Microbiological diagnosis of Staphylococcus aureus. Microscopy of Staphylococcus aureus. Isolation of Staphylococcus aureus. Coagulase test. Staphylococcus and staph infections

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 located in asymmetrical clusters. The cell wall contains a large amount of peptidoglycan, teichoic acids associated with it, and protein A. They are Gram-positive. They do not form spores and have no flagella. In some strains a capsule can be found. Can form L-forms.

Cultural properties: Staphylococci are facultative anaerobes. Grows well on simple media. 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, and coagulase.

Staphylococci are flexible and quickly become resistant to antibacterial drugs. A significant role in this is played by plasmids, transmitted with the help of 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 cell wall of staphylococcus 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 (phagotype) 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 and promotes microbial adhesion; cell wall components – stimulate the development of inflammatory processes. Aggression enzymes: catalase – protects bacteria from the action of phagocytes, β-lactamase – destroys antibiotic molecules.

Resistance. Resistance in the environment and sensitivity to disinfectants is common.

Pathogenesis. The source of staphylococcal infection is humans and some species of animals (patients or carriers). Transmission mechanisms: respiratory, household contact, nutritional.

Immunity: P ostinfectious – cellular-humoral, unstable, uncharged.

Clinic. About 120 clinical forms manifestations that 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, and digestive system (intoxication).

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

Bacterioscopic method: Smears are prepared from the test material (except blood) and stained with Gram. The presence of gram “+” cluster-shaped cocci, located in the form of clusters.

Bacteriological method: The material is inoculated in a loop onto blood and yolk-salt agar plates to obtain isolated colonies. The 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. On FSA, S. aureus forms golden, round, convex, opaque colonies. Around colonies of staphylococci with lecithinase activity, zones of turbidity with a pearlescent tint are formed. To definitively establish the type of staphylococcus, 2-3 colonies are subcultured into test tubes with slanted nutrient agar to obtain pure cultures, followed by their determination differential features. S.aureus – “+”: formation of plasmacoagulase, leticinase. Fermentation: glk, minnita, formation of a-toxin.

To establish the source of a hospital infection, pure cultures of staphylococcus are isolated from patients and bacteria carriers, after which they are phage-typed using a set of standard 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 in a loop onto squares (according to the number of phages included in the set), previously marked with a pencil on the bottom of the Petri dish. The crops are incubated at 37 °C. The results are assessed the next day based on 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. The antibody titer to riboteichoic acid (cell wall component) is determined.

Treatment and prevention. Broad-spectrum antibiotics (beta-lactamase-resistant penicillins). In cases of severe staphylococcal infections that cannot be treated with antibiotics, antitoxic antistaphylococcal plasma or immunoglobulin immunized with adsorbed staphylococcal toxoid can be used. Identification and treatment of patients; conducting a routine examination of medical staff, vaccination with staphylococcal toxoid.

Staphylococcal toxoid: obtained from the native toxoid by precipitation with trichloroacetic acid and adsorption on aluminum oxide hydrate.

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

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

56. Types and mechanisms of bacterial nutrition.

Types of food. Microorganisms need carbohydrate, nitrogen, sulfur, phosphorus, potassium and other elements. Depending on the carbon sources for nutrition, bacteria are divided into autotrophs using carbon dioxide C0 2 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 being oxidized, called an electron or hydrogen donor, microorganisms are divided into two groups. Microorganisms that use inorganic compounds as hydrogen donors are called lithotrophic (from the Greek lithos - stone), and microorganisms that use organic compounds as hydrogen donors are called organotrophs.

Considering the energy source, among bacteria there are phototrophs, i.e. photosynthetic (for example, blue-green algae, which use light energy), and chemotrophs, which require chemical sources of energy.

Mechanisms of nutrition. The entry of various substances into the 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 entry of substances The cell contains the cytoplasmic membrane. Conventionally, four mechanisms for the penetration of nutrients into the bacterial cell can be distinguished:: These are 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 occurs without energy consumption.

Facilitated diffusion also occurs as a result of the 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 that are localized in the cytoplasmic membrane and have specificity. Each transporter transports a corresponding substance across the membrane or transfers it to another component of the cytoplasmic membrane - the transporter itself. Carrier proteins can be permeases, the site of synthesis of which is the cytoplasmic membrane. Facilitated diffusion occurs without energy consumption; substances move from higher to lower concentrations.

Active transport occurs with the help of permeases and is aimed at transferring substances from lower concentrations towards higher ones, 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, phosphorylated.

The release of substances from the cell occurs through diffusion and with the participation of transport systems.

Staphylococci. general characteristics

Staphylococcus usually found in clusters resembling a bunch of grapes. Individual cocci, approximately 1 μm in diameter, tend to unite into clusters, since their division occurs in three mutually perpendicular planes and the daughter cells maintain a peculiar spatial group arrangement. Under special conditions, they can be arranged singly, in pairs, or in the form of short chains. They are gram-positive, non-motile, do not form spores and grow actively on almost all artificial media, usually forming opaque, smooth, shiny colonies.

Because the staphylococci produce catalase, hydrogen peroxide, which is formed as a metabolite under aerobic conditions, is not toxic to them, and, for the most part, they grow better in the presence of oxygen. However, they easily tolerate the absence of oxygen, and some of them are even strict anaerobes. They grow best at temperatures of 25-35°C, but can grow at 8°C and at temperatures above 48°C.

When cultivated on blood agar under aerobic conditions, pigments are formed - from golden to lemon yellow and white. The golden pigment gave the name to one of the types of staphylococcus - Staphylococcus aureus. However, at the same time, some strains Staphylococcus aureus can also produce white pigment.

Staphylococcus more resistant to heat, light, drying than other bacteria, extreme temperatures and chemical agents. They can withstand 60°C for an hour, and some strains can even withstand 80°C for 30 minutes, although most vegetative forms of bacteria die when exposed to 60°C for 30 minutes.

Due to its resistance to drying out staphylococci can be carried with dust particles, can persist for weeks and months in dried pus or sputum. Another feature of staphylococci is their stability in a salt environment (they do not die at NaCl concentrations of up to 15%). In this regard, they can be preserved in canned foods (preserves). In food products preserved by salting, staphylococci can grow and produce enterotoxin.

These microbes are resistant to phenol and most other disinfectants and are sensitive to basic dyes. They tend to develop resistance to sulfonamides and antibiotics. About 80% of Staphylococcus aureus strains are resistant to penicillin.

The genus Staphylococcus is represented by three species:

1. Staphylococcus aureus;

2. Staphylococcus epidermidis;

3. Staphylococcus saprophyticus.

Species differ mainly in biochemical properties and enzymes produced. Staphylococcus aureus ferments mannitol under anaerobic conditions and produces coagulase, whereas the other two species lack these properties.

Staphylococcus saprophyticus is primarily saprophytic, as evidenced by its name. It appears to be potentially pathogenic and has limited invasiveness. Can cause urinary tract infection.

Staphylococci. Toxic products

Staphylococcus produce many products with pronounced toxic properties. Probably no other microbe produces them in such quantities. Among them are extracellular toxins, hemolysins (staphylolysins), enzymes. All of them, to one degree or another, determine the pathogenicity and virulence of the microbe. No single strain is capable of producing all toxic products at once.

Hemolysins- these are exotoxins that act directly on the cell membrane, as a result of which lysis of red blood cells, leukocytes, platelets of macrophages occurs and damage to many tissues develops. The action of hemolysin probably explains the fatal outcome of many cases of staphylococcal infections.

When a staphylococcus culture grows on blood agar, hemolysis manifests itself in the form of a clearing zone (p-hemolysis). Unlike streptococcus, staphylococcus does not cause partial hemolysis (a-hemolysis). It should be noted that greek letters are used to designate immunologically different types of staphylococcal hemolysins, while in streptococci these letters indicate the type of hemolysis - complete or incomplete. For example, hemolysin, designated as a-hemolysin (a-lysine, a-toxin) in staphylococci, means the presence of a light zone around the colonies on blood agar.

Cytotoxin- one of the most important factors virulence of staphylococci, causes platelet aggregation and selectively acts on the smooth muscles of small veins.

Leukocidin- a non-hemolytic exotoxin that destroys white blood cells. It causes degranulation of polymorphonuclear neutrophil leukocytes and macrophages.

Enterotoxin- an extracellular toxin that is produced by about 50% of coagulase-positive strains and which causes most cases of food poisoning. The toxin acts directly on the vomiting center of the central nervous system. Toxin production is due to phage conversion. There are 5 types of toxin - A, B, C, D, E. The accumulation of enterotoxin in contaminated food leads to food poisoning with gastroenteritis syndrome, which is not an infection in the usual sense of the term, it is rather toxemia. The presence of a toxin in a suspected food can be determined immunologically, for example, in the precipitation reaction.

Exfoliatin(exfoliative toxin) is a toxin whose production is determined by a plasmid. Selectively damages certain skin cells in such a way that large areas of the skin can peel off completely. Newborns and young children are especially sensitive to the effects of the toxin. This lesion is called "scalded skin syndrome."

Coagulase, an important extracellular enzyme produced only by some staphylococci (coagulase-positive), causes the formation of a blood plasma clot. In the laboratory, the determination of coagulase is used as the only reliable evidence of the pathogenicity of the isolated strain. In non-virulent strains, attempts to detect coagulase usually end in failure. These strains are designated as coagulase-negative. In the presence of this enzyme and the manifestation of its action, individual cocci are covered with a layer of fibrin and, thus, they are reliably protected from attack by phagocytes.

The ability to produce coagulase correlates with the presence of other toxic products in these strains. Coagulase + staphylococci can also have an adhesive factor - this is a cell-associated, but antigenically different form of coagulase that causes rapid adhesion of cells emulsified in a drop of plasma.

Staphylococcal lipases- enzymes that destroy lipids cellular structures and blood lipoproteins. Staphylococci utilize metabolites of skin structures and therefore are able to intensively colonize (populate) the surface of the skin. The formation of lipase gives this microbe the ability to invade healthy skin n subcutaneous tissue with the formation of local abscesses. Strains without lipase are more often associated with generalized infection.

Hyaluronidase(distribution factor, invasion), which is produced by more than 90% of pathogenic staphylococci, increases tissue permeability for cocci and their toxic substances. Causes degradation of hyaluronic acid, which connects tissue cells.

Nuclease, present in 90-96% of S.aureus breaks down DNA and RNA. The nuclease of S. aureus is thermostable, the nuclease of coagulase-negative staphylococci is thermolabile.

Staphylokinase dissolves fibrin clots and, accordingly, promotes the spread of local, initially limited, infection.

Staphylococci. Pathogenicity.

Some staphylococci non-pathogenic, others (S. aureus) cause severe infections, S. epidermidis, although sometimes causing mild, limited lesions, is generally considered non-pathogenic, except in some unusual medical situations, such as when introduced into the body for medicinal purposes technical devices foreign to body tissues. In general, S. epidermidis is a common cause of endocarditis that develops during cardiac valve replacement and infections complicating orthopedic replacement or neurosurgical bypass surgery.

Well known staphylococcal infections of the skin and surface tissues of the body, such as pyoderma, boils, abscesses, carbuncles, paronychia, impetigo contagiosa and infectious complications surgical wounds. The clinical picture of skin infection depends on the age of the patient. For example, “scalded skin syndrome” is observed in young children.

Staphylococcus are also capable of causing diseases of entire systems and can affect almost all organs and tissues. They can be one of the causes of pneumonia, purulent pleurisy, endocarditis, meningitis, brain abscess, puerperal fever, phlebitis, cystitis and pyelonephritis. Staphylococcal pneumonia is a fatal complication of epidemic influenza. Staphylococcus are the most common cause of osteomyelitis. Staphylococcal illnesses often occur in hospitals, especially in patients who are already seriously ill. For example, staphylococcal pneumonia is a superinfection that threatens patients who have taken large doses of antibiotics.

Staphylococcal septicemia occurs in two forms. The first is fulminant deep toxemia, which leads to death after a few days. Another, more common form, lasts longer, is accompanied by the development of metastatic abscesses in different parts of the body, but is not irreversible.

Staphylococcal septicemia may be primary, but more often it is the result of secondary penetration of microorganisms into the bloodstream from a local source of infection. Often the process begins as a trivial inflammation of the hair follicle. The microbe can enter the bloodstream from an infected wound, from a source of pneumonia, or from an infected intravenous catheter. The catheter should not be left in the vein for more than 3-4 days due to the risk of serious infection. Superficial ulcers; boils around the nose and lips are easily complicated by septicemia, so it is better to avoid injuring them.

So-called "dangerous triangle"- This is a triangular area of ​​the face from the corners of the mouth, upper lip and to the top of the nose. From this area, infection can spread directly into the cranial cavity, which can be life-threatening. This complication is associated with the peculiarities of the anatomical structure of this part of the body: the absence of reliable mechanical barriers, veins that do not have valves that would impede the reverse flow of blood, the presence of muscles that are constantly in motion.

Even with such manipulations as ear piercing (for cosmetic purposes), carried out without maintaining the necessary sterility, there is a danger of developing secondary staphylococcal infection, which from open wound can penetrate into the bloodstream and sometimes lead to the development of sepsis.

Staphylococcus may cause enteritis and enterocolitis, complicating antibiotic therapy. They are the most common cause of food poisoning, as the toxin can accumulate in large quantities in food.

Staphylococcus can sometimes cause purulent-inflammatory processes in cows and horses. If mastitis develops, the microbe can get into the milk. Pustular lesions on the skin of the hands of milkmaids may occur. Infection of other animals in the herd is possible.

Staphylococci. Hospital infection

Man is closely related to the ubiquitous staphylococci (ubiquitous microorganism), and therefore staphylococcal infection poses a threat anywhere and at any time. An example of this is hospital-acquired infection, which has been a serious problem for hospitals around the world for many years.

There are a number of issues associated with this problem that require discussion. First of all, staphylococcal hospital infection is a complication of the widespread use of antibiotics. These important antimicrobial agents are very widely and freely prescribed, but their action is predominantly bacteriostatic rather than bactericidal. Staphylococcus are endowed with good adaptability and antibiotic-resistant strains arise among them. Secondly, the features of health insurance and medical services contribute to the early detection of the disease and early hospitalization of a larger number of patients. Thirdly, expanding the scope and number of surgical interventions. Sophisticated surgical techniques allow tissue to be left open longer. Fourth, certain types of therapy can cause suppression of the immune system (immunosuppression). For example, tissue and organ transplantation against the background of the prescription of anti-rejection drugs. In these cases, resistance to infection is suppressed.

The following lesions are observed with the highest frequency in hospital staphylococcal infections:

1. Pyoderma is a term denoting purulent lesions of the skin and subcutaneous tissues. More often in newborns. Dangerous complications such as pneumonia and septicemia are possible. A newborn can infect a mother who develops an abscess. mammary gland- mastitis.

2. Wound infection, especially a surgical wound.

3. Secondary staphylococcal infection of the elderly and persons incapable of maintaining hygiene standards.

4. Gastroenteritis in persons with suppressed (for example, antibiotics) intestinal microflora.

Staphylococci. Pathological anatomy

The most noticeable manifestation staphylococcal infection - abscess, the main type of damage. The formation of an abscess is based on the pyogenic activity of staphylococcus and its rather limited ability to spread,

Since the microbe constantly lives on the skin, it is most often affected. A boil is a skin abscess. The formation of an abscess is determined by the location and level of spread. Staphylococcal pneumonia is multiple abscesses in the lungs. Pie-lonephritis- these are multiple abscesses of the excretory system of the kidneys. Staphylococcal septicemia - education multiple abscesses all over the body. In this case, the more appropriate term is pye-mia, which literally means “pus in the blood.” When staphylococci Infect the wound, they cause the formation of pus in it. Staphylococcus are the most important cause of wound infection. In general, they cause up to 80% of all purulent processes in humans.

Staphylococci. Epidemiology

Staphylococcus normally live on human skin, as well as on the mucous membrane oral cavity, pharynx and nose. They can be here constantly until one day they overcome the skin or mucous barrier and cause the development of the disease. In another case, they penetrate through intact skin into the hair follicles and ducts of the sebaceous glands.

Normally, the ability of staphylococcus to invade and the resistance of the host are well balanced, so the infection does not develop until a situation arises when a highly virulent microbe or a macroorganism with reduced resistance is encountered.

As a rule, a local process develops - an abscess or furuncle, without the spread of infection. But in some cases, the microbe goes beyond the local infection, enters the bloodstream and affects various tissues and organs of the body.

The mechanism of transmission of infection is predominantly contact. For example, through the hands of staff in a hospital. Personnel are at risk of becoming carriers, in which case staphylococcus may remain on their nasal mucosa for a long time. Carriers can become sources of infection.

Staphylococci. Bacteriological diagnostics

Bacteriological diagnosis staphylococcal infection is not particularly difficult if blood and other body fluids, which are normally sterile, are examined. Selected culture staphylococcus must be identified as pathogenic, as opposed to a normal microbe that lives on the skin. A number of tests are used for this. Freshly isolated crops staphylococcus characterized as pathogenic by such characteristics as the production of yellow pigment, hemolysin, mannitol fermentation, production of DNAse and coagulase.

Phagotyping. Bacteriophages are viruses that infect bacteria and, in some cases, cause their lysis. The action of phages is specific. Only a single phage or group of phages infects a specific strain of bacteria, which makes it possible to use phages for typing bacteria (phage typing). For this purpose, a suspension of a known phage is added to a dish with a fresh culture of the microbe to be typed. If this microbe is sensitive to the action of this phage, it is lysed and does not grow on the medium. Transparent zones, so-called “plaques” or “negative colonies,” appear on the plate.

Phagotyping of staphylococci deserves special mention. It was found that specific bacteriophages (staphylophages, Table 1) react with 60% of coagulase+ staphylococci. Coagulase-staphylococci are not so sensitive. Since this process is specific, it is used to determine the phage products of isolated staphylococci. For convenience, bacteriophages are assigned specific numbers, and staphylococcal strains lysed by a specific phage have the number of this phage and are designated as the corresponding phage. This only applies to S. aureus strains. Most other staphylococci are non-typeable.

By decision of the international subcommittee on phage typing of staphylococci, an international classification of S. aureus phages was proposed (Table 1)

Table 1. International classification of S. aureus phages

Groups	Individual phages	Common phages S.aureus
	29, 52, 52A, 79, 80	29.52/52A; 52/52A/80/81; 80
	3A, 3B, 3C, 55.71	3A/3B/3C; 3С/55
	6, 7, 42E, 47, 53, 54, 75	6/7/47/53/54/75/77
Mixed

Phage typing is used for epidemiological purposes to identify the source of infection, since phage typing is a highly specific process. In cases, for example, of nosocomial infections, the method can accurately indicate the carrier of the pathogenic strain (the same phage product).

Staphylococci. Immunity against infection

Humans have significant natural immunity to Staphylococcus. Specific antibodies are found in the blood serum of most people. Their appearance is associated with previous so-called “minor” staphylococcal infections of the skin and mucous membranes. Acquired immunity can perform a certain protective function, but practically cannot serve as serious protection against staphylococcal infection. Special group at risk are persons with reduced protective reactions, for example due to diabetes or viral infections.

Staphylococci. Infection prevention and control

The basis for controlling staphylococcal infections in hospitals (and anywhere else) is careful adherence to hygiene standards and strict adherence to aseptic techniques. Circulating pathogenic strains of staphylococcus are just as sensitive to disinfectants as ordinary non-pathogenic strains.

An important activity is to identify carriers of pathogenic staphylococcus (on the nasal mucosa) among staff, especially in neonatal units.

Phagotyping of coagulase+ staphylococci helps to identify the source in case of nosocomial infections

Conclusion

1. Staphylococcus , streptococci and pneumococci are the main gram-positive cocci that belong to two genera: Staphylococcus and Streptococcus. Formally, pneumococcus is currently called Streptococcus pneumoniae.

2. Staphylococcus - aerobic cocci, grow well on all nutrient media, some varieties produce catalase. On blood agar, staphylococci produce pigments that can be golden, lemon yellow or white. Staphylococci have significant resistance to physical and chemical agents. They quickly develop resistance to antibiotics; about 80% of strains are resistant to penicillin.

3. The most reliable sign of the pathogenicity of Staphylococcus strains is their ability to produce coagulase.

4. The most important toxic metabolic products of staphylococci are hemolysins, enterotoxin, lipases, and nuclease. Some strains produce coagulase.

5. The main manifestation of staphylococcal infection is an abscess.

6. Phagotyping is a valuable epidemiological technique that allows one to determine the source of pathogenic staphylococcus, which is especially important for hospital infections.

7. The key measures to prevent the intrahospital spread of staphylococcal infection are careful adherence to hygienic standards, strict control over aseptic technique, identification of carriage of staphylococcus among staff of neonatal departments and surgical hospitals, removal of permanent carriers from the “risk zone” for patients .

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Staphylococcosis in hospitals 40-60%

Currently, the genus Staphylococcus no longer belongs to the Micrococcaceae family (since the early 90s). 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.

When researching fusobacteria, rightella, bacteroides (strict anaerobes) in an oxygen-free environment, if you do not use a medium with antibiotics (aminoglycosides), then staphylococci (and enterobacteria) will kill everything!!!

Exhaustive seeding method

Staphylococci are facultative anaerobes. Micrococcus – strict aerobes

Differentiation - test with aerobic and anaerobic breakdown of glucose. All Staphylococcus glucose+ without gas under Vaseline oil. Micrococcus - no.

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

The limit of halotolerance is an environment with 7.5% or more NaCl.

Micrococcus - still growing at 6.5%, and only Staphylococcus is higher. (ZhSA – 10% NaCl). Some types 15% NaCl.

Milk-salt agar – micrococci grow because NaCl is lower there.

The bourgeoisie uses Chapman's medium - mannitol-salt agar (1% mannitol) - 7.5% NaCl. Mannitol=mannitol, like glycerol=glycerol

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

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

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

Each person has 7-10 types of micrococci on the skin (and there are about 15 species in total), M. luteus predominates - large yellow colonies on non-inhibitory media (the old name is air sarcina, true sarcina do not grow in aerobic conditions!!!). They stain evenly according to Gram. In the form of piles, notebooks, packages. Others are white, colorless, etc. M.livi – enamel-white. M.cristi - red colonies.

Staphylococcus stains unevenly according to Gram (calico stain). Clusters – most often produced by S. aureus, as well as piles, tetrads, bags, etc. Staphylococci with liquid steadies - single, pairs, groups.

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

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

Angelina Hess - introduced agar into media in the mid-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 in vivo treatment is very difficult. Sepsis in patients with cardiac bypass surgery. If they end up in a heart-lung machine.

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

Stomatococci – 1 species. They grow well on KA (anything can grow on KA), but they do not grow in salt media. They have no particular clinical significance. UPMF. Gingivitis and stomatitis are very rarely caused (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. More than 86 nosological forms of diseases.

S. aureus and S. epidermidis – 45% of all staphylococcosis each. 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 sections of the nose are the main biotope. There's more of everything per unit area.

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

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

Plasmids are transmitted at a speed of about 2 hours (staphylococcus spent 2 hours on the skin of another person and received a new plasmid).

Carriage.

In maternity institutions, routine testing for staphylococcus is not carried out (only during outbreaks) - order 345. The surgical profile still has 720 orders (not carried out anywhere in the world). The number of staphylococcal infections is the same among the “bourgeoisie” and among us, regardless of sanitation.

Using test systems, it is possible to differentiate up to 12-15 types of staphylococci and several types of micrococci.

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

The planned examination for staphylococcus carriage has been cancelled.

There are currently 5 types of coagulase-positive staphylococci.

	coagulase				maltose	flocculation	Hemolysis on KA with human blood
			Hourly day				Almost 100%
						± less than 50%
	B/W night

S. aureus does not produce coagulase very rarely - during primary cultures from a patient with massive antibiotic therapy. Therefore, we re-culture the joint and then add plasma again. If not, it is not S. aureus.

Pigment on media with a high protein content and light (WSA, whey protein media)

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

Acetoin=acetylmethylcarbinol – VP (Voges-Proskauer) reaction

His medium with glucose is semi-liquid. When inoculated from a saline medium, they are always glucose positive, according to at least, at the top of the column.

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

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

Semi-liquid medium with mannitol was excluded in 1974 - the final negative answer was only on the 5th day. Placed only on the cup - the answer is the next morning.

Solid medium with glycerin (1%) (can be replaced with any sugar and alcohol). Liquid and semi-liquid media are never used for differentiation within the genera Staphylococcus and Micrococcus. Indicator – bromothymol blue, pH – neutral; or bromocresol red, bromothymol red. We sow in plaques - up to 10 crops per cup. Radial strip – up to 6 crops per cup. There is yellowing around the staphylococcus. Micrococci do not utilize glycerin.

S. aurecularis in test systems is 95-99% similar to S. aureus and can only be distinguished by the plasma coagulation reaction.

Coagulase-negative staphylococci

Under certain conditions they can produce pigment.

In staphylococci, differentiation to species cannot be carried out based on 1-2 tests.

There are only 6-7 species that grow in plasma in the form of Mannitol+ flakes.

	coagulase	Alkaline phosphatase			trehalose
						More than 20% of strains
						5-10% strains
						95-100% strains
						5% strains
S. saprophyticus						5% strains

100% of S. aureus has hemolytic activity.

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

Coagulase-negatives cannot become coagulase-positive.

S. epidermidis is never coagulase-positive, no matter what is written in order 720

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.

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

Phenolphthalein phosphate → phenolphthalein (colorless). We alkalize with 3-10% ammonia solution (drop it onto the lid of the cup). The colonies are crimson.

You can buy 10% phenolphthalein phosphate solution. Keeps in the refrigerator for years. 0.5 ml of solution per 100 ml of 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 - of human origin - produce lecitovitelase. Lecithin-negative S. aureus – from animals.

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

LSA cups for transmission - clearing zone (protease) - it is not important to us.

Turbidity - lipases predominate - are also not important.

Bourgeois blood media are made from sheep erythrocytes. Human strains of S. aureus were thought to produce only α-hemotoxin, but now there is also a β-hemotoxin. This can only be determined using sheep red blood cells.

β-hemotoxin gives a zone of incomplete hemolysis after 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) - found only in S. aureus. It is not destroyed by boiling or even short-term autoclaving.

And according to our orders, only thermolabile DNase is detected (no point).

In heated food (while hiding the fact of PTI), staphylococcus is destroyed, but toxins remain. The presence of thermostable DNase can determine whether S. aureus was present.

According to orders, highly polymerized DNA is 1-2 mg per 1 ml of medium (pH = 8.0). Sowing a plate with plaques (or an injection). 24 hours. We develop it by filling the cup with 3N HCl, a DNA precipitate with HCl is formed, and where the DNase has worked, we see a 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 S. aureus culture – 7-8 mm.

S. epidermidis produces only heat-labile DNase, S. aureus produces both DNases—the zone is larger.

Staphylococcus aureus

Staphylococcus aureus- facultative anaerobic gram-positive cocci, nonmotile, catalase- and coagulase-positive. Some strains S. aureus produce staphylococcal enterotoxins (SEs), which cause food poisoning. Staphylococci are present in the air, dust, wastewater, 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 nasal and throat cavities, as well as on the hair and skin of 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. The foods that most often cause staphylococcal food poisoning are meat and meat products, poultry, eggs, salads (containing eggs, tuna, chicken, potatoes, pasta), confectionery (for example, cream pies, chocolate eclairs), sandwich fillings, milk and dairy products. Thus, products that are manually processed for a significant period of time during the preparation process and subsequently stored warm can be a real source of staphylococcal food poisoning.

Intoxication due to 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. A patient's susceptibility to toxins is determined by the patient's condition, the concentration of the toxin, and the amount of contaminated food 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 aversions, 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 spread to others; patients need to take plenty of fluids.

Pathogenicity, extent of the disease

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

According to recent studies in developed countries, of all known infections, one of the most famous, “ordinary” bacteria most often leads to death - Staphylococcus aureus (Staphylococcus aureus; all strains of staphylococci that produce coagulase are called golden). This opportunistic microorganism is isolated from 15-30% of healthy people, and carriage in most of them is limited to several months. When the patient has a deficiency of immunity, the pathogen causes numerous life-threatening zoonotic 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 sepsis caused by Staphylococcus aureus died. Dermatovenerologists in many countries are concerned about the increase in the number of staphylococcal infections and deaths due to staphylococcus. Bacteria cause spoilage food products, including so-called “non-perishable” products. When bacteria multiply, for example, in hard cheese and salami, they produce toxins. They are the most common cause of serious food poisoning. During cooking, staphylococcus dies, but its heat-stable toxins are not destroyed. Staphylococci are the cause of many human diseases, including heart diseases (endocarditis and pericarditis), and musculoskeletal diseases (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 common infectious skin diseases. According to WHO, Staphylococcus aureus tops the list of bacteria that are most often infected in medical institutions. There is a high risk of staphylococcus infection when using intravenous catheters and other medical devices that come into contact with the internal environment of the body. A risk factor is artificial ventilation lungs. Staphylococcus infections can also occur when normal hospital hygiene practices are not followed. In total, S. аureus accounts for about 30% of all “hospital infections.” Staphylococci that survive under conditions of constant use of disinfectants and the use of antibiotics are a serious risk factor for personnel and the basis of hospital infections.

Taxonomy

Staphylococci are a genus of spherical, non-motile, asporogenous, gram-positive, chemoorganotrophic, facultative anaerobic or aerobic bacteria from the family Micrococcaceae. There are about 30 species in the genus Staphylococcus, with 14 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; the species-specific antigens are teichoic acids.

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 glycerinteichic acid. Staphylococci form smooth colonies colored yellow or orange by carotenoids. However, pigmentation is not a species characteristic. When grown on yolk-salt agar, cloudy, round, smooth 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 sediment, which turns into a viscous mass.

Physiological and biochemical characteristics

Staphylococci are facultative anaerobes, but grow rapidly and abundantly in the presence of oxygen. Chemoorganotrophs with oxidative and enzymatic metabolism. Biochemically very active. They produce catalase; in a medium with glucose under anaerobic conditions, most strains produce acetoin ( positive reaction Voges-Proskauer). They release ammonia when growing on argenine 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. The 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). Optimum growth was noted at pH 7.0-7.5, but growth is possible in the range pH=4.2÷9.3. Can withstand elevated temperatures well osmotic pressure Therefore, the elective substrate for them is media with a high concentration of sodium chloride - yolk-salt or milk-salt agar. Staphylococci tolerate drying well, while maintaining virulence; die when exposed to direct sunlight for 10-12 hours. Quite 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 to 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 require amino acids and vitamins; when grown under anaerobic conditions, they require additional uracil and additional carbon sources. Several types can form on blood agar hemolysins- substances that damage red blood cells, leukocytes and other cells. They also produce fibrinolysin, phosphatase, and bacteriocins; individual strains produce coagulase, DNAase, H 2 S and enterotoxins (up to 10 species characterized by lethal, hemolytic or necrotic effects). Some carbohydrates are fermented, releasing acid without gas. Resistant to lysozyme, sensitive to various antibiotics. Species pathogenic to humans have one of 4 toxicity factors, such as: 1) exotoxin, 2) enterotoxin, 3) leukocidin, 4) coagulase (pathogenicity enzyme). Antibiotic cross-resistance is controlled by the R plasmid. This resistance factor is transmitted to other prokaryotes by transduction.

Sources and factors of transmission of infection

Staphylococci are conditionally pathogenic microbes. They are representatives of the normal microflora of humans and animals - they densely colonize various biotopes of the body (skin, mucous membrane of the nose and pharynx, abdominal cavity and axillary areas). Staphylococci enter the body through skin and mucous membranes by airborne droplets and airborne dust. The mechanism of infection is usually associated with the transfer of the pathogen from sites of colonization to a traumatized surface (for example, skin). Close contacts with carriers and persons suffering from staphylococcal lesions also play a significant role. Staphylococcus is resistant to many external influences, therefore it is found in the external environment - air (dust), soil, and on household items. The 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 - sick animals, for example, (in case of 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. Staphylococcal infection is characterized by a multiplicity of mechanisms, pathways and transmission factors. Bacteria can be transmitted contact through a non-sterile medical instrument, the hands of medical staff, nutritionally with dairy products and confectionery products, aerogenic and parenterally(for injections).

Pathogenesis

Susceptibility to staphylococci is very low in persons with normal immune status and increased in immunocompromised hosts. Very often, staphylococcal infection develops against the background 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 the adhesion of microbes and their spread throughout tissues. When growing in vitro it is not formed. Cell wall components stimulate development inflammatory reactions and neutralize immunoglobulins, immobilize phagocytes. Teichoic acids trigger a cascade compliment(a component of immunological reactions) via an alternative pathway. 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 various localizations 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 of 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 organs(angina, 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) genitourinary organs(cystitis, mastitis, etc.), 8) staphylococcal sepsis. These diseases can be acute or chronic. Food poisoning clinically manifests itself in the form of vomiting and watery diarrhea within 2-6 hours after eating infected foods - creamy confectionery, canned food, meat and vegetable salads, etc.

Detection methods

As a rule, only colonies of staphylococci grow on selective differential media. Isolation of staphylococcus in pure culture allows us to assume the staphylococcal etiology of the disease. The biomaterial for research is selected depending on clinical picture diseases. In S. aureus strains isolated during food staphylococcal intoxication, the presence of enterotoxins is determined in biological and immunological tests. Using traditional methods, preliminary results on the identification of staphylococcal infection can be obtained in 1-2 days.

Classic method

The method for detecting S. aureus is based on sowing a sample of the product and/or dilutions of its sample into a liquid selective medium, incubating the crops, reseeding the cultural liquid onto the surface of an agar selective diagnostic medium, and confirming by biochemical characteristics that the isolated typical and/or atypical colonies belong to S. aureus.

Staphylococcus aureus bacteria are pathogens to both man and other mammals. They are gram positive bacteria that are small round in shape (cocci) and occur as clusters appearing like a bunch of grapes on electron microscopy.

Microbiology of Staphylococcus:-

Coagulase reaction

Staphylococcus were earlier divided into two groups on the basis of their ability to clot blood plasma. The coagulase-positive staphylococci constitute the most pathogenic species S aureus. The coagulase-negative staphylococci (CNS) are now known to comprise over 30 other species. It is the CNS that is present as harmless bacteria on skin but some of these may cause infections as well.

These days coagulase reaction is no longer used to classify S.aureus. This is because coagulase is a marker for S aureus but there is no direct evidence that it is a virulence factor. However, the term is still in widespread use among clinical microbiologists.

Proteins and virulence

S. aureus expresses certain proteins and polysaccharides on its surface. This is correlated with virulence. Virulence is the effect of many factors expressed during infection. The bacteria also produce certain toxins. In the body antibodies neutralize staphylococcal toxins and enzymes.

Taxonomy and naming conventions

At least 30 species of staphylococci have been recognized by biochemical analysis. This is especially so with DNA-DNA hybridization. Of these, 11 are found in humans as harmless bacteria on skin, in the nose and throat. These can cause disease and infections in certain situations.

Identifications under the microscope

These bacteria are Gram-positive cocci about 0.5 - 1.0 μm in diameter. They are present as grape like clusters. They may also occur in pairs and occasionally in short chains. The clusters arise because staphylococci divide in two planes. This clustering helps to distinguish staphylococci from streptococci, which usually grow in chains.

Growing on solid medium colonies of S. aureus these appear as golden clumps.

Catalase test

This test helps to distinguish between streptococci (which is catalase-negative) and staphylococci (which are catalase positive). On an agar slant or broth culture of the bacteria several drops of 3% hydrogen peroxide are applied. Catalase-positive cultures bubble at once. This cannot be done on blood agar since blood itself will produce bubbles.

Identification of S.aureus

After sample from the lesions are taken, they can be stained with Gram stain. S. aureus is Gram positive. The organism from the clinical specimen from blood culture or pus is then streaked over solid media such as blood agar, tryptic soy agar or heart infusion agar. If the specimen is suspected to be contaminated it is plated on mannitol salt agar containing 7.5% sodium chloride.

Another test is production of thermostable deoxyribonuclease. S aureus can be confirmed by testing colonies for agglutination with latex particles coated with immunoglobulin G and fibrinogen which bind protein A and the clumping factor, respectively.

Microscopically cells occur singly and in pairs, short chains, and grape-like clusters. The cell wall of the bacteria contains teichoic acid. Ribitol teichoic acid (Polysaccharide A) is present in Staphylococcus aureus. Protein A uniformly coats surface of S. aureus and is usually oxidase negative.