Streptococcal infection microbiology. Microbiological diagnosis of streptococcal infections
Morphological and tinctorial properties. Streptococci - Streptococcus (first described by Ogston in 1881) have the appearance of cocci located in chains. The length of the chains is very variable. In pathological material and on dense nutrient media, they are short of 4-6 individual cocci; on liquid nutrient media, unusually long chains are found, which include many tens of individual cocci (see Fig. 60) - sometimes the chains consist of paired cocci that have slightly elongated shape (diplostreptococci). The diameter of individual individuals varies between 0.5-1 microns. They do not form spores or capsules, they do not have flagella. There are such varieties of streptococci that have a capsule in the pathological material. Streptococci stain well with aniline dyes and are Gram-positive.
Cultural and biochemical properties. Streptococci are a large group of bacteria that includes many varieties that differ from each other in cultural, biological and pathogenic properties. Streptococci grow in aerobic conditions or as facultative anaerobes. On simple nutrient media, they either do not develop at all, or grow extremely poorly, especially pathogenic species.
Rice. 64. Streptococcus colonies on sugar agar.
For the cultivation of streptococci, nutrient media are used, adding 1% glucose, 5-10% blood, 10-20% serum or ascitic fluid to them. The reaction of the medium is slightly alkaline (pH 7.2-7.6). The optimum temperature is 37°.
After 24 hours of growth, small grayish-white, slightly hazy colonies develop on the agar. Under a microscope at low magnification, they have a granular appearance. Larger colonies on blood agar. In some strains, they are surrounded by a light zone of hemolysis (Fig. 64). In others, a green color appears around the colony, and finally, in others, no changes are observed.
On the broth, streptococci grow in the form of a characteristic near-bottom, parietal, finely crumbly sediment, leaving the medium transparent. Some streptococci give diffuse growth.
Streptococci can decompose lactose, glucose, sucrose and sometimes mannitol with the formation of acids (without gas). Some streptococci have a reducing ability.
resistance. Streptococci show considerable resistance to physical and chemical influences. In the dried state, especially surrounded by a protein coat, they remain viable at room temperature for several months. When heated in a humid environment to 70 °, some species die no earlier than an hour later. Disinfectants kill streptococci in the following terms: 1-5% phenol solution - within 15-20 minutes, depending on the concentration of the drug, 0.5% Lysol solution - within 15 minutes. Under the action of rivanol at a dilution of 1: 100,000 and vucine at a dilution of 1: 80,000, streptococci die.
Toxin formation and pathogenicity for animals. The picture of the disease in streptococcal infections leaves no doubt that streptococci act on the human body through the toxic products they secrete. On liquid nutrient media, streptococci secrete exotoxin-type poisons found in culture filtrates. Exotoxins include 1) hemotoxin (streptolysin O and streptolysin S), which dissolves red blood cells. This poison exhibits its effect both in vivo and in vitro; 2) erythrogenic toxin (erythrogenin), which is a specific scarlet fever toxin. With the introduction of this toxin intradermally, a vascular reaction in the form of redness appears in persons sensitive to scarlet fever. This toxin consists of two fractions. Fraction A is thermolabile, has antigenic properties and is neutralized by antitoxic anti-scarlet fever serum. Fraction B is thermostable and is an allergen - 3) leukocidin, which destroys leukocytes and 4) necrotoxin, which causes tissue necrosis. Enzymes include fibrinolysin (streptokinase) and hyaluronidase.
Along with exotoxins, toxic substances such as endotoxin were found in streptococci. Of laboratory animals, rabbits are most sensitive to streptococci, and to a lesser extent guinea pigs and white mice.
Depending on the virulence of the cultures, streptococci may cause local inflammation or sepsis in a susceptible animal.
Determination of fibrinolysin (streptokinase). To 10 ml of human blood add 1 ml of 2% sodium citrate solution. After settling, unstained plasma is separated, 
Rice. 65. Hemolytic streptococcus. Growth on blood agar.
diluted with sterile saline 1:3 and add 0.5 ml of 18-20-hour broth culture of the test streptococcus. The tubes are gently shaken and placed in a water bath at 42° for 20-30 minutes. At this time, a fibrin clot is formed. The test tubes are left for 20 minutes in a water bath; in the presence of fibrinolysin, the clot dissolves within 20 minutes. Some strains of streptococci dissolve fibrin very slowly, therefore, 2 hours after standing in a water bath, the test tubes are transferred to a thermostat and the result of the experiment is taken into account the next day.
Streptococcus classification. Initially, streptococci were classified according to the length of the chains (Streptococcus longus, Streptococcus brevis). Such a division turned out to be untenable, since this sign is very unstable.
More rational is the classification of Schottmuller, which is based on the ratio of streptococci to erythrocytes. Depending on the nature of growth on blood agar, the following types of streptococci are distinguished:
- hemolytic streptococcus - Streptococcus haemolyticus dissolves red blood cells (Fig. 65);
- greenish streptococcus - Streptococcus viridans forms greenish-gray colonies on blood agar, which are surrounded by opaque areas of olive-greenish color;
- non-hemolytic streptococcus - Streptococcus anhaemolyticus on blood agar does not cause any changes.
Rice. 63. Staphylococcus in pus. Gram stain.
Rice. 66. Streptococcus in pus. Gram stain.
The liquid material is applied to a glass slide with a loop or a Pasteur pipette. If the material is thick, it is rubbed on the glass in a drop of saline. The material from the swab is applied to a sterilized glass slide.
When gram-positive cocci located in chains are detected under a microscope, the streptococcal etiology of the disease is tentatively established.
The material should then be plated on sugar and blood agar plates to obtain isolated colonies and isolate a pure culture. Small (0.5 mm), flat, dryish, grayish, transparent colonies of streptococcus make it possible to differentiate the type of streptococcus (hemolytic, green, non-hemolytic).
To determine the reducing ability of streptococci, 0.1 ml of an 18-hour test broth culture is inoculated into 5 ml of milk with methylene blue (the medium consists of sterile skim milk, to which a 1% aqueous solution of methylene blue is added in an amount of 2 ml per 100 ml of milk) and put in a thermostat at 37 ° for 24 hours. With a positive reaction, the milk becomes colorless, with a negative reaction, the color of the medium does not change.
To determine the virulence and toxigenicity of streptococci, a rabbit is injected intradermally with 200-400 million microbial bodies. After 24-48 hours, an inflammatory reaction of varying degrees appears at the injection site, with or without necrosis.
Identification of hemolytic streptococci is also carried out using the agglutination and precipitation reaction.
Agglutination reaction. One drop of saline and a drop of agglutinating group sera A, B, C, D (whole or diluted with saline 1:2 or 1:10) are applied to the glass slide with separate Pasteur pipettes, into which the studied broth culture is added drop by drop. If the culture is not very granular and does not give spontaneous agglutination, then within half an hour it is possible to determine the group of streptococcus. In addition to the group, it is possible to determine the type of streptococcus within group A. Typing is also carried out using the agglutination test on glass with type-specific sera and according to the same method as the group determination.
Specific prevention and therapy. Vaccination and vaccine therapy are not widely used in streptococcal diseases. More often use polyvalent antistreptococcal serum. Sulfanilamide preparations are distinguished by high activity in the treatment of streptococcal diseases. These drugs, both when taken orally and when applied topically or parenterally, have a sharply inhibitory effect on streptococci. For the treatment of streptococcal infections, antibiotics - penicillin, tetracycline, etc. - are used with great success.
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streptococci(Streptococcus) - causative agents of a large number of human and animal infections, 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: AND, AT,
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.
DOMAIN → Bacteria; TYPE → Firmicutes; CLASS → Vasilli; ORDER → Lactobacillales;
FAMILY → Streptococcaceae; GENUS → Streptococcus; SPECIES → Streptococcus species (up to 50 species)
The main features of the genusStreptococcus:
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 genusStreptococcus:
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 byS. 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 causedS. 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.
Microbiology: lecture notes Tkachenko Ksenia Viktorovna
2. Streptococci
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).
1) etiotropic antibiotic therapy;
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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