Microbiology: lecture notes Tkachenko Ksenia Viktorovna
1. Staphylococci
Family Staphilococcoceae, genus Staphilicoccus.
They are the causative agents of staphylococcal pneumonia, newborn staphylococcus, sepsis, pemphigus.
These are small gram-positive cocci. In smears, they are located in clusters, often uviform. They do not form a dispute, they are motionless. Form microcapsules. They are facultative anaerobes.
Undemanding to nutrient media, grow well on simple media, give pigment colonies. The elective medium for staphylococci is yolk-salt agar, less often - milk-salt agar.
Staphylococci are resistant to high concentrations of sodium chloride.
Unlike micrococci, staphylococci are able to decompose glucose under anaerobic conditions, glycerol under aerobic conditions. They are sensitive to lysostaphin, since their cell wall contains special teichoic acids - ribitol teichoic.
Staphylococci are biochemically active, have proteolytic and saccharolytic activity. According to their biochemical properties, they are divided into types:
1) St. aureus (has many factors of pathogenicity, may have a variety of localization of lesions);
2) St. epidermidis (affects the skin);
Three tests are used to differentiate these three types:
1) fermentation of mannitol under anaerobic conditions;
2) production of plasmacoagulase;
3) sensitivity to the antibiotic novobiocin.
For St. aureus all three tests are positive, for St. saprophiticus all three tests are negative, St. epidermidis is sensitive to novobiocin.
Staphylococcal antigens are divided into:
1) extracellular (variant-specific proteins of exotoxins and exoenzymes);
2) cellular:
a) superficial (glycoproteins) - variant-specific;
b) deep (teichoic acids) - group-specific.
Pathogenicity factors of staphylococci.
1. The role of adhesins is performed by complexes of cell wall surface proteins with teichoic acids.
2. Hyaluronidase is a factor of tissue invasion into the intercellular spaces of cells.
3. Enzymes of aggression:
1) plasmacoagulase;
2) fibrinolysin;
3) lecithinase;
4) phosphatase;
5) phosphotidase;
6) exonucleases;
7) proteases.
4. Toxins:
1) hematolysins (a, b, g, d, e); cause hemolysis of human erythrocytes, have a dermatonecrotic effect;
2) hemotoxins; responsible for the development of toxic shock;
3) leukocidin; consists of two factions; for one, the targets are macrophages, for the other, polymorphonuclear leukocytes;
4) exofolative exotoxin; causes multiple skin lesions;
5) enterotoxins (A, B, C, D, E); with the alimentary route of infection, they cause food toxicosis or food poisoning in children, damage enterocytes.
Diagnostics:
1) bacteriological examination. Wednesday - blood, yolk-salt agar;
2) serodiagnostics. Antibodies to α-hemotoxin are detected in the reaction of toxin neutralization.
1. Chemotherapy - antibiotics, sulfonamides, nitrofurans.
2. Phage therapy - polyvalent phages.
Taxonomy: belong to the department Firmicutes, family Micrococcacae, genus Staphylococcus. This genus includes 3 species: S. aureus, S. epidermidis and S. saprophyticus.
Morphological properties: All types of staphylococci are round cells. In the smear, they are arranged in asymmetrical bunches. The cell wall contains a large amount of peptidoglycan, teichoic acids associated with it, protein A. Gram-positive. They do not form a dispute, they do not have flagella. In some strains, a capsule can be found. May form L-shapes.
Cultural properties: Staphylococci are facultative anaerobes. Grow well in simple environments. On dense media, they form smooth, convex colonies with various pigments that have no taxonomic significance. Can grow on agar with high NaCl content. They have saccharolytic and proteolytic enzymes. Staphylococci can produce hemolysins, fibrinolysin, phosphatase, lactamase, bacteriocins, enterotoxins, coagulase.
Staphylococci are plastic, quickly acquire resistance to antibacterial drugs. An essential role in this is played by plasmids transmitted by transducing phages from one cell to another. R-plasmids determine resistance to one or more antibiotics through the production of β-lactamase.
Antigenic structure... About 30 antigens, which are proteins, polysaccharides and teichoic acids. The staphylococcus cell wall contains protein A, which can firmly bind to the Fc-fragment of the immunoglobulin molecule, while the Fab-fragment remains free and can bind to a specific antigen. Sensitivity to bacteriophages (phage type) is due to surface receptors. Many strains of staphylococci are lysogenic (the formation of some toxins occurs with the participation of a prophage).
Pathogenicity factors: Conditionally pathogenic. The microcapsule protects against phagocytosis, promotes the adhesion of microbes; cell wall components - stimulate the development of inflammatory processes. Enzymes of aggression: catalase - protects bacteria from the action of phagocytes, β-lactamase - destroys antibiotic molecules.
Resistance. Environmental resistance and sensitivity to disinfectants are common.
Pathogenesis. The source of staphylococcal infection is humans and some species of animals (sick or carriers). Transmission mechanisms - respiratory, contact-household, alimentary.
Immunity: P ostinfectious - cellular-humoral, unstable, not stressed.
Clinic. About 120 clinical manifestations that are local, systemic or generalized. These include purulent-inflammatory diseases of the skin and soft tissues (boils, abscesses), lesions of the eyes, ear, nasopharynx, urogenital tract, digestive system (intoxication).
Microbiological diagnostics . Research material - pus, blood, urine, sputum, stool.
Bacterioscopic method: smears are prepared from the test material (except for blood), stained according to Gram. The presence of gram "+" uviform cocci, located in the form of clusters.
Bacteriological method: The material is inoculated with a loop on blood and yolk-saline agar plates to obtain isolated colonies. Crops are incubated at 37C for 24 hours. The next day, the grown colonies are examined on both media. The presence or absence of hemolysis is noted on blood agar. S. aureus forms golden round convex opaque colonies on the JSA. Around the colonies of staphylococci with lecithinase activity, opacification zones with a pearlescent hue are formed. For the final establishment of the type of staphylococcus, 2-3 colonies are subcultured into tubes with slant nutrient agar to obtain pure cultures with subsequent determination of their differential characteristics. S.aureus - "+": formation of plasma coagulase, leticinase. Fermentation: glc, minnita, a-toxin formation.
To establish the source of the nosocomial infection, pure cultures of staphylococcus are isolated from patients and carriers of bacteria, after which they are phage typed using a set of typical staphylophages. Phages are diluted to the titer indicated on the label. Each of the studied cultures is inoculated on nutrient agar in a Petri dish with a lawn, dried, and then a drop of the corresponding phage is applied with a loop on the squares (according to the number of phages included in the set), previously marked with a pencil on the bottom of the Petri dish. Crops are incubated at 37 ° C. The results are evaluated the next day by the presence of lysis of the culture.
Serological method: in cases of chronic infection, the titer of anti-a-toxin in the blood serum of patients is determined. Determine the titer of AT to riboteichoic acid (component of the cell wall).
Treatment and prevention... Broad-spectrum antibiotics (β-lactamase-resistant penicillins). In the case of severe staphylococcal infections that cannot be treated with antibiotics, antitoxic anti-staphylococcal plasma or immunoglobulin immunized with adsorbed staphylococcal toxoid can be used. Identification, treatment of patients; conducting a routine examination of the medical staff, vaccination with staphylococcal toxoid.
Staphylococcal toxoid: is obtained from native toxoid by precipitation with trichloroacetic acid and adsorption on aluminum oxide hydrate.
Staphylococcal vaccine: suspension of coagulase-positive staphylococci, inactivated by heating. Used to treat long-term current diseases.
Immunoglobulin human anti-staphylococcal : gamma-globulin fraction of blood serum, contains staphylococcal toxoid. Prepared from human. blood, high in antibodies. It is used for specific treatment.
56. Types and mechanisms of bacteria nutrition.
Types of food. Microorganisms need carbohydrates, nitrogen, sulfur, phosphorus, potassium and other elements. Depending on the carbon sources for nutrition, bacteria are divided into autotrophs using carbon dioxide CO2 and other inorganic compounds to build their cells, and heterotrophs feeding on ready-made organic compounds. Autotrophic bacteria are nitrifying bacteria found in the soil; sulfur bacteria living in water with hydrogen sulfide; iron bacteria living in water with ferrous iron, etc.
Depending on the substrate to be oxidized, called an electron or hydrogen donor, microorganisms are divided into two groups. Microorganisms using inorganic compounds as hydrogen donors are called lithotrophic (from the Greek lithos - stone), and microorganisms using organic compounds as hydrogen donors are called organotrophs.
Given the energy source, phototrophs are distinguished among bacteria, i.e. photosynthetic (for example, blue-green algae that use light energy), and chemotrophs, which require chemical energy sources.
Nutritional mechanisms. The entry of various substances into a bacterial cell depends on the size and solubility of their molecules in lipids or water, the pH of the medium, the concentration of substances, various factors of membrane permeability, etc. The cell wall allows small molecules and ions to pass through, retaining macromolecules weighing more than 600 D. The main regulator of the intake of substances the cell is the cytoplasmic membrane. Conventionally, four mechanisms can be distinguished for the penetration of nutrients into a bacterial cell.: it is simple diffusion, facilitated diffusion, active transport, group translocation.
The simplest mechanism for the entry of substances into the cell is simple diffusion, in which the movement of substances occurs due to the difference in their concentration on both sides of the cytoplasmic membrane. Substances pass through the lipid part of the cytoplasmic membrane (organic molecules, drugs) and, less often, through water-filled channels in the cytoplasmic membrane. Passive diffusion takes place without energy consumption.
Facilitated diffusion also occurs as a result of a difference in the concentration of substances on both sides of the cytoplasmic membrane. However, this process is carried out with the help of carrier molecules localized in the cytoplasmic membrane and possessing specificity. Each carrier transports the corresponding substance through the membrane or transfers to another component of the cytoplasmic membrane - the carrier itself. Carrier proteins can be permeases, the place of synthesis of which is the cytoplasmic membrane. Facilitated diffusion proceeds without energy consumption, substances move from a higher concentration to a lower one.
Active transport occurs with the help of permeases and is aimed at transferring substances from a lower concentration to a higher one, i.e. as if against the flow, therefore, this process is accompanied by the expenditure of metabolic energy (ATP), formed as a result of redox reactions in the cell.
Transfer (translocation) groups is similar to active transport, differing in that the transferred molecule is modified during the transfer process, for example, it is phosphorylated.
The release of substances from the cell is carried out by diffusion and with the participation of transport systems.
STYLAB offers test systems for analyzing the content of Staphylococcus aureus in food and the environment by microbiological methods, as well as for determining the DNA of this bacterium using PCR.
Staphylococcus aureus ( Staphylococcusaureus) is a ubiquitous gram-positive immobile facultatively anaerobic non-spore-forming bacterium belonging to cocci - globular bacteria. This microorganism is part of the normal microflora of the skin and mucous membranes in 15-50% of healthy people and animals.
Some strains of this bacterium are resistant to. The most famous of these is methicillin-resistant Staphylococcus aureus (MRSA). For a long time, it was considered the causative agent of nosocomial infections, but since the mid-1990s, it has been known about diseases in people who were not in hospitals. Most often, these were purulent lesions of the skin, however, when scratching the lesions, MRSA entered the bloodstream and affected other organs. Methicillin-resistant Staphylococcus aureus was susceptible to vancomycin, a toxic antibiotic that nevertheless eradicates the organism.
Another antibiotic-resistant bacterium is vancomycin-resistant Staphylococcus aureus (VRSA). Doctors and scientists have been expecting this organism since they learned about the existence of MRSA and vancomycin-resistant enterococcus (VRE), a non-pathogenic organism that lives in the intestines, since horizontal transfer allowed the exchange of genes between these bacteria. VRSA was first discovered in 2002 and was indeed resistant to all powerful antibiotics that existed at that time. However, his weak point was his sensitivity to the old sulfonamide - bactrim.
Staphylococcus aureus is found in soil and water, often contaminates food and can affect all tissues and organs: skin, subcutaneous tissue, lungs, central nervous system, bones and joints, etc. This bacterium can cause sepsis, purulent skin lesions and wound infections.
The optimum temperature for Staphylococcus aureus is 30-37 ° C. It can withstand heating up to 70-80 ° C for 20-30 minutes, dry heat - up to 2 hours. This bacterium is resistant to drying out and salinization and is able to grow on media with 5-10% table salt content, including fish and meat balyk and other products. Most disinfectants kill Staphylococcus aureus.
Staphylococcus aureus secretes a wide variety of toxins. Membranotoxins (hemolysins) of four types provide hemolysis; in addition, membranotoxin α in experiments causes skin necrosis, and when administered intravenously, death of animals. Exfoliatins of two types damage skin cells. Leukocidin (Panton-Valentine's toxin) causes disturbances in the water-electrolyte balance in the cells of leukocytes, especially macrophages, neutrophils and monocytes, which leads to their death.
In accordance with TR CU 021/2011 and other documents, the content of coagulase-positive staphylococci is also limited in food. These are bacteria that produce coagulase, an enzyme that causes blood plasma to clot. In addition to S. aureus These include S. delphini, S. hyicus, S. intermedius, S. lutrae, S. pseudintermedius and S. schleiferi subspecies. coagulans... According to some reports, S. leei is also coagusal positive.
To determine Staphylococcus aureus in samples, both microbiological methods, including selective media, and DNA analysis using the PCR method are used.
Staphylococcus aureus was discovered in 1878 by R. Koch and in 1880 by L. Pasteur in purulent material. L. Pasteur, having infected a rabbit, finally proved the role of staphylococcus as a causative agent of purulent inflammation. The name "staphylococcus" was given in 1881 by A. Ogston (because of the characteristic arrangement of cells), and in 1884 F. Rosenbach described its properties in detail. Staphylococci are gram-positive, regular geometric spherical cells with a diameter of 0.5 - 1.5 microns, usually located in the form of clusters (see color incl., Fig. 92), catalase-positive, reduce nitrates to nitrites, actively hydrolyze proteins and fats, ferment glucose under anaerobic conditions to form acid without gas. They can usually grow in the presence of 15% NaCl and at 45 ° C. The content of G + C in DNA is 30 - 39 mol%. Staphylococci do not have flagella, do not form spores. They are widespread in nature. Their main reservoir is the skin of humans and animals and their mucous membranes, communicating with the external environment. Staphylococci are facultative anaerobes, only one species ( Staphylococcus saccharolyticus) Is a strict anaerobic. Staphylococci are not demanding on nutrient media, grow well on ordinary media, the optimum temperature for growth is 35 - 37 ° C, pH 6.2 - 8.4. Colonies are round, 2 - 4 mm in diameter, with smooth edges, convex, opaque, colored in the color of the formed pigment. Growth in a liquid culture is accompanied by uniform turbidity, and a loose precipitate forms over time. When growing on conventional media, staphylococci do not form capsules, however, when inoculated into a semi-liquid agar with plasma or serum, most strains S. aureus forms a capsule. Capsule-free strains in semi-liquid agar grow in the form of compact colonies, capsule strains - form diffuse colonies.
Staphylococci have high biochemical activity: they ferment with the release of acid (without gas) glycerin, glucose, maltose, lactose, sucrose, mannitol; form various enzymes (plasma coagulase, fibrinolysin, lecithinase, lysozyme, alkaline phosphatase, DNase, hyaluronidase, tellurite reductase, proteinase, gelatinase, etc.). These enzymes play an important role in the metabolism of staphylococci and largely determine their pathogenicity. Enzymes such as fibrinolysin and hyaluronidase are responsible for the high invasiveness of staphylococci. Plasma coagulase is the main factor in their pathogenicity: it protects against phagocytosis and converts prothrombin into thrombin, which causes fibrinogen to coagulate, as a result of which each cell is covered with a protein film that protects against phagocytes.
Classification. Genus Staphylococcus includes more than 20 species, which are divided into two groups - coagulase-positive and coagulase-negative staphylococci. Various characters are used to differentiate species (Table 22).
Table 22
Differential signs of the main types of staphylococci
Note. (+) - positive sign; (-) - the sign is negative; + (-) - is a fickle sign ;? - unknown.
I. Coagulase-positive staphylococci:
1.S. aureus***.
2.S. intermedius**.
3.S. hyicusa.
II. Coagulase-negative staphylococci:
* Pathogenic for humans only.
** Pathogenic for animals only.
*** Pathogenic for humans and animals.
a Not all strains S. hyicus have coagulase.
Pathogenic for humans are mainly coagulase-positive staphylococci, but many coagulase-negative staphylococci are also capable of causing diseases, especially in newborns (neonatal conjunctivitis, endocarditis, sepsis, urinary tract diseases, acute gastroenteritis, etc.). S. aureus depending on who is its main bearer, it is divided into 10 ekovars ( hominis, bovis, ovis and etc.).
More than 50 types of antigens have been found in staphylococci, antibodies to each of them are formed in the body, many of the antigens have allergenic properties. By specificity, antigens are subdivided into generic (common to the entire genus Staphylococcus); cross-reacting - antigens common with isoantigens of human erythrocytes, skin and kidneys (autoimmune diseases are associated with them); species and type-specific antigens. By type-specific antigens detected in the agglutination reaction, staphylococci are divided into more than 30 serovariants. However, the serological method of typing staphylococci has not yet received widespread use. Species-specific include protein A, which forms S. aureus... This protein is located superficially, it is covalently bound to peptidoglycan, its molecular weight is about 42 kDa. Protein A is especially actively synthesized in the logarithmic growth phase at 41 ° C, is thermolabile, and is not destroyed by trypsin; its unique property is the ability to bind to the Fc-fragment of IgG immunoglobulins (IgG 1, IgG 2, IgG 4), to a lesser extent to IgM and IgA. On the surface of protein A, several regions have been identified that can bind to the region of the immunoglobulin polypeptide chain located at the border of the CH 2 and CH 3 domains. This property has found wide application in the coagglutination reaction: staphylococci loaded with specific antibodies, in which active centers remain free, give a rapid agglutination reaction when interacting with the antigen.
The interaction of protein A with immunoglobulins leads to dysfunctions of the complement systems and phagocytes in the patient's body. It has antigenic properties, is a strong allergen and induces the multiplication of T and B lymphocytes. Its role in the pathogenesis of staphylococcal diseases has not yet been fully elucidated.
Strains S. aureus differ in sensitivity to staphylococcal phages. For typing S. aureus use an international set of 23 temperate phages, which are divided into four groups:
1st group - phages 29, 52, 52A, 79, 80;
2nd group - phages 3A, 3C, 55, 71;
3rd group - phages 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85;
4th group - phages 94, 95, 96;
outside the groups - phage 81.
The relation of staphylococci to phages is peculiar: one and the same strain can be lysed either by one phage, or simultaneously by several. But since their sensitivity to phages is a relatively stable sign, phage typing of staphylococci is of great epidemiological significance. The disadvantage of this method is that no more than 65 - 70% can be typed. S. aureus... In recent years, sets of specific phages have been obtained for typing S. epidermidis.
Staphylococcus pathogenicity factors. Staphylococcus aureus is a unique microorganism. It can cause more than 100 different diseases belonging to eleven classes according to the International Classification of 1968. Staphylococci can affect any tissue, any organ. This property of staphylococci is due to the presence of a large complex of pathogenic factors in them.
1. Adhesion factors - the attachment of staphylococci to tissue cells is due to their hydrophobicity (the higher it is, the stronger the adhesive properties are), as well as the adhesive properties of polysaccharides, possibly also protein A, and the ability to bind fibronectin (the receptor of some cells).
2. Various enzymes that play the role of factors of "aggression and defense": plasma coagulase (the main factor of pathogenicity), hyaluronidase, fibrinolysin, DNase, lysozyme-like enzyme, lecithinase, phosphatase, proteinase, etc.
3. Complex of secreted exotoxins:
erythrocytes, necrosis when administered intradermally to a rabbit, destruction of leukocytes, death of a rabbit when administered intravenously. However, it turned out that this effect is caused by the same factor - a membrane-damaging toxin. It has a cytolytic effect on various types of cells, which is manifested as follows. Molecules of this toxin first bind to unknown receptors of the target cell membrane or are nonspecifically absorbed by lipids contained in the membrane, and then form a mushroom heptamer of 7 molecules, consisting of 3 domains. The domains that form the “cap” and “edge” are located on the outer surface of the membranes, and the domain of the “stem” serves as a transmembrane pore channel. Through it, the entry and exit of small molecules and ions occurs, which leads to swelling and death of cells with a nucleus, and osmotic lysis of erythrocytes. Found several types of membrane-damaged from humans, it lyses the erythrocytes of humans, rabbits and rams. The lethal effect in rabbits is caused by intravenous administration after 3 - 5 minutes. Hemolysinizes erythrocytes of humans and many animal species. The lethal effect on a rabbit when administered intravenously causes after 16 - 24 - 48 hours. Very often, in staphylococci, ob
b) exfoliative toxins A and B are distinguished by antigenic properties, relation to temperature (A - thermostable, B - thermolabile), localization of genes that control their synthesis (A is controlled by a chromosomal gene, B - by a plasmid gene). Often in the same strain S. aureus both exfoliatins are synthesized. Associated with these toxins is the ability of staphylococci to cause pemphigus in newborns, bullous impetigo, scarlet fever;
c) true leukocidin - a toxin that differs from hemolysins in antigenic properties, selectively acts on leukocytes, destroying them;
d) exotoxin causing toxic shock syndrome (TSS). It has the properties of a superantigen. TSS is characterized by an increase in temperature, a decrease in blood pressure, skin rashes with subsequent peeling on the hands and feet, lymphocytopenia, sometimes diarrhea, kidney damage, etc. More than 50% of strains are capable of producing and secreting this toxin S. aureus.
4. Strong allergenic properties, which are possessed by both components of the cell structure and exotoxins and other waste products secreted by bacteria. Staphylococcal allergens can cause both delayed-type hypersensitivity (HPH) and immediate-type hypersensitivity (HPN) reactions. Staphylococci are the main culprits for skin and respiratory allergies (dermatitis, bronchial asthma, etc.). The peculiarity of the pathogenesis of staphylococcal infection and its tendency to transition into a chronic form are rooted in the effect of the GHZ.
5. Cross-reacting antigens (with isoantigens of erythrocytes A and B, kidneys and skin - induction of autoantibodies, development of autoimmune diseases).
6. Factors inhibiting phagocytosis. Their presence can be manifested in the suppression of chemotaxis, protection of cells from absorption by phagocytes, in providing staphylococci with the ability to multiply in phagocytes and blocking the "oxidative burst". Phagocytosis is inhibited by the capsule, protein A, peptidoglycan, teichoic acids, toxins. In addition, staphylococci induce the synthesis of suppressors of phagocytic activity by some cells of the body (for example, splenocytes). Inhibition of phagocytosis not only prevents the body from cleansing staphylococci, but also disrupts the function of processing and presentation of antigens to T- and B-lymphocytes, which leads to a decrease in the strength of the immune response.
The presence of a capsule in staphylococci increases their virulence for white mice, makes them resistant to the action of phages, does not allow typing with agglutinating sera, and masks protein A.
Teichoic acids not only protect staphylococci from phagocytosis, but, obviously, play an essential role in the pathogenesis of staphylococcal infections. It has been established that in children with endocarditis, antibodies to teichoic acids are found in 100% of cases.
7. Mitogenic effect of staphylococci on lymphocytes (protein A, enterotoxins and other products secreted by staphylococci have this effect).
8. Enterotoxins A, B, C1, C2, C3, D, E. They are characterized by antigenic specificity, thermal stability, resistance to the action of formalin (do not convert to toxoid) and digestive enzymes (trypsin and pepsin), are stable in the pH range from 4, 5 to 10.0. Enterotoxins are low molecular weight proteins with a molecular weight of 26 to 34 kDa with properties of superantigens.
It has also been established that there are genetically determined differences in sensitivity to staphylococcal infection and the nature of its course in humans. In particular, severe staphylococcal purulent-septic diseases are more often found in people with blood groups A and AB, less often in people of 0 and B groups.
The synthesis of enterotoxins is associated with the ability of staphylococci to cause food poisoning such as intoxication. Most often they are caused by enterotoxins A and D. The mechanism of action of these enterotoxins is poorly understood, but it differs from the action of other bacterial enterotoxins, which disrupt the function of the adenylate cyclase system. All types of staphylococcal enterotoxins cause a similar pattern of poisoning: nausea, vomiting, pain in the pancreas, diarrhea, sometimes headache, fever, muscle spasm. These features of staphylococcal enterotoxins are due to their superantigenic properties: they induce excessive synthesis of interleukin-2, which causes intoxication. Enterotoxins stimulate intestinal smooth muscles and increase gastrointestinal motility. Poisoning is most often associated with the use of dairy products infected with staphylococcus (ice cream, pastries, cakes, cheese, cottage cheese, etc.) and canned food with butter. Infection of dairy products can be associated with mastitis in cows or with pyoinflammatory diseases in humans related to food production.
Thus, the abundance of various factors of pathogenicity in staphylococci and their high allergenic properties determine the peculiarities of the pathogenesis of staphylococcal diseases, their nature, localization, severity of the course and clinical manifestations. Avitaminosis, diabetes, decreased immunity contribute to the development of staphylococcal diseases.
Staphylococcus resistance. Among bacteria that do not form spores, staphylococci, like mycobacteria, have the greatest resistance to external factors. They tolerate drying well and remain viable and virulent for weeks and months in dry fine dust, a source of dust infection. Direct sunlight kills them only for many hours, while diffused light acts very weakly. They are also resistant to high temperatures: heating up to 80 ° C withstand about 30 minutes, dry heat (110 ° C) kills them within 2 hours; they tolerate low temperatures well. Sensitivity to chemical disinfectants varies greatly, for example, a 3% solution of phenol kills them within 15 - 30 minutes, and a 1% aqueous solution of chloramine - in 2 - 5 minutes.
Features of epidemiology. Since staphylococci are permanent inhabitants of the skin and mucous membranes, the diseases caused by them can be either autoinfection (with various injuries of the skin and mucous membranes, including microtraumas), or an exogenous infection caused by contact-household, airborne droplets, airborne dust or alimentary (for food poisoning) methods of infection.
Carriage of pathogenic staphylococci is of particular importance, since carriers, especially in medical institutions (various surgical clinics, maternity hospitals, etc.) and in closed groups, can cause staphylococcal infections. The carriage of pathogenic staphylococci can be temporary or intermittent, but persons in whom it is permanent (resident carriers) pose a particular danger to others. In such people, staphylococci persist for a long time and in large numbers on the mucous membranes of the nose and throat. The reason for the long-term carriage is not entirely clear. It may be a consequence of a weakening of local immunity (lack of secretory IgA), dysfunction of the mucous membrane, an increase in the adhesive properties of staphylococcus, or due to any of its other properties.
Features of pathogenesis and clinical picture. Staphylococci easily enter the body through the smallest damage to the skin and mucous membranes and can cause a variety of diseases - from juvenile acne (acne) to severe peritonitis, endocarditis, sepsis or septicopyemia, in which the mortality rate reaches 80%. Staphylococci cause boils, hydradenitis, abscesses, phlegmon, osteomyelitis; in wartime - frequent culprits of purulent complications of wounds; staphylococci play a leading role in purulent surgery. Possessing allergenic properties, they can cause psoriasis, hemorrhagic vasculitis, erysipelas, nonspecific polyarthritis. Staphylococcal infection of food is a common cause of food poisoning. Staphylococci are the main culprits of sepsis, including in newborns. Unlike bacteremia (bacteria in the blood), which is a symptom of the disease and is observed in many bacterial infections, sepsis (septicemia - putrefaction) is an independent disease with a definite clinical picture, which is based on damage to the organs of the reticuloendothelial system (system of mononuclear phagocytes - SMF ). With sepsis, there is a purulent focus, from which the pathogen periodically enters the bloodstream, spreads throughout the body and affects the reticuloendothelial system (SMF), in the cells of which it multiplies, releasing toxins and allergens. In this case, the clinical picture of sepsis weakly depends on the type of pathogen, but is determined by the defeat of certain organs.
Septicopyemia is a form of sepsis in which the pathogen causes purulent foci in various organs and tissues, i.e., it is sepsis complicated by purulent metastases.
Bacteremia in sepsis and septicopyemia can be short-term and long-term.
Post-infectious immunity exists, it is due to both humoral and cellular factors. An important role in it is played by antitoxins, antimicrobial antibodies, antibodies against enzymes, as well as T-lymphocytes and phagocytes. The intensity and duration of immunity against staphylococci have not been sufficiently studied, since they have too diverse antigenic structure, and there is no cross-immunity.
Laboratory diagnostics. The main method is bacteriological; developed and implemented serological tests. If necessary (in case of intoxication), a biological sample is used. The material for bacteriological research is blood, pus, mucus from the pharynx, nose, discharge from wounds, sputum (with staphylococcal pneumonia), feces (with staphylococcal colitis), in case of food intoxication - vomit, feces, gastric washings, suspicious foods. The material is inoculated on blood agar (hemolysis), on milk-salt (milk-yolk-salt) agar (the growth of foreign bacteria is inhibited due to NaCl, pigment and lecithinase are better detected). The isolated culture is identified by species characteristics, the presence of the main signs and factors of pathogenicity (golden pigment, mannitol fermentation, hemolysis, plasma coagulase) is determined, the sensitivity to antibiotics is checked, if necessary, phage typing is performed. Among serological reactions, RPHA and IFM are used to diagnose purulent-septic diseases, in particular, to determine antibodies to teichoic acid or to species-specific antigens.
To determine the enterotoxigenicity of staphylococci, three methods are used:
1) serological - with the help of specific antitoxic sera in the precipitation reaction in the gel, enterotoxin is detected and its type is established;
2) biological - intravenous administration of staphylococcus broth culture filtrate to cats in a dose of 2 - 3 ml per 1 kg of body weight. Toxins cause vomiting and diarrhea in cats;
3) indirect bacteriological method - isolation from a suspicious product of a pure culture of staphylococcus and determination of its pathogenicity factors (the formation of enterotoxin correlates with the presence of other pathogenic factors, in particular RNAase).
The simplest and most sensitive is the serological method for the detection of enterotoxin.
Treatment. For the treatment of staphylococcal diseases, beta-lactam antibiotics are mainly used, to which the sensitivity should first of all be determined. In severe and chronic staphylococcal infections, specific therapy gives a positive effect - the use of an autovaccine, toxoid, anti-staphylococcal immunoglobulin (human), antistaphylococcal plasma.
Specific prophylaxis. To create artificial immunity against staphylococcal infection, staphylococcal toxoid (liquid and tableted) is used, but it creates antitoxic immunity only against staphylococci, lysed mainly by group I phages. The use of vaccines from killed staphylococci or their antigens, although it leads to the emergence of antimicrobial antibodies, but only against those serovariants from which the vaccine is made. The problem of finding a highly immunogenic vaccine effective against many types of pathogenic staphylococci is one of the most important problems of modern microbiology.
Staphylococci - spherical gram-positive: immobile asporogenic bacteria of the genus Staphylococcus from the family Micrococcaceae. Discovered in 1880 independently of each other by L. Pasteur and A. Ogston and studied in more detail by F. Rosenbach in 1884.
In 1976, the following three species were officially approved by the International Committee for Taxonomy of Staphylococci: S. aureus, S. epidermidis and S. saprophyticus. To date, 19 species of staphylococci isolated from animals and humans have been described.
Staphylococci are important in the infectious pathology of animals: almost any organ and any tissue can be affected by these microbes. They cause boils, abscesses, phlegmon, osteomyelitis, mastitis, endometritis, bronchitis, pneumonia, meningitis, pyemia and septicemia, enterocolitis, food toxicosis, staphylococcosis of birds.
Morphology. Staphylococci are spherical cells with a diameter of 0.5-1.5 microns. In preparations from pus and young broth cultures, they are located singly, in pairs, in short chimneys or in small heaps; in smears from agar cultures - and in the form of separate clusters of irregular shape, resembling a bunch of grapes. They do not have flagella and capsules, do not form spores. They are well stained with aniline dyes, gram-positive; in old cultures, individual cells are stained with gram-negative.
Cultivation. Optional anaerobes. Grow well on universal nutrient media. at a temperature of 35-40 ° C (growth is possible in the range of 6.5-46 ° C), the optimum pH is 7.0-7.5. Adding glucose or blood to the nutrient medium accelerates the growth of staphylococci. A characteristic property of most strains is the ability to grow in the presence of 15% sodium chloride or 40% bile. On MPA, round colonies with smooth edges, 2–5 mm in diameter, are formed slightly above the agar surface. Colonies can be colored because staphylococci produce water-insoluble carotenoid pigments. The most intense pigments are formed on agar with 10% skim milk after 24-hour incubation at 37 ° C and on potatoes at a temperature of 20-25 ° C under aerobic conditions in the light. S. aureus synthesizes golden or orange pigments, pigment-free strains are also found; S.epidermidis typically synthesizes white or yellow pigment; most S. saprophyticus strains lack pigment.
When growing with MP6, staphylococci first cause diffuse turbidity followed by the precipitation of a loose flocculent sediment. Grows characteristically in a column of gelatin. After 18-26 hours, along with the abundant growth on the injection, an initial dilution of the medium is outlined, which then increases, and by the 4th-5th day along the course of the injection, a funnel filled with liquid is formed. Pathogenic strains of staphylococci form a significant hemolysis zone on blood agar.
Biochemical properties. Staphylococci ferment with the formation of acid without gas glucose, maltose, fructose, sucrose, xylose, glycerin, mannitol and do not decompose dulcite, salicin, inulin, raffinose. Allocate ammonia and. hydrogen sulfide, do not form indole, reduce nitrates to nitrites; produce catalase, phosphatase, urease; pathogenic strains - arginase. Coagulate and peptolize milk, liquefy gelatin, sometimes coagulated blood serum.
However, proteolytic activity in staphylococci can vary greatly.
Toxin formation. Pathogenic staphylococci synthesize and secrete highly active exotoxins and enzymes. Among exotoxins, there are four types of hemotoxins (staphylolysins), leukocidin and enterotoxins.
Hemotoxins include alpha, beta, gamma and delta hemolysins.
Alpha-hemolysin causes lysis of erythrocytes of sheep, pigs, dogs, has a lethal and dermatonecrotic effect, destroys leukocytes, aggregates and lyses platelets.
Beta-hemolysin lyses erythrocytes of humans, sheep, cattle, and is lethal for rabbits.
Gamma-hemolysin is found in strains isolated from humans, its biological activity is low.
Delta-hemolysin causes lysis of erythrocytes in humans, horses, sheep, rabbits, and destroys leukocytes.
All staphylococcal hemolysins are membrane toxins: they are able to lyse the membranes of eukaryotic cells.
Leukocidin is a non-hemolytic exotoxin that causes degranulation and destruction of leukocytes.
Entrotoxins - thermostable polypeptides, are formed during the reproduction of enterotoxigenic staphylococci in nutrient media, food (milk, cream, cottage cheese, etc.), intestines. Resistant to the action of digestive enzymes. Six antigenic variants are known. Enterotoxins cause food toxicosis in humans, cats, especially kittens, and puppies of dogs are sensitive to them.
The factors of pathogenicity of staphylococci also include the enzymes coagulase, hyaluronidase, fibrinolysin, DNase, lecitovitellase, etc. Coagulase is a bacterial proteinase that coagulates the blood plasma of animals. The presence of coagulase is one of the most important and constant criteria for the pathogenicity of staphylococci.
Antigenic structure. In staphylococci, the best studied antigens of the cell wall: peptidoglycan, teichoic acids and protein A. Peptidoglycan is a common species antigen for staphylococci. Teichoic acids are species-specific polysaccharide antigens. S. aureus contains ribitol teichoic acid (polysaccharide A), S. epidermidis contains glycerol teichoic acid called polysaccharide B. Protein A is found in Staphylococcus aureus. It is a low molecular weight protein that binds to Fc fragments of mammalian IgG. Strains that produce large amounts of protein A are more resistant to phagocytosis. In mucoid strains of Staphylococcus aureus, a capsular polypeptide antigen was also identified.
Stability. Staphylococci are relatively resistant microorganisms. Direct sunlight will only kill them after a few hours. In dust they remain for 50-100 days, in dried pus - more than 200 days, in broth culture - 3-4 months, on semi-liquid agar - 6 months. In a liquid medium at 70 ° C, they die after 1 hour, at 85 ° C - after 30 minutes, at 100 ° C - in a few seconds. From disinfectants 1% formalin solution and 2% sodium hydroxide solution kill them within 1 hour, 1% chloramine solution - after 2-5 minutes. Staphylococci are highly sensitive to brilliant green and pyoctanine.
Many strains are sensitive to benzylpenicillin, semi-synthetic penicillins, streptomycin, chloramphenicol, tetracycline, fuzidin and other antibiotics, as well as nitrofuran drugs. However, there are also many antibiotic-resistant strains. They are usually characterized by multidrug resistance, which is controlled by the R-plasmid and can be spread by transduction. Staphylococci that synthesize penicillinase (beta-lactamase) are capable of destroying some penicillins. Staphylococci are very resistant to sulfonamides.
Pathogenicity. The main role in infectious pathology of animals and humans belongs to S. aureus. Staphylococcal infections can also be caused by S. epidermidis and S. saprophyticus. Pigment formation and breakdown of carbohydrates cannot serve as a criterion for the pathogenicity of staphylococci. The main factors determining the pathogenicity of these bacteria is the ability to produce exotoxins and enzymes coagulase, fibrinolysin and hyaluronidase.
Horses, cattle and small ruminants, pigs, ducks, geese, turkeys, chickens, from laboratory animals - rabbits, white mice, kittens are sensitive to staphylococci. When a culture of pathogenic staphylococci is administered intradermally to rabbits, inflammation and then skin necrosis develops, with intravenous injection of culture filtrate in rabbits, acute poisoning and death occurs after a few minutes.
Pathogenesis. Staphylococci enter the body through damaged skin and mucous membranes, enterotoxins - with food.
Staphylococcal infections develop more often and are more severe in conditions of a decrease in the body's natural resistance and in immunodeficiency states. In the pathogenesis of staphylococcal processes, the leading role belongs to exotoxins and pathogenic enzymes. Allergies can also be important. All these factors together and determine whether local purulent-inflammatory foci, systemic diseases of internal organs, sepsis or food toxicosis will occur.
Laboratory diagnostics. Examine wound exudate, pus of abscesses, wounds, milk for mastitis, discharge from the genitals with endometritis, blood from the jugular vein with septicemia.
Smears are prepared from the pathological material, stained according to Gram, and microscoped. Direct microscopy allows only a preliminary answer to be given. Simultaneously sow the material into blood, milk-salt and yolk-salt agar plates.
Pathogenic strains on blood agar form a hemolysis zone around the colonies. Pigment formation is counted on milk-salt agar plates. On yolk-salt agar, most pathogenic staphylococci cause a lecitovitellase reaction, manifested in the formation of a cloudy zone around the colony with a rainbow corolla along the periphery. To obtain a pure culture and further study, material from a characteristic colony is screened out on MPA. A pure culture is microscoped, after which a plasma coagulation reaction is performed with citrated rabbit blood plasma. In the presence of the enzyme coagulase, the plasma is clotted. Additionally, DNAse and mannitol degradation are determined under anaerobic conditions.
The lethal properties of the culture on rabbits are revealed and a dermatonecrotic test is carried out. For this purpose, 0.2 ml of a 2 billion suspension of culture is injected intradermally into the shaved area of the rabbit's skin. In the positive case, an infiltration forms at the injection site and necrosis occurs.
S. aureus, unlike other species, ferments mannitol under anaerobic conditions. Pathogenic staphylococci, in addition to hemolytic and lecithinase activity, have the ability to coagulate plasma, cause skin necrosis and destroy DNA.
The death of a rabbit indicates the presence of a lethal effect of the toxin.
If it is necessary to establish the source of staphylococcal infection and the ways of its spread, the isolated cultures are subjected to phage typing. The international set of staphylococcal phages consists of 22 types, divided into 4 groups. Enterotoxins in food and crops are determined in RDP with staphylococcal antisera to enterotoxins A, B, C, D, E, F.
Due to the wide distribution of drug-resistant staphylococcal strains, the sensitivity of the isolated cultures to antibiotics is determined on a solid medium by the method of paper disks or replicas. This is very important for choosing rational chemotherapy.
Immunity. Healthy animals are naturally resistant to staphylococcal infections. It is due to the barrier function of the skin, mucous membranes, phagocytosis and the presence of specific antibodies synthesized as a result of latent immunization. The inflammatory reaction at the site of introduction of the pathogen also prevents the spread of microbes in the body.
Immunity in staphylococcal infections is predominantly antitoxic, low tension and short-lived. Therefore, frequent relapses are not excluded. Nevertheless, high titers of antitoxins in the blood of animals increase their resistance to recurrent diseases. Antitoxins not only neutralize exotoxins, but also cause rapid mobilization of phagocytes.
Staphylococci also induce delayed-type hypersensitivity. It is known that repeated staphylococcal skin lesions lead to more pronounced destructive changes.
Biologicals. The proposed purified adsorbed staphylococcal toxoid and autovaccine - heated at 70-75 ° C washout of agar culture of staphylococcus isolated from the body of a sick animal. Sometimes phage and antivirus filtrate of a 2-3-week culture of staphylococcus are used locally.
