account:
- source of selection;
- nature of hemolysis;
- the ability to form soluble hemolysis;
- resistance to various temperatures;
- feature to grow in milk with methylene blue;
- fermentation of sugars;
- liquefaction of gelatin.
Serological serotypes: By agglutination on glass, strains of beta-hemolytic streptococcus isolated from scarlet fever and other streptococcal infections and from healthy carriers were divided into 50 serological types. Cultures of 46 types are assigned to group A, types 7, 20, 21 - to group C, and type 16 - to group D.
The division of streptococci into types is also carried out using the precipitation reaction. The results of type determination by the agglutination reaction and in the precipitation reaction usually give the same results. Scarlet fever usually predominates
1 or 2-3 types. Common antigenic substances were found in strains belonging to groups A, C, Q.
Streptococcal (with scarlet fever) toxin contains
2 factions:
- thermolabile or true scarlatinal toxin;
- thermostatic, which has the properties of an allergen.
The true erythrogenic toxin is a protein. It is a streptococcal exotoxin that causes the Dick reaction in people susceptible to scarlet fever. Purified erythorogenic toxin is used for skin tests to determine the level of antitoxic immunity (Dick reaction).
- For bacteriological examination, the material collected with a swab from the mucous membrane of the pharynx and nose is inoculated on a Petri dish with blood agar, placed in a thermostat for 3-4 hours at 37 °C. In the presence of streptococci, characteristic rods grow on the agar in a day. For microscopic examination, an isolated colony is subcultured into a liquid nutrient medium (meat-peptone broth with whey) and after 24 hours of cultivation in a thermostat, it is subjected to research. Smears are stained according to Gram or methylene blue according to Loeffler. Then, the biochemical properties of the cultures are studied and the type of streptococcus is determined using an agglutination test on glass and a precipitation test with typical sera. Of the serological reactions, the complement fixation reaction (CFR) is used with the serum of an immunized rabbit.
2. Streptococci
They belong to the family Streptococcaceae, genus Streptococcus.
These are gram-positive cocci, arranged in chains or in pairs in smears. They are facultative anaerobes. Do not grow on nutrient media. On blood agar, small-dotted, pigmentless colonies are produced, surrounded by a zone of hemolysis: a - green, b - transparent. The disease is often caused by b-hemolytic streptococcus. In sugar broth, near-wall growth is given, and the broth itself remains transparent. Grow at 37°C. Streptococci are able to break down amino acids, proteins, carbohydrates. According to biochemical properties, 21 species are distinguished. Most of them are conditionally pathogenic.
The most important in the development of infectious diseases are:
1) S. pyogenus, the causative agent of a specific streptococcal infection;
2) S. pneumonia, the causative agent of pneumonia, can cause a creeping corneal ulcer, otitis, sepsis;
3) S. agalactia, may be part of the normal microflora of the vagina; infection of newborns leads to the development of sepsis and meningitis in them;
4) S. salivarius, S. mutans, S. mitis, are part of the normal microflora of the oral cavity; in oral dysbiosis are the leading factors in the development of caries.
streptococcal antigens.
1. Extracellular - proteins and exoenzymes. These are variant-specific antigens.
2. Cellular:
1) surface proteins are represented by surface proteins of the cell wall, and in S. pneumonia, by capsule proteins. They are variant-specific;
2) deep - teichoic acids, peptidoglycan components, polysaccharides. They are group specific.
pathogenicity factors.
1. Complexes of teichoic acids with surface proteins (play the role of adhesins).
2. M-protein (possesses antiphagocytic activity). This is a superantigen, that is, it causes polyclonal activation of immune system cells.
3. OF-protein - an enzyme that causes hydrolysis of blood serum lipoproteins, reducing its bactericidal properties. The OF protein is important for adhesion. According to the presence or absence of this protein, there are:
1) OF+ strains (rheumatogenic); the entrance gate is the pharynx;
2) OF-strains (nephritogenic); primary adhesion to the skin.
4. Enzymes of aggression and defense:
1) hyaluronidase;
2) streptokinase;
3) streptodornasis;
4) proteases;
5) peptidases.
5. Exotoxins:
1) hemolysins:
a) O-streptolysin (has a cardiotoxic effect, a strong immunogen);
b) S-streptolysin (weak immunogen, does not have a cardiotoxic effect);
2) erythrogenin (has a pyrogenic effect, causes capillary paresis, thrombocytolysis, is an allergen, occurs in strains that cause complicated forms of infection, in pathogens of scarlet fever, erysipelas).
There is no specific prevention.
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No. 8 Streptococcus. Taxonomy. Characteristic. Microbiological diagnosis of streptococcal infections.
Treatment.
Taxonomy. Streptococci belong to the Firmicutes division of the Streptococcus genus. The genus consists of more than 20 species, among which there are representatives of the normal microflora of the human body and pathogens of severe infectious epidemic human diseases.
Morphological and cultural properties. Streptococci are small spherical cells arranged in chains, gram-positive, do not form spores, immobile. Most strains form a capsule consisting of hyaluronic acid. The cell wall contains proteins (M-, T- and R-antigens), carbohydrates (group-specific) and peptidoglycans. Easily transform into L-shapes. Pathogens grow on media enriched with carbohydrates, blood, serum, ascitic fluid. On dense media, they usually form small gray colonies. Capsular strains of group A streptococci form mucous colonies. On liquid media, streptococci usually grow near the bottom. Streptococci are facultative anaerobes. According to the nature of growth on blood agar, they are divided into cultural variants: a-hemolytic (green), b-hemolytic (complete hemolysis) and non-hemolytic.
resistance. Sensitive to physical and chemical environmental factors, they can remain viable for a long time at low temperatures. Antibiotic resistance develops slowly.
Pathogenicity. Based on the polysaccharide antigen, they are divided into serogroups (A, B, C ... O). Group A streptococci produce more than 20 substances that are antigenic and aggressive. On the surface of the cell there is a protein antigen M, which is closely associated with virulence (prevents phagocytosis). This protein determines the type of streptococci. Pathogenicity factors include streptokinase (fibrinolysin), DNase, hyaluronidase, erythrogenin. The most pathogenic for humans are group A hemolytic streptococci, called S. pyogenes. This species causes many diseases in humans: scarlet fever, erysipelas, tonsillitis, acute endocarditis, postpartum sepsis, chronic tonsillitis, rheumatism.
Immunity: post-infection unstable, not stressed.
Microbiological diagnostics.
Material for research - pus, urine, blood, sputum.
Bacterioscopic method: Gram stain of smears from pathological material. With a positive result, chains of gram "+" cocci are found.
Bacteriological method: The test material is seeded on blood agar in a Petri dish. After incubation at 37 °C for 24 hours, the nature of the colonies and the presence of hemolysis zones around them are noted. From a part of the material taken from the colonies, a smear is prepared, stained according to Gram and microscoped. To obtain a pure culture, 1-3 suspicious colonies are subcultured into test tubes with slant blood agar and sugar broth. On blood agar, Streptococcus pyogenes forms small, hazy round colonies. In the broth, streptococcus gives a near-wall growth in the form of flakes, leaving the medium transparent. According to the nature of hemolysis on blood agar, streptococci are divided into three groups: 1) non-hemolytic; 2) a-hemolytic; 3) ?-hemolytic, forming a completely transparent zone of hemolysis around the colony. The final stage of bacteriological research is the identification of the isolated culture by antigenic properties. On this basis, all streptococci are divided into serological groups (A, B, C, D, etc.). The serogroup is determined in a precipitation reaction with polysaccharide precipitinogen C. The serovar is determined in an agglutination reaction. The identified culture of streptococcus is tested for sensitivity to antibiotics using the disk method.
Serodiagnostics: establish the presence of specific antigens in the patient's blood using RSK or precipitation reaction. Antibodies to O-streptolysin are determined to confirm the diagnosis of rheumatism.
Treatment: Broad-spectrum antibiotics (penicillins resistant to β-lactamase). With the isolation of streptococcus A - penicillin. Chemotherapy with antibiotics, to which the sensitivity of the microbe is revealed - levomycetin, rifampicin.
Prevention: specific - no. Nonspecific - identification, treatment of patients; conducting a routine examination of medical staff, vaccination streptococcal bacteriophage (liquid)
- Streptococcus phagolysate filtrate. Applied externally, intradermally, intramuscularly ., O-streptolysin dry
(lyophilized filtrate of the broth culture of streptococcus - an active producer of O-streptolysin. It is used for setting serological reactions - determining anti-O-streptolysin in the blood serum of patients).
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 that 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. Those who were not sick around the injection site develop hyperemia and edema, which is characterized as a positive reaction (absence 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.
Control 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.
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 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 investigated 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.
Control 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).
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