1. Staphylococci, their properties. Classification, factors of pathogenicity of staphylococci. Diseases caused by staphylococci. Epidemiology and features of hospital strains of staphylococci. Laboratory diagnostics, specific prevention and therapy .

The most famous:

    Staphylococcus aureus (Staphylococcus aureus), as the most pathogenic for humans. Named for its ability to form a golden pigment. It can cause purulent inflammatory processes in almost all organs and tissues in humans.

    Staphylococcus epidermidis (Staphylococcus epidermidis) - often found on the skin and mucous membranes of humans, can cause sepsis, endocarditis, conjunctivitis, purulent infection of wounds and purulent infections of the urinary tract.

    Staphylococcus saprophyticus (Staphylococcus saprophyticus) - can cause acute cystitis and urethritis.

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 rounded cells. In the smear are arranged in asymmetrical clusters. The cell wall contains a large amount of peptidoglycan associated with it teichoic acids, protein A. Gram-positive. They do not form spores, they do not have flagella. In some strains, a capsule can be found. Can form L-shapes.

cultural properties: Staphylococci are facultative anaerobes. They grow well on simple media. On dense media, they form smooth, convex colonies with various pigments that have no taxonomic significance. Can grow on high NaCl agar. Possess saccharolytic

kimi 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. As part of cell wall Staphylococcus aureus contains protein A, which can bind tightly 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).

Pathogenic factors: Conditionally pathogenic. The microcapsule protects against phagocytosis, promotes adhesion of microbes; components of the cell wall - stimulate the development of inflammatory processes. Enzymes of aggression: catalase - protects bacteria from the action of phagocytes, β-lactamase - destroys antibiotic molecules.

resistance. Environmental stability and sensitivity to disinfectants are common.

Pathogenesis. The source of staphylococcal infection is humans and some animal species (sick or carriers). Transmission mechanisms - respiratory, contact-household, alimentary.

Immunity: P ostinfectious - cellular-humoral, unstable, unstressed.

Clinic. About 120 clinical forms of manifestation, which are local, systemic or generalized. These include purulent-inflammatory diseases of the skin and soft tissues (boils, abscesses), damage to the eyes, ear, nasopharynx, urogenital tract, digestive system (intoxication).

Microbiological diagnostics . Material for research - pus, blood, urine, sputum, feces.

Bacterioscopic method: smears are prepared from the test material (except for blood), stained according to Gram. The presence of gram "+" grape-shaped cocci, located in the form of clusters.

Bacteriological method: The material is seeded in a loop on blood and yolk-salt agar plates to obtain isolated colonies. The cultures are incubated at 37C for 24 hours. The next day, grown colonies are examined on both media. On blood agar, the presence or absence of hemolysis is noted. On LSA, S. aureus forms golden, round, raised, opaque colonies. Around the colonies of staphylococci with lecithinase activity, cloudy zones with a pearly tint are formed. For the final determination of the type of staphylococcus, 2-3 colonies are inoculated into test tubes with slanted nutrient agar to obtain pure cultures, followed by the determination of their differential characteristics. S.aureus - "+": the formation of plasmacoagulase, lethicinase. Fermentation: glk, minnita, formation of a-toxin.

To establish the source of a nosocomial infection, pure cultures of staphylococcus aureus are isolated from patients and bacteria carriers, 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 seeded on nutrient agar in a Petri dish with a lawn, dried, and then a drop of the corresponding phage is applied in a loop to the squares (according to the number of phages included in the set), previously marked with a pencil on the bottom of the Petri dish. The cultures are incubated at 37°C. The results are evaluated the next day by the presence of culture lysis.

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 antibodies to riboteichoic acid (component of the cell wall).

Treatment and prevention. Broad-spectrum antibiotics (penicillins resistant to β-lactamase). In the case of severe staphylococcal infections that do not respond to antibiotic treatment, antitoxic anti-staphylococcal plasma or immunoglobulin immunized with adsorbed staphylococcal toxoid can be used. Identification, treatment of patients; conducting a scheduled examination of medical staff, vaccination with staphylococcal toxoid. Staphylococcal toxoid: obtained from native toxoid by precipitation with trichloroacetic acid and adsorption on alumina hydrate.

Staphylococcal vaccine: a suspension of heat-inactivated coagulase-positive staphylococci. Used to treat long-term diseases.

Immunoglobulin human antistaphylococcal : gamma-globulin fraction of blood serum, contains staphylococcal toxoid. Prepared from human. blood, with a high content of antibodies. Used for specific treatments.

Staphylococcus aureus

Staphylococcus aureus- facultative anaerobic Gram-positive cocci, non-motile, catalase- and coagulase-positive. Some strains S. aureus produce staphylococcal enterotoxins (SEs) that cause food poisoning. Staphylococci are present in the air, dust, sewage, water, milk, food, as well as on food production equipment, on various surfaces in the environment, on the skin of people and animals. It is people and animals that are the main reservoir of infection. Staphylococci are present in the nose and throat, as well as on the hair and skin in at least 50% of healthy people. Staphylococcus aureus able to grow in a wide temperature range from 7 to 48.5°C (optimum 30 - 37°C); pH 4.2 - 9.3 (optimum pH 7.0-7.5) and at a high concentration of sodium chloride (up to 15% NaCl). These properties allow bacteria to colonize a wide variety of foods. Foods most commonly associated with staph food poisoning are meat and meat products, poultry, eggs, salads (containing eggs, tuna, chicken, potatoes, pasta), confectionery (eg, cream pies, chocolate eclairs), sandwich fillings, milk and dairy products. Thus, foods that are processed manually during cooking and subsequently kept warm can be a real source of staphylococcal food poisoning.

Intoxication with staphylococcal infection

The cause of the disease is produced Staphylococcus aureus toxins, so the disease is characterized by a very short incubation period - usually from 0.5 to 6 hours. The patient's susceptibility to toxins is determined by the patient's condition, the concentration of the toxin, and the amount of contaminated product eaten. The infectious dose may be less than 1.0 µg, which corresponds to approximately 100,000 cfu/g of product. The most common symptoms of the disease are nausea, vomiting, food aversion, abdominal cramps, and diarrhea. The rehabilitation period usually takes 1-3 days, but in severe cases, full recovery may take longer. The disease is not transmitted to others, patients must take a large number liquids.


Pathogenicity, scale of the disease

Coagulose-positive staphylococci are gram-positive, catalase-positive microorganisms that form typical colonies on selective diagnostic nutrient media that are coagulase-positive (S. aureus; S. aureus spp. anaerobius) or rabbit plasma-specific reaction on rabbit plasma fibrinogen agar determined by the standard method. Staphylococcus aureus (Staphylococcus aureus) are coagulose-positive staphylococci that form acetoin and fermenting maltose under aerobic conditions during the determination of these biochemical tests by the standard method.

According to recent studies in developed countries of all known infections, one of the most famous, "ordinary" bacteria, Staphylococcus aureus (Staphylococcus aureus) most often leads to death; all strains of staphylococci that produce coagulase are called golden). This conditionally pathogenic microorganism is isolated from 15-30% of healthy people, and the carriage in most of them is limited to several months. With a deficiency of immunity in a patient, the pathogen is the cause of numerous life-threatening zooanthroponotic infections. The most common toxic staphylococcal disease is food poisoning. Many strains of Staphylococcus aureus produce enterotoxin. This poison causes diarrhea, vomiting, pain and abdominal cramps. According to a New Zealand study, 8.6% of children hospitalized with Staphylococcus aureus sepsis die. Dermatovenereologists in many countries are concerned about the increase in the number of staph infections and deaths due to staph. Bacteria cause food spoilage, including so-called "non-perishable" foods. Breeding, for example, in hard cheese and salami, bacteria release toxins. They are the most common cause of serious food poisoning. During cooking, staphylococcus dies, but its thermostable toxins are not destroyed. Staphylococci are the cause of many human diseases, including cardiac (endocarditis and pericarditis), musculoskeletal (osteomyelitis and infectious arthritis). On the skin and in soft tissues, staphylococcus causes the so-called flaky skin syndrome And cellulites, which are the most frequent infectious diseases skin. According to WHO, Staphylococcus aureus tops the list of bacteria that are most commonly infected in healthcare facilities. There is a high risk of staphylococcal infection when using intravenous catheters and other medical devices that come into contact with internal environment organism. Artificial ventilation is also a risk factor. Staphylococcus infection can also occur when normal hygiene practices in hospitals are violated. In total, S. aureus accounts for about 30% of all "hospital infections". Staphylococci surviving under conditions of constant use of disinfectants and antibiotics are the most serious risk factor for personnel, the basis of hospital infection.


Taxonomy

Staphylococci are a genus of spherical, immobile, asporogenic, Gram-positive, chemoorganotrophic, facultative anaerobic or aerobic bacteria from the Micrococcaceae family. There are about 30 species in the genus Staphylococcus, of which 14 are found on human skin and mucous membranes, including S. aureus, S. epidermidis, S. saprophyticus. For species identification, mainly 3 tests are used: plasmacoagulase production, anaerobic fermentation of mannitol and glucose. The antigenic structure of the bacterium is complex; teicic acids are species-specific antigens.

Morphology of bacteria and colonies

Bacterial cells with a diameter of 0.5-1.5 microns divide asymmetrically in several planes, forming clusters resembling bunches of grapes. There are single cells, pairs and tetrads. The cell wall contains peptidoglycan and glycerinteic acid. Staphylococci form smooth colonies, stained yellow or orange by carotenoids. However, pigmentation is not a specific feature. When growing on yolk-salt agar, cloudy, round, even colonies of cream, yellow or orange color are formed. When cultivated in liquid media, bacteria cause their uniform turbidity, and then the formation of a loose precipitate, which turns into a viscous mass.


Physiological and biochemical characteristics

Staphylococci are facultative anaerobes, but grow rapidly and profusely in the presence of oxygen. Chemoorganotrophs with oxidative and enzymatic metabolism. Biochemicals are very active. They produce catalase, on a medium with glucose under anaerobic conditions, most strains form acetoin (positive Voges-Proskauer reaction). They release ammonia during growth on argenin broth, reduce nitrates to nitrites or molecular nitrogen, actively hydrolyze proteins, and under aerobic conditions break down many carbohydrates to acetic acid and carbon dioxide. A generic feature is the fermentation of glucose under anaerobic conditions, which distinguishes staphylococci from microcci. Bacteria grow on basic media at 37°C (optimum 35-40°C), but can grow in a wider temperature range (6.5÷46°C). The growth optimum was noted at pH 7.0-7.5, but growth is possible in the range of pH=4.2÷9.3. They withstand high osmotic pressure well, therefore, media with a high concentration of sodium chloride - yolk-salt or milk-salt agar - serve as an elective substrate for them. Staphylococci tolerate desiccation well while maintaining virulence; die on direct impact. sunlight within 10-12 hours. Pretty resistant to heat: at 70-80 o C they die in 20-30 minutes, at 150 o C - in 10 minutes; dry heat kills them in 2 hours. Bacteria are resistant low temperatures, repeated freezing and thawing does not kill them. Staphylococci are less resistant to the action of disinfectants (hydrogen peroxide, etc.), but resistant to the effects of pure ethanol. When grown under aerobic conditions, bacteria need amino acids and vitamins, while anaerobic conditions require additional uracil and additional sources of carbon. Several species can form on blood agar. hemolysins- substances that affect erythrocytes, leukocytes and other cells. They also produce fibrinolysin, phosphatase, bacteriocins; some strains produce coagulase, DNase, H 2 S and enterotoxins (up to 10 species characterized by lethal, hemolytic or necrotic action). Some carbohydrates are fermented with the release of acid without gas. Resistant to lysozyme, sensitive to various antibiotics. Species pathogenic for humans have one of 4 toxicity factors, such as: 1) exotoxin, 2) enterotoxin, 3) leukocidin, 4) coagulase (pathogenicity enzyme). Cross-resistance to antibiotics is controlled by the R-plasmid. To other prokaryotes, this resistance factor is transferred by transduction.


Sources and factors of infection transmission

Staphylococci are opportunistic pathogens. They are representatives of the normal microflora of humans and animals - they densely colonize various biotopes of the body (skin, nasal mucosa and oral pharynx, abdominal cavity and axillary areas). Staphylococci are introduced into the body through the skin and mucous membranes by airborne droplets and airborne dust. The mechanism of infection is usually associated with the transfer of the pathogen from the areas of colonization to the traumatized surface (eg, skin). Close contacts with carriers and persons suffering from staphylococcal lesions also play an important role. Staphylococcus is resistant to many external influences, therefore it is found in external environment- air (dust), soil, on household items. Sources and factors of transmission of staphylococcal infection are multiple. The main sources are patients with erased forms of staphylococcal infection or carriers, less often - diseased animals, for example, (with food staphylococcal poisoning and enterocolitis) cows with mastitis. The greatest epidemiological danger is posed by medical personnel who may be carriers of hospital strains of staphylococcus aureus. Staphylococcal infection is characterized by a multiplicity of mechanisms, routes and transmission factors. bacteria can be transmitted contact through a non-sterile medical instrument, the hands of medical staff, alimentary with dairy products and confectionery, aerogenic and parenterally(when injected).

Pathogenesis

Susceptibility to staphylococci is very low in immunocompromised individuals and increased in immunocompromised hosts. Very often, staphylococcal infection develops against the background of secondary immunodeficiencies. The pathogenicity factors of the pathogen are the microcapsule, cell wall components, aggression enzymes and toxins. The microcapsule protects bacteria from phagocytosis, promotes adhesion of microbes and their spread through tissues. When growing in vitro it does not form. The components of the cell wall stimulate the development of inflammatory reactions and neutralize immunoglobulins, immobilize phagocytes. Teicic acids trigger a cascade compliment(component of immunological reactions) by an alternative route. The staphylococcal aggression enzyme coagulase exists in three antigenic forms and causes blood serum to clot. In a number of pathological conditions of the body, staphylococci leave their usual biotopes, overcome tissue barriers and are introduced by the bloodstream into the internal sterile environment of the body. There they cause a typical pathological reaction - inflammation. It manifests itself in the form of purulent-inflammatory processes of various localization and severity, up to sepsis and septicopyemia. Staphylococci are the etiological factor of diseases, the vast majority of which are purulent-inflammatory in nature. Bacteria can infect almost any tissue in the body. Infections caused by S. aureus are diverse and include more than 100 nosological forms, including diseases: 1) skin and subcutaneous tissue(abcesses, felons, furunculosis, etc.), 2) respiratory system(tonsillitis, pleurisy, pneumonia, etc.), 3) nervous system And sense organs(meningitis, otitis, conjunctivitis, etc.), 4) digestive organs(stomatitis, peritonitis, enteritis, enterocolitis, food intoxication, etc.), 5) musculoskeletal system And connective tissue(arthritis, osteomyelitis), 6) blood circulation(endocarditis, phlebitis, etc.), 7) urinary organs(cystitis, mastitis, etc.), 8) staphylococcal sepsis. These diseases can be acute or chronic. food poisoning clinically manifest in the form of vomiting and watery diarrhea already 2-6 hours after eating infected foods - confectionery with cream, canned food, meat and vegetable salads, etc.


Detection methods

On selective differential media, as a rule, only colonies of staphylococci grow. Assume the staphylococcal etiology of the disease allows the isolation of staphylococcus in pure culture. Biomaterial for research is selected depending on the clinical picture of the disease. In strains of S. aureus isolated from food staphylococcal intoxication, the presence of enterotoxins is determined in biological and immunological tests. Traditional methods preliminary results by identification of staphylococcal infection can be obtained in 1-2 days.

Classic method

The method for detecting S. aureus is based on inoculation of a sample of the product and/or dilutions of its sample into a liquid selective medium, incubation of the inoculations, subculturing of the culture liquid on the surface of an agar selective diagnostic medium, confirmation by biochemical signs that the identified typical and/or atypical colonies belong to S. aureus.


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PRIVATE MEDICAL MICROBIOLOGY
PATHOGENIC COCCI
Pathogenic cocci that cause various clinical manifestations of diseases in humans include: 1) staphylococci, 2) streptococci, 3) pneumococci, 4) meningococci and 5) gonococci.
According to the tinctorial sign (staining), they can be divided into two subgroups: gram-positive cocci (staphylococci, streptococci and pneumococci), gram-negative cocci (meningococci and gonococci).
characteristic feature all pathogenic cocci is the ability to cause purulent inflammatory processes, which is why they are also called pyogenic (pyogenic) cocci.
Most sharply and constantly pyogenic properties are expressed in staphylococci.
STAPHYLOCOCCUS
Morphology and tinctorial properties. Staphylococci - Staphylococcus (first isolated by Pasteur in 1880) have the shape of regular balls with an average diameter of 0.8-0.9 microns. In a smear from a pure culture, staphylococci are located in the form of heaps and clusters, resembling a bunch of grapes (Fig. 60). In pus and other pathological material, one can often find single cocci, paired and even short chains. They stain well with basic aniline dyes, are Gram-positive, do not form spores, do not form capsules, and are immobile.
Cultural and biochemical properties. Staphylococci grow well on conventional nutrient media both in the presence and in the absence of free oxygen. The temperature optimum is 37°.

Rice. 60. Staphylococcus (a) and streptococcus (b) in pure culture.


Rice. 61. Staphylococcus colonies on meat peptone agar. SW. 20.

Rice. 62. Sowing by injection into gelatin.

Individual colonies on the surface of the agar are rounded disks. medium size, convex, wet, opaque, homogeneous or fine-grained (see fig. 01). The color of the colonies depends on the pigment produced by staphylococci and can be golden (Staphylococcus aureus), lemon yellow (Staphylococcus citreus) and white (Staphylococcus albus). When sowing with a stroke on a slant agar, an abundant growth of staphylococcus, pigmented, is formed.
Staphylococcus pigments are lipochromes, are insoluble in water and stain only the culture, but not the nutrient medium. Pigment formation is intense when microorganisms are grown in the presence of oxygen, at room temperature, in the light.
When sowing by injection into gelatin on the 2-3rd day of growth, a zone of liquefaction is noticeable on the surface of the medium. As the culture liquefies, it falls to the bottom in the form of a cottony sediment (Fig. 62).
Staphylococci grow profusely in broth. After 24 hours in the thermostat, the broth becomes cloudy and a precipitate forms at the bottom of the tube.
Staphylococci quickly curdle milk, giving it an acidic reaction.
Fermented (without gas) lactose, glucose, maltose, mannitol. The ability to break down mannitol usually characterizes pathogenic strains of staphylococcus aureus.
Pathogenic staphylococci on blood agar plates, they form colonies surrounded by a hemolysis zone and coagulate citrated blood plasma.
resistance. Staphylococcus is one of the most resistant microbes. A temperature of 80° kills it when wet within 10 minutes. The temperature of 70 ° staphylococci can withstand up to 1 hour. Carbolic acid (3-5% solution) kills them within 15-30 minutes. In pus, staphylococcus persists for a long time.
Toxins, enzymes of staphylococci, their pathogenicity for animals. Pathogenic staphylococci produce a number of toxins and enzymes. Staphylococcal hemolychin dissolves red blood cells. This toxin is thermolabile (destroys at 55°C), belongs to exotoxins, and when animals are immunized with it, an antitoxin can be obtained. The filtrate of staphylococcus cultures also has a necrotic and lethal effect. It has now been proven that hemolysin, necrotoxin and lethal toxin are components of a single staphylococcal exotoxin. These toxins are inseparable from each other, and the detection of one of them serves as evidence of the pathogenicity of the isolated staphylococcus aureus. Some of the hemolytic strains of Staphylococcus aureus produce an enterotoxin that causes acute gastroenteritis. In the filtrates of staphylococcus cultures, leukocidin, a poison that destroys leukocytes, is often found. Of the enzymes, pathogenic staphylococci secrete: 1) plasmocoagul and z y - coagulating plasma, 2) fibrinolysin - dissolving fibrin clots, 3) hyaluronidase - dissolving hyaluronic acid, a substance that glues connective tissue elements, 4) penicillinase - inactivating penicillin and 5) lecithinase - forms cloudy zone around the colonies on yolk-salt agar (Chistovich's medium).
Of laboratory animals, rabbits are most sensitive to staphylococcus and, to a lesser extent, Guinea pigs and mice. In rabbits with intradermal injection of culture, pronounced necrosis appears, with subcutaneous injection, abscesses develop, and infection with highly virulent strains of staphylococcus leads to sepsis and death of animals. To staphylococcal enterotoxin, suckling kittens and young mice are most sensitive.
Classification of staphylococci. The old classification of staphylococci according to the pigment they secrete (golden, white and lemon yellow) does not make it possible to correctly determine their pathogenicity. Therefore, at present, the classification of staphylococci proposed by Gross is considered the most correct. According to this classification, all staphylococci are divided into three groups: pathogenic, slightly pathogenic and non-pathogenic.
The first group - pathogenic staphylococci:
a) on plates with 5% blood agar form a clear zone of hemolysis;
b) when administered intradermally to a rabbit, they cause phenomena of pronounced necrosis, sometimes ending in the death of the animal;
c) produce the enzyme plasmocoagulase, which causes plasma coagulation within 2 hours;
d) are isolated mainly from patients with sepsis or acute purulent infection.
The second group - weakly pathogenic staphylococci:
a) on cups with blood agar form a zone of mild incomplete hemolysis;
b) when administered intradermally to a rabbit, reddening of the skin is caused at the injection site, sometimes an infiltrate, more often without necrosis;
c) when inoculated into citrate plasma, they cause clotting within 6 hours and later;
d) are excreted from the surface of the skin in case of folliculitis, from the mucous membranes in case of catarrh of the upper respiratory tract, from the surface of wounds, but not from their depths.
The third group - non-pathogenic staphylococci:
a) hemolysis is not detected on blood agar plates;
b) when administered intradermally to a rabbit, necrosis and infiltration are not observed, in some cases redness of the skin appears;
c) plasma coagulation reaction is negative;
d) are found constantly on healthy skin, on the mucous membranes of the pharynx, nose and in the air.
Pathogenesis and diseases in humans. Staphylococci are very common microbes. They are found in air, water and soil, in humans - almost constantly on the skin and mucous membranes. This explains the fact that staphylococcus is the most common causative agent of all kinds of suppuration. Damage to the skin and mucous membranes or a decrease in their resistance opens the "gate" for staphylococcal infection.
The skin is most often affected. Staphylococci are the causative agents of boils - purulent inflammation of the hair follicles. If boils become multiple, the disease is called furunculosis. When several boils merge together, forming a more extensive purulent infiltrate, a carbuncle occurs.
Staphylococci are found in abscesses (abscesses), in festering wounds, with catarrh of the mucous membranes, septicemia and septicopyemia (sepsis with the formation of purulent foci in the internal organs). Enterotoxic strains of Staphylococcus aureus can cause food poisoning. The latter are observed as a result of the use of staphylococcus-infected milk and other dairy products, cream cakes, etc., since staphylococcus finds optimal conditions for its reproduction in these products. Pathogenic staphylococci can get into milk either from cows with mastitis or from the hands of milkmaids suffering from pyoderma.
Immunity. There is no innate immunity to staphylococcus aureus in humans. However, a person has significant resistance to this microorganism, which is explained by the protective role of the skin and the presence of antibodies in the blood.
Apparently, acquired immunity can take place, as evidenced by an increase in antibody titer in patients and recovered patients. However, this immunity is unstable. Moreover, it has been noted that many people who have had furunculosis have an increased susceptibility to staphylococcus aureus.
Resistance to staphylococcus can be weakened due to various reasons - diabetes, injury, debilitating diseases, etc.
Microbiological diagnostics. The study of pus. The most common material for microbiological research is pus. For this purpose, with open lesions, after preliminary cleaning of the wound surface with a sterile cotton swab, the discharge is removed from the deeper parts of the wound. In closed processes (furuncle, abscess), for the same purpose, after disinfection of the skin, a puncture of the focus of suppuration is performed with a sterile syringe. From the resulting pus, smears are made, stained with blue and Gram, and microscoped.
According to the location of cocci and tinctorial properties, it is difficult to differentiate between staphylococci and streptococci, therefore, they proceed to bacteriological examination, i.e., to sowing pus on plates with simple, blood agar, on yolk-salt agar (see section "Sanitary microbiology" p. 454) and on the enrichment medium (sugar broth). The seeded cups, turned upside down, and the broth are placed in a thermostat at 37 ° for 1-2 days.
On blood agar plates, the colonies form zones of hemolysis, often several times the diameter of the colony. These clear, colorless areas of hemolysis were formed due to the dissolution of red blood cells caused by staphylococcus hemotoxin. On plates with yolk-salt agar, pathogenic staphylococci grow with the formation of a golden pigment and a turbidity zone - in the form of an iridescent corolla around the colony. A preparation is prepared from one of the colonies, stained by Gram and microscoped. The detection of gram-positive cocci in the preparation, arranged in the form of bunches of grapes, gives the right to issue an indicative positive result. The remainder of the colony is then subcultured onto agar slant to obtain a pure culture. It is necessary to differentiate pathogenic Staphylococcus aureus from non-pathogenic, i.e., study growth on blood agar, yolk-salt agar, inoculate in nitrate plasma and determine toxigenicity in animals.
Plasma coagulation test. For the plasma coagulation reaction, G. V. Vygodchikov recommends the following method. The rabbit takes 10 ml of blood from the heart, placed in a test tube with 1 ml of 5% sodium citrate solution. The blood is centrifuged and the plasma is aspirated. The resulting plasma before the experiment is diluted with saline 1:4 and poured into sterile tubes of 0.5 ml. Staphylococcal colonies are seeded with a loop in test tubes with nitrate rabbit plasma, placed in a thermostat at 37 ° and plasma coagulation is checked every 2-3 hours. Pathogenic staphylococci coagulate plasma within 2-10 hours, most often within 4 hours.
Toxigenicity test. Toxigenicity of staphylococcal cultures is determined by experience on rabbits, and enterotoxin - on suckling kittens. A suspension of microbes in physiological saline is prepared from rounds of staphylococcus on agar slant and set according to an optical standard with the calculation of 2 billion microbial bodies in 1 ml of suspension. On the side or back of the rabbit, the wool is plucked and 0.2 ml of microbial suspension is injected intradermally into the animal. With a positive reaction at the injection site, an infiltrate is formed, and then necrosis.
To detect a lethal toxin, a staphylococcus broth culture filtrate is injected intravenously into a rabbit at the rate of 0.75 ml per 1 kg of body weight. The death of the animal occurs after 15 minutes.
To determine enterotoxin, kittens 1.5-2 months of age (weighing 400 g) are fed with the original infected product or milk culture of staphylococcus aureus. Signs of poisoning (diarrhea, sometimes vomiting) occur after 30-60 minutes. Observation is carried out for 4-6 hours, and if during this time the kittens do not have any reaction, the biological test is considered negative.
Blood study. If sepsis is suspected in a patient, 10 ml of blood is sterilely taken from the cubital vein with a syringe and inoculated in 100-200 ml of sugar broth. Sowing is placed for enrichment for 24-48 hours in a thermostat. In a positive case, the medium becomes cloudy, and Gram-positive cocci are found in smears from it, which have a characteristic shape and location. The broth is inoculated into a blood agar plate and further research is carried out as described above. The broth is kept in a thermostat for 10 days, inoculating every 2 days on 5% blood agar.
Study of other materials. When examining materials contaminated big amount foreign microbes (feces, sputum), it is recommended to use agar with 7.5% sodium chloride, to which 10% warm skimmed milk is added (Petrovich medium). On such agar, the growth of foreign microflora is significantly suppressed.
Phage typing. Phage typing uses a set of typical staphylococcal phages, consisting of 21 phages, divided into four groups. Each typical phage is able to reproduce only on the corresponding strain of staphylococcus. Phage typing of staphylococci special meaning at sa-
sanitary and bacteriological examination of food products to identify the source of infection and establish epidemic links.
specific therapy and chemotherapy. The staphylococcal vaccine is widely used to treat recurrent and chronic infections, such as furunculosis. Toxoid is used to prevent these diseases.
In the treatment of staphylococcal infections, chemotherapeutic drugs of the sulfanilamide series (norsulfazol, sulfodimesin, etc.), antibiotics (penicillin in large doses, erythromycin, oxacillin, etc.), staphylococcal bacteriophage (for skin lesions) are also successfully used. In severe staphylococcal sepsis, hyperimmune anti-staphylococcal plasma and gamma globulin obtained from donors immunized with toxoid are recommended.

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Staphylococcosis in hospitals 40-60%

At present, the Staphylococcus genus no longer belongs to the Micrococcaceae family (since the early 1990s). The family Micrococcaceae does not exist (because there is no genetic relationship between its components). They were united only on the basis of gram-positive aerobic and facultative anaerobic cocci, dividing in several planes.

Micrococcus is a genetic relative of actinomycetes.

Staphylococcus is a genetic relative of Clostridia.

In studies for fusobacteria, pravotella, bacteroids (strict anaerobes) on an oxygen-free medium, if you do not use a medium with antibiotics (aminoglycosides), then staphylococci (and enterobacteria) will kill everything !!!

Depleted Seeding Method


Staphylococci are facultative anaerobes. Micrococcus - strict aerobes

Differentiation is a test with aerobic and anaerobic breakdown of glucose. All Staphylococcus glucose+ without gas under vaseline oil. Micrococcus is not.

Staphylococcus - more than 30 species, with subspecies - 44. They are differentiated by 33 tests.

The boundary of halotolerance is a medium with 7.5% or more NaCl.

Micrococcus are still growing by 6.5%, and only Staphylococcus is higher. (HSA - 10% NaCl). Some types of 15% NaCl.

Milk-salt agar - micrococci grow, because there is NaCl below.

The bourgeois have Chapman's medium - mannitol-salt agar (1% mannitol) - 7.5% NaCl. Mannitol = mannitol, as glycerol = glycerol

Targeted isolation of Staphylococcus (for example, in sanitary and bacteriological studies). Yellowing zone due to acid formation. As its alternative, we have JSA.

Those. if we isolate cocci on a medium with ≥ 7.5 NaCl, then they are always glucose +, because it is Staphylococcus and Micrococcus does not grow

The morphology of micrococci is slightly different from staphylococci - both colonies and cells larger than staphylococci.

Each person has 7-10 species of micrococci on the skin (and only about 15 species), M. luteus - prevails - large yellow colonies on non-inhibitory media (the old name is air sarcins, true sarcins do not grow under aerobic conditions !!!). Gram stain evenly. In the form of heaps, notebooks, packages. Others are white, colorless, etc. M.livi - enamel white. M.cristi - red colonies.

Staphylococcus Gram stains unevenly (calico color). Clusters - more often gives S. aureus, and so heaps, tetrads, packages, etc. Staphylococci from liquid steda - single, pairs, heaps.

On media without yolk, the S. aureus pigment is not formed and the colonies are gray.

Carotenoids do not dissolve in water and the environment around the colony never stains in Staphylococcus and Micrococcus.

Angelina Hess - introduced agar into media in the middle of the 19th century

Micrococcus is also UPMF - sepsis. In vitro sensitive to everything. In animals, penicillin - 5 units per injection - at the level of the invention of penicillin - treats sepsis. But it is very difficult to treat in vivo. Sepsis in patients with cardiac bypass surgery. If they enter the heart-lung machine.

Planococcus - we are not interested - in sea ​​water and not dangerous to humans

Stomatococci - 1 species. They grow well on KA (everything grows on KA), but they do not grow on salt media. They have no special clinical significance. UPMF. Very rarely cause gingivitis and stomatitis (but more often these diseases are caused by anaerobes and viruses).

S. aureus subsp. aureus

S. aureus subsp. anaerobius - does not grow under aerobic conditions (strict anaerobe)

There are no saprophytes among staphylococci - all opportunistic pathogens. More than 86 nosological forms of diseases.

S. aureus and S.epidermidis - 45% of all staphylococcosis. Food poisoning - only S. aureus (S. epidermidis - can only cause in infants). Enterotoxins are produced only by S. aureus.

Each person has 10-12 types of staphylococci living on the skin.

The anterior parts of the nose are the main biotope. There is the most per unit area.

S. epidermidis can also, along with S. aureus, cause outbreaks of nosocomial infections.

In newborns: S. epidermidis conjunctivitis earlier in Moscow in the 70s; hospital pneumonia S. epidermidis now.

Plasmids are transferred at a rate of about 2 hours (2 hours a staphylococcus aureus was on the skin of another person and received a new plasmid).

Carrying.

In obstetric institutions, a planned study for staphylococcus aureus is not carried out (only in case of outbreaks) - order 345. The surgical profile still has order 720 (it is not carried out anywhere in the world). The number of staphylococcal infections is the same among the “bourgeois” and among us, regardless of sanitation.

By test systems, up to 12-15 types of staphylococci and several types of micrococci can be differentiated.

Order 691 - for maternity hospitals cancelled. The new order 345 does not have a bacteriological part, therefore everyone is still guided by order 691 in bacteriology.

Scheduled screening for staphylococcus carriage has been cancelled.

There are now 5 types of coagulase-positive staphylococci.

coagulase

maltose

flocculation

Hemolysis on KA with human blood

H/D day

Almost 100%

± less than 50%

H/h night

S. aureus does not give coagulase very rarely - with primary crops from a patient with massive antibiotic therapy. Therefore, we transfer to the jamb and then put the plasma again. If not, it is not S. aureus.

Pigment on media with a high protein content and light (HSA, serum medium with protein)

Among coagulase-negative staphylococci, some can also form pigment. S promagenes - forms a yellow pigment on any medium

Acetoin=acetylmethylcarbinol - VP reaction (Voges-Proskauer)

His medium with glucose is semi-liquid. When sowing from saline medium, they are always glucose-positive, at least in the upper part of the column.

The final negative response to the semi-liquid His medium with glucose is issued only on the 5th day.

If gas - we assume that it is mixed or not staphylococcus at all.

The semi-liquid medium with mannitol was excluded in 1974 - the final negative answer was only on the 5th day. It is put only on a cup - the answer is in the morning.

Solid medium with glycerol (1%) (can be replaced by any sugar and alcohol). Liquid and semi-liquid media for differentiation within the genera Staphylococcus and Micrococcus are never used. Indicator - bromthymol blue, pH - neutral; or bromcresol red, bromthymol red. Sow in plaques - up to 10 cultures per cup. Radial stripe - up to 6 cultures per plate. Around staphylococcus - yellowing. Micrococci do not utilize glycerin.


S. aurecularis on test systems, it gives 95-99% similarity to S. aureus, and it can be distinguished only by the plasma coagulation reaction.

Coagulase-negative staphylococci

Under certain conditions, they can give a pigment.

In staphylococci, it is impossible to differentiate to the species by 1-2 tests.

It grows in plasma in the form of Mannitol + flakes, only 6-7 species.

coagulase

Alkaline phosphatase

trehalose

More than 20% strains

5-10% strains

95-100% strains

5% strains

S.saprophiticus

5% strains

100% of S. aureus has hemolytic activity.

S. saprophiticus is very rare. Cystitis and urethritis in women young age- sensitive to antibiotics and chemotherapy drugs

Coagulase-negative cannot become coagulase-positive.

S. epidermidis is never coagulase positive, no matter what order 720 says

A non-spore cell can live 10-12 days.

Alkaline phosphatase is characteristic only of S. epidermidis.

Alkaline phosphatase - paranitrophenol phosphate or phenolphthalein phosphate from tests for ALP in biochemistry - 50 mg per 100 ml of agar up to 20 strains per plate.

Paranitrophenolphosphate → paranitrophenol (yellow) and the color of the medium is lemon yellow.

Phenolphthalein phosphate → phenolphthalein (colorless). We alkalize with 3-10% ammonia solution (we drop it on the lid of the cup) Raspberry colonies.

You can buy 10% solution of phenolphthalein phosphate. Keeps in the refrigerator for years. 0.5 ml solution per 100 ml agar.

S. epidermidis is naturally sensitive to novobiocin.

Lecitovitelase (yolk factor) - in international classification not used, only with us.

20-25% of S. epidermidis are lecithinase positive.

80% of S. aureus - human origin - give lecitovitelase. Lecithinase-negative S. aureus - from animals.

Other staphylococci can also give. That is, if lecitovitelase is +, then it is 80% S. aureus, 20% other staphylococci.

Cups of HSA in the light - the zone of enlightenment (protease) - it is not important to us.

Turbidity - dominated by lipases - is also not important.

Bourgeois blood media - from ram erythrocytes. It was believed that human strains of S. aureus produce only α-hemotoxin, but now there is also β-hemotoxin. This can only be disassembled on ram erythrocytes.

β-hemotoxin gives a zone of incomplete hemolysis in a day, and if you then put it in the refrigerator until the end of the working day, the zone will completely clear up.

S. aureus produces 2 enzymes

1) thermolabile DNase (nuclease) - all staphylococci and most other microbes have it.

2) Thermostable DNase (nuclease) - only S. aureus has it. Will not be destroyed by boiling and even short-term autoclaving.

And according to our orders, only thermolabile DNase is determined (it makes no sense).

In heated food (while hiding the fact of PTI), staphylococcus is destroyed, and toxins remain. By the presence of thermostable DNase, it can be determined whether S. aureus was present.

According to the orders of highly polymerized DNA 1-2 mg per 1 ml of medium (pH=8.0). Sowing on a cup with plaques (or injection). 24 hours. We develop by filling the cup with 3N HCl, a precipitate of DNA with HCl is formed, and where the DNase has worked, we see the clearing zone. In S. aureus, its radius is 10-12 mm; in S. epidermidis, its radius is 4-5 mm. Around the hole with the killed culture of S. aureus - 7-8 mm.

S. epidermidis forms only thermolabile DNase, S. aureus forms both DNases - the zone is larger.

Table of contents of the subject "Gram-positive cocci. Staphylococci.":









Microbiological diagnosis of Staphylococcus aureus. Microscopy of Staphylococcus aureus. Isolation of Staphylococcus aureus. coagulase test.

Microscopy of Staphylococcus aureus. Identification of accumulations of gram-positive cocci and polymorphonuclear leukocytes in the study of stained smears of clinical material can serve as the basis for a preliminary diagnosis. It should be remembered that the results of microscopy cannot be considered sufficient for issuing a final conclusion.

Isolation of Staphylococcus aureus

culture of Staphylococcus aureus carried out on simple nutrient media, usually on a thio-glycol medium and KA. If there is a risk of sample contamination, differential diagnostic media are used. The most commonly used milk-salt (or milk-yolk-salt) agar and salt agar with mannitol, on which the growth of contaminating microflora is inhibited by a high concentration of NaCl. In addition, on milk-salt agar (MCA), the ability to pigment formation and decomposition of lecithin (lecitovitelase activity) is well manifested. IN Lately agar with colistin and nalidixic acid has found widespread use as a differential diagnostic medium.

After 18-24 hours Staphylococcus aureus (S. aureus) forms smooth, raised, cloudy colonies about 4 mm in diameter. Bacteria synthesize a yellow pigment, the color of the colonies varies from white to orange. On CA, S. aureus colonies are surrounded by a zone of complete hemolysis (Fig. 1, see color insert).

Staphylococci grow well on the broth, first causing it to become evenly cloudy, and then forming a loose, flocculent sediment. They give a very characteristic growth in gelatin; after 24-28 hours (along with abundant growth along the puncture of the microbiological needle), an initial liquefaction of the medium is observed, and on the 4th-5th day a funnel open downwards is formed, filled with a liquefied medium.

For intraspecific differentiation Staphylococcus aureus (S. aureus) use a coagulase test (for the presence of a clotting factor), positive in 95% of the isolates (Fig. 12-3). There are several other distinguishing features.

The ability to ferment mannitol under anaerobic conditions.
Lecitovitelase activity - the formation of a pearly precipitate - "corolla" surrounding the colonies grown on media with the addition of egg yolk. The precipitate consists of phosphorylcholine formed from egg yolk lecithin by the action of an enzyme.
The ability to synthesize thermostable DNase.
The ability to agglutinate sensitized ram erythrocytes (the latter test detects protein A, clotting factor, or both).

For Express Identification Staphylococcus aureus (S. aureus) apply a latex agglutination test using commercial kits of latex particles loaded with AT, such as "Staphylatex" (American Microscan).