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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 Micrococcaceae family does not exist (since there is no genetic relationship between its components). They united only on the basis of gram-positive aerobic and facultative anaerobic cocci, dividing in several planes.
Micrococcus is genetically related to actinomycetes.
Staphylococcus is genetically related to Clostridia.
When researching fusobacteria, pravotella, bacteroids (strict anaerobes) in an oxygen-free environment, if you do not use a medium with antibiotics (aminoglycosides), then staphylococci (and enterobacteria) will kill everything !!!
Depletion sowing method
Staphylococci are facultative anaerobes. Micrococcus - strict aerobes
Differentiation is a test with aerobic and anaerobic glucose breakdown. All Staphylococcus glucose + still under vaseline oil. Micrococcus is not.
Staphylococcus - more than 30 species, with subspecies - 44. They are differentiated by 33 tests.
The halotolerance limit is an environment with 7.5 and more% NaCl.
Micrococcus - still growing by 6.5%, and only Staphylococcus is higher. (LSA - 10% NaCl). Some types are 15% NaCl.
Milk-salt agar - micrococci grow, because there NaCl below.
The bourgeoisie - Chapman's medium - mannitol-salt agar (1% mannitol) - 7.5% NaCl. Mannitol = mannitol like glycerol = glycerin
Targeted isolation of Staphylococcus (for example, during sanitary and bacteriological studies). Yellowing zone due to acid formation. As its alternative, we have - ZhSA.
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 in staphylococci.
Each person has 7-10 species of micrococci on the skin (and there are about 15 species in total), M. luteus prevails - large yellow colonies on non-inhibitory media (the old name is air sarcins, true sarcins do not grow in aerobic conditions !!!). They are evenly colored according to Gram. In the form of heaps, notebooks, packages. Others are white, colorless, etc. M.livi - enamel white. M.cristi - red colonies.
Staphylococcus aureus according to Gram stains unevenly (chintz color). Bunches - often produces S. aureus, as well as heaps, notebooks, packages, etc. Staphylococci with liquid steads - solitary, pairs, heaps.
On media without yolk, S. aureus pigment does not form and 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 to Wednesdays in the middle of the 19th century
Micrococcus is also UPMF - sepsis. In vitro is 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 get into a heart-lung machine.
Planococcus - we are not interested - they are not dangerous in sea water and to humans
Stomatococci - 1 type. They grow well on CA (anything grows on CA), but they do not grow on salt media. They have no particular 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 anaerobic)
There are no saprophytes among staphylococci - all opportunistic... 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 live on the skin.
The anterior parts of the nose are the main biotope. There is most of all per unit area.
S. epidermidis can also cause outbreaks of nosocomial infections along with S. aureus.
In newborns: conjunctivitis S. epidermidis earlier in Moscow in the 70s; hospital pneumonia S. epidermidis now.
Plasmids are transmitted at a rate of about 2 hours (Staphylococcus aureus stayed on the skin of another person for 2 hours and received a new plasmid).
Carrier.
In obstetric institutions, a routine study for staphylococcus is not carried out (only in case of outbreaks) - order 345. The surgical profile has 720 orders so far (it is not carried out anywhere in the world). The number of staphylococcal infections is the same in both the "bourgeois" and in our country, regardless of sanitation.
According to test systems, up to 12-15 types of staphylococci and several types of micrococci can be differentiated.
691 orders - canceled for maternity hospitals. The new 345 order does not have a bacteriological part, therefore, in bacteriology, everyone is still guided by 691 orders.
Routine examination for staphylococcus carriage has been canceled.
Coagulase-positive staphylococci are now 5 types.
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coagulase |
maltose |
flocculation |
Hemolysis on CA with human blood |
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W / w day |
Almost 100% |
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± less than 50% |
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W / W night |
S. aureus does not give coagulase very rarely - with primary cultures from a patient with massive antibiotic therapy. Therefore, we reseed on the jamb and then put the plasma back on. If not, it is not S. aureus.
Pigment on media with a high protein content and light (YSA, whey medium with protein)
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 with saline, always glucose positive, 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 gas - we assume that 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 in the morning.
Dense medium with glycerin (1%) (can be replaced with any sugar and alcohol). Liquid and semi-liquid media for differentiation within the genera Staphylococcus and Micrococcus are never used. Indicator - bromothymol blue, pH - neutral; or bromcresol red, bromothymol red. We sow with plaques - up to 10 cultures per dish. Radial stripe - up to 6 cultures per dish. Around staphylococcus - yellowing. Micrococci do not utilize glycerin.
S. aurecularis on test systems, it gives 95-99% similarity to S. aureus and only by the plasma coagulation reaction can it be distinguished.
Coagulase-negative staphylococci
Under certain conditions, they can produce pigment.
In staphylococci, it is impossible to differentiate to species by 1-2 tests.
It grows in plasma in the form of Mannitol + flakes, only 6-7 species.
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coagulase |
Alkaline phosphatase |
trehalose |
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More than 20% strains |
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5-10% strains |
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95-100% strains |
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5% strains |
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S. saprophiticus |
5% strains |
100% of S. aureus has hemolytic activity.
S. saprophiticus is very rare. Cystitis and urethritis in young women - sensitive to antibiotics and chemotherapy
Coagulase-negative ones cannot become coagulase-positive.
S. epidermidis is never coagulase positive, whatever is written in order 720
A non-spore cell can live for 10-12 days.
Alkaline phosphatase is characteristic only of S. epidermidis.
Alkaline phosphatase - paranitrophenol phosphate or phenolphthalein phosphate from ALP tests in biochemistry - 50 mg per 100 ml of agar up to 20 strains per plate.
Para-nitrophenol phosphate → para-nitrophenol (yellow) and the color of the medium is lemon yellow.
Phenolphthalein phosphate → phenolphthalein (colorless). Alkalize 3-10% ammonia solution (drip on the cup lid) Raspberry colonies.
You can buy 10% phenolphthalein phosphate solution. Stored 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) - is not used in the international classification, only here.
20-25% S. epidermidis lecithinase-positive.
80% of S. aureus - of human origin - gives lecitovitelase. Lecithinase-negative S. aureus - from animals.
Other staphylococci can also give. That is, if lecitovitelase +, then it is 80% S. aureus, 20% other staphylococci.
Luminal ALCA cups - the clearing zone (protease) - it is not important to us.
Turbidity - lipases predominate - are also not important.
Bourgeois blood media - from sheep erythrocytes. Human strains of S. aureus were thought to produce only α-hemotoxin, but there is now β-hemotoxin as well. It can only be disassembled on sheep 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, then the zone will completely brighten.
S. aureus produces 2 enzymes
1) thermolabile DNase (nuclease) - it is found in all staphylococci and in most other microbes.
2) Thermostable DNase (nuclease) - available only in S. aureus. Will not be destroyed by boiling or even short-term autoclaving.
And according to our orders, only thermolabile DNase is determined (it makes no sense).
In heated food (when the fact of PTI is hidden), staphylococcus is destroyed, but toxins remain. By the presence of thermostable DNase, it can be determined whether S. aureus was present.
By orders of highly polymerized DNA 1-2 mg per 1 ml of medium (pH = 8.0). Plaque culture (or injection). 24 hours. We develop, pouring 3N HCl into a cup, a precipitate of DNA with HCl is formed, and where 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 - both DNases - the zone is larger.
Page 38 of 91
PRIVATE MEDICAL MICROBIOLOGY
PATHOGENIC COCKI
Pathogenic cocci that cause diseases in humans of various clinical manifestations include: 1) staphylococci, 2) streptococci, 3) pneumococci, 4) meningococci, and 5) gonococci.
They can be divided into two subgroups by tinctorial trait (coloration): gram-positive cocci (staphylococci, streptococci and pneumococci), gram-negative cocci (meningococci and gonococci).
A characteristic feature of all pathogenic cocci is the ability to cause purulent inflammatory processes, which is why they are also called pyogenic (pyogenic) cocci.
The most sharply and constantly pyogenic properties are expressed in staphylococci.
STAPHYLOCOCCI
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 arranged in 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 paint well with basic aniline paints, are gram-positive, do not form spores, do not form capsules, and are motionless.
Cultural and biochemical properties. Staphylococci grow well on conventional nutrient media both in the presence and absence of free oxygen. Temperature optimum 37 °.
Rice. 60. Staphylococcus (a) and streptococcus (b) in pure culture.
Rice. 61. Colonies of staphylococcus on mesopatamia agar. Uv. twenty.
Rice. 62. Sowing by injection into gelatin.
Individual colonies on the surface of the agar are round discs of medium size, convex, moist, opaque, homogeneous or fine-grained structure (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 streaked on agar slant, abundant growth of Staphylococcus aureus is formed, pigmented.
Staphylococcus pigments are lipochromes, insoluble in water and stain only the culture, not the culture medium. Pigment formation is intense when microorganisms are grown in the presence of oxygen, at room temperature, in the light.
When sowing with an injection into gelatin on the 2-3rd day of growth, a liquefaction zone 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).
In broth, staphylococci give abundant growth. After 24 hours in the thermostat, the broth becomes cloudy, and a precipitate forms at the bottom of the test tube.
Staphylococci quickly curd milk, giving it an acidic reaction.
Ferments (without gas) lactose, glucose, maltose, mannitol. The ability to break down mannitol usually characterizes pathogenic strains of staphylococcus.
Pathogenic staphylococci on blood agar plates form colonies surrounded by a hemolysis zone, and coagulate the citrated blood plasma.
Resistance. Staphylococcus aureus is one of the very persistent microbes. A temperature of 80 ° kills him when wet within 10 minutes. The temperature of 70 ° staphylococcus is maintained for up to 1 hour. Carbolic acid (3-5% solution) kills them within 15-30 minutes. In pus, staphylococcus aureus persists for a long time.
Toxins, enzymes of staphylococci, their pathogenicity for animals. Pathogenic staphylococci produce a number of toxins and enzymes. Staphylococcal hemolichin dissolves red blood cells. This toxin is thermolabile (decomposes at 55 °), belongs to exotoxins, and when immunizing animals with it, 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. Some of the hemolytic strains of Staphylococcus aureus produce enterotoxin, which causes acute gastroenteritis. In the filtrates of staphylococcal cultures, leukocidin, a poison that destroys leukocytes, is often found. Pathogenic staphylococci are isolated from enzymes: 1) plasma coagul az y - clotting plasma, 2) fibrinolysin - dissolving fibrin clots, 3) hyaluronidase - dissolving hyaluronic acid, a substance that adheres connective tissue elements, 4) penicillinase - inactivating - and 5) penicillinase cloudiness zone around colonies on yolk-salt agar (Chistovich's medium).
Of laboratory animals, rabbits and, to a lesser extent, guinea pigs and mice are most sensitive to staphylococcus. 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. Suckling kittens and young mice are most sensitive to staphylococcal enterotoxin.
Classification of staphylococci. The old classification of staphylococci by the pigment secreted by them (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) a clear hemolysis zone is formed on plates with 5% blood agar;
b) when administered intradermally to a rabbit, they cause phenomena of pronounced necrosis, sometimes ending with the death of the animal;
c) produce the enzyme plasmacoagulase, which causes plasma coagulation within 2 hours;
d) are allocated mainly from patients with sepsis or acute purulent infection.
The second group is weakly pathogenic staphylococci:
a) a zone of mild incomplete hemolysis is formed on blood agar plates;
b) when injected intradermally, the rabbit causes reddening of the skin at the injection site, sometimes infiltration, more often without necrosis;
c) when inoculated into citrate plasma, they cause clotting within 6 hours and later;
d) are secreted from the surface of the skin during folliculitis, from the mucous membranes during catarrh of the upper respiratory tract, from the surface of wounds, but not from their depths.
The third group is non-pathogenic staphylococci:
a) no hemolysis was detected on blood agar plates;
b) with intradermal administration to a rabbit, the phenomena of necrosis and infiltration are not observed, in some cases, reddening of the skin appears;
c) the reaction of plasma coagulation 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 aureus is the most frequent 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.
Most often, the skin is affected. Staphylococci are the causative agents of boils - purulent inflammation of the hair follicles. If boils become multiple in nature, the disease is called furunculosis. When several boils merge together, forming a more extensive purulent infiltrate, a carbuncle occurs.
Staphylococci are found with 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 consumption of milk and other dairy products infected with staphylococcus, cakes with cream, etc., since in these products staphylococcus finds optimal conditions for its reproduction. 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 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 the titer of antibodies in patients and those who have recovered. However, this immunity is unstable. Moreover, it has been noted that many people who have had furunculosis have an increased susceptibility to staphylococcus.
Resistance to staphylococcus can be weakened due to various reasons - diabetes, trauma, debilitating diseases, etc.
Microbiological diagnostics. Study of pus. Most often, pus is the material for microbiological research. For this purpose, with open lesions, after preliminary cleaning of the wound surface with a sterile cotton swab, the discharge from the deeper parts of the wound is removed. 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 staphylococci and streptococci, therefore, they proceed to bacteriological research, that is, to inoculation of 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, colonies form hemolysis zones, the diameter of which is often several times the diameter of the colony. These transparent colorless zones of hemolysis were formed due to the dissolution of red blood cells caused by staphylococcal hemotoxin. On plates with yolk-salt agar, pathogenic staphylococci grow with the formation of a golden pigment and a cloudy zone - in the form of a rainbow 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, located in the form of bunches of grapes, gives the right to issue an approximate positive result. Then the remainder of the colony is subcultured onto agar slant to obtain a pure culture. It is necessary to differentiate pathogenic staphylococcus from non-pathogenic, i.e., study the growth on blood agar, yolk-salt agar, inoculate in nitrate plasma and determine the toxicity in animals.
Plasma coagulation test. For the reaction of plasma coagulation G.V. Vygodchikov recommends the following technique. 10 ml of blood is taken from the heart of a rabbit, 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. Colonies of staphylococci are inoculated with a loop into tubes with nitrate rabbit plasma, placed in a thermostat at 37 ° and plasma clotting is checked every 2-3 hours. Pathogenic staphylococci coagulate plasma within 2-10 hours, most often within 4 hours.
Toxigenicity test. The toxicity of staphylococcal cultures is determined by experience on rabbits, and enterotoxin - on suckling kittens. A suspension of microbes in saline is prepared from a round of staphylococcus on a slant agar 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 the animal is injected intradermally with 0.2 ml of microbial suspension. With a positive reaction, an infiltrate forms at the injection site, and then necrosis.
To detect the lethal toxin, the rabbit is injected intravenously with the filtrate of the broth culture of staphylococcus at the rate of 0.75 ml per 1 kg of body weight. The death of the animal occurs in 15 minutes.
To determine enterotoxin, kittens 1.5-2 months of age (weighing 400 g) are fed with the original contaminated product or staphylococcus dairy culture. Signs of poisoning (diarrhea, sometimes vomiting) occur within 30-60 minutes. The observation is carried out for 4-6 hours, and if during this time the kittens have no reaction, the biological test is considered negative.
Blood test. If a patient suspects sepsis, 10 ml of blood is taken from the ulnar vein using a syringe and inoculated into 100-200 ml of sugar broth. Sowing is placed for enrichment for 24-48 hours in a thermostat. In the positive case, the medium becomes cloudy, and in the smears from it, gram-positive cocci with a characteristic shape and location are found. The broth is subcultured into a plate with blood agar and further research is carried out as described above. The broth is kept in a thermostat for 10 days, sowing on 5% blood agar every 2 days.
Researching other materials. When examining materials contaminated with a large number of foreign microbes (feces, sputum), it is recommended to use agar with 7.5% sodium chloride, to which 10% of warm skim milk is added (Petrovich medium). On such agar, the growth of extraneous 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 capable of multiplying only on the corresponding staphylococcus strain. Phage typing of staphylococci is of particular importance in
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. For the prevention of these diseases, toxoid is used.
In the treatment of staphylococcal infections, chemotherapeutic drugs of the sulfonamide series (norsulfazole, sulfodimezin, etc.), antibiotics (penicillin in large doses, erythromycin, oxacillin, etc.), staphylococcal bacteriophage (for skin lesions) are also successfully used. In severe staphylococcal sepsis, hyperimmune antistaphylococcal plasma and gamma globulin obtained from donors immunized with toxoid are recommended.
Widespread staphylococcal infections, starting with upper respiratory tract disease, can attack the entire body. Due to the predominant lesion of certain organs, the material for research in staphylococcal infections can be sputum, pus, blood, rinsing nasopharyngeal lavages, urinary tract discharge, food (mainly dairy and confectionery products), washings from infected surfaces, vomit, exudates , selected in strict accordance with the rules of aspect.
When analyzing the material, microscopic, bacteriological (isolation of a pure culture of microbes and their identification) and biological methods are used.
I. MICROSCOPIC METHOD
The microscopic method is of independent importance only for aseptic work with materials that are sterile in a healthy person (for example, blood, cerebrospinal fluid). The detection of staphylococci in this case has an independent diagnostic value. In other cases, the microscopic method is used as a preliminary, approximate. When using it, it is necessary to pay attention to the number of microorganisms in each field of vision (with staphylococcal diseases, the pathogenic pathogen can displace the rest of the microflora and be found in smears in huge quantities), the size of the bunches (with high pathogenicity, staphylococcus is intensively divided, individuals do not have time to disperse and give large clusters - clusters), the size of individual individuals (pathogenic staphylococci are mostly very small).
II. BACTERIOLOGICAL METHOD
The bacteriological method is the isolation of a pure culture of pathogens and their identification.
Staphylococci are among the most common microorganisms. They are also found in a healthy person. Therefore, for the diagnosis of the disease, the establishment of its staphylococcal nature, it is very important to prove the pathogenicity of the isolated bacteria. At the same time, solving the diagnostic problem is closely related to clarifying the issues of epidemiology, treatment and prevention of this infection. On this basis, the bacteriological method consists of several stages and directions.
All of the above stages of the study are reflected in the diagram:
Isolation of a pure culture of the pathogen should be carried out taking into account its cultural characteristics of halophilicity (good development in the presence of an excess content of table salt with simultaneous suppression of other microflora), a high need for proteins and carbohydrates. This is achieved through the use of elective nutrient media that simultaneously perform the functions of differential diagnostic.
COMPOSITION OF NUTRITIONAL MEDIUM FOR STAPHYLOCOCCI
When performing the analysis, it is necessary to take into account the following deviations from the typical characteristics of staphylococci.
DETERMINATION OF BASIC INDICATORS OF VIRULENCE
The main indicators of the virulence of staphylococci are hemolytic activity, the production of the plasma coagulase enzyme and necrotoxicity.
When assessing the degree of pathogenicity of cultures, Gross tests are widely used, according to which all staphylococci can be divided into three groups. The first group of undoubtedly pathogenic staphylococci include bacteria that have (sharp hemolyzing activity, clotting citrate plasma within 1-2 hours and having pronounced necrotizing properties. The second group of opportunistic or moderately pathogenic staphylococci are stamps that give insignificant hemolysis on agar with 5% rabbit or lamb blood, clotting plasma after 6 hours, and when administered intradermally to a rabbit causing redness and infiltration The third group of non-pathogenic staphylococci include cultures that do not hemolyze erythrocytes, do not coagulate plasma and do not have necrotizing properties.
Thus, the assessment of the virulence of the isolated staphylococcus is based on a comprehensive test of three indicators of the pathogenic effect.
At the same time, there are official indications that when staphylococci are isolated from lactic acid products, especially those that have been stored in the refrigerator for a long time, individual signs of pathogenicity may disappear while maintaining the ability to form toxins as a whole. Consequently, staphylococci in which one of the signs of pathogenicity drops out should be considered pathogenic (Instruction letter of the Erisman Institute of 1967).
DETERMINATION OF HEMOTOXIN is performed by direct inoculation of the culture on blood MPA containing 5-10% defibrinated rabbit or lamb blood. The addition of human blood is undesirable, since staphylococci secreting alpha-hemolysin do not destroy human erythrocytes and, therefore, the judgment about the pathogenicity of this strain isolated from a sick person will be unreliable.
Sometimes, as a result of the variability of staphylococci, as well as during long-term storage of cultures in unfavorable conditions, the hemolytic activity of the latter is weakened or completely disappears. To restore the hemolyzing ability of bacteria, it is advisable to add to the medium on which hemotoxicity is tested, a reducing mixture at the rate of 0.015 g for every 10 ml of medium. The mixture consists of one part Na 2 SO 3 (sodium sulfate) and two parts Na HSO 3 (sodium bisulfate). The reconstitution mixture is stored in the dark and added to the ex tempore molten medium. In order to preserve the hemolyticity of exotoxin in staphylococcal culture, it is also recommended to add 100 ml of saturated solution of Na 2 S 2 0 3 (reducing agent) and 250 ml of saturated solution of hydroquinone (stabilizer) to 100 ml of culture filtrate. VI Ioffe suggests adding only 0.1 ml of 1% sodium sulfate Na 2 SO 3 solution for every 0.4 ml to restore hemolytic activity.
DETERMINATION OF PLASMOCOAGULASE is carried out by inoculating a culture of staphylococcus in a narrow tube with 0.5 ml of 5% rabbit or human citrate plasma. Crops are placed in a thermostat for 6-10 hours with the registration of results after 1, 2, 3 and 6 hours. Human blood plasma gives inconsistent results, and donor plasma with glucose and merthiolate is generally not suitable. If it is impossible to replace human plasma, it is used only after 10-fold dilution with saline.
Along with the classical method for determining plasma coagulase, the plasma coagulation reaction on glass or an accelerated "slid test" is also used. This method is based on the ability of coagulase-active staphylococci to stick together with blood plasma and coagulate it. Coagulase-negative strains do not possess this property. To carry out the reaction, take a drop of water, suspend the test culture in it, and then add one drop of diluted rabbit or human blood plasma. After 15-60 seconds, a plasma clot is formed. A later (later than a minute) reaction is considered doubtful, and a reaction that occurred after 3 minutes is considered negative.
DETERMINATION OF NECROTOXIN is carried out by intradermal injection of 0.2 ml of suspension of 2 billion, daily agar culture of staphylococcus in saline to a rabbit. The observation of the animal is carried out within 24-48 hours. Only an infiltrate with a yellowish center, a dark rim and a bright red border along the periphery with subsequent phenomena of necrosis is regarded as a positive reaction.
DETERMINATION OF ADDITIONAL INDICATORS OF STAPHYLOCOCCUS PATHOGENICITY
DETERMINATION OF THE HYALURONIDASE ENZYME is carried out by testing the culture on a substrate containing hyaluronic acid. As the latter, an extract from the umbilical cord of a newborn is used. For this, the umbilical cords in the amount of 3-5 pieces, collected in a 0.5% solution of carbolic acid, are thoroughly washed from the blood with distilled water, cleaned of blood vessels, finely chopped and passed through a meat grinder twice. Then the resulting minced meat is weighed and poured with one and a half volume of distilled water, after which it is left at room temperature for 30 minutes, shaking occasionally. Then all this mass is poured into a funnel with 2-3 layers of sterile gauze, filtered, squeezed, thrown into boiling water for a minute, quantitatively corresponding to the initial weight of the crushed ropes. Let it boil. The liquid is quickly filtered through a double layer of sterile gauze into sterile tubes and a few drops of chloroform are added to each of them for conservation and substrate. Then the tubes are closed with a cotton stopper and placed in the cold to preserve. The extract remains almost unchanged under these conditions for several months.
The hyaluronidase test is performed in two stages. The first of them is the determination of the working dose of hyaluronic acid (the experiment is performed only once after preparation of the extract and is repeated 1-2 times a month during long-term storage), the second is the detection of the hyaluronidase enzyme.
| Scheme for determining the working dose and titer of hyaluronic acid | ||||||
| Reagents | Test tubes and their contents in ml | |||||
| 1 | 2 | 3 | 4 | 5 | 6 | |
| Hyaluronic acid extract | 0,1 | 0,2 | 0,3 | 0,4 | 0,5 | 0,6 |
| Diet. water or nat. solution | 0,9 | 0,8 | 0,7 | 0,6 | 0,5 | 0,4 |
| Into the thermostat at +37 ° C for 15 min. | ||||||
| 15% solution of vinegar. acid (indicator) | 2 to | 2 to | 2 to | 2 to | 2 to | 2 to |
| Result (clot formation | - | + | + | + + | + + + | + + + + |
The tubes are placed in a thermostat at 37 ° C for 15 minutes, after which 2-3 drops of a 15% acetic acid solution are added, gently shaken and the results are read by the formation of a clot. The titre of hyaluronic acid is considered to be its minimum amount, which, under the action of acetic acid, gives a clear clot. In this example, the titer of the hyaluronic substrate is 0.2 ml. The same amount is taken as a working dose.
Schemes for determining the hyaluronidase activity of cultures, see below.
The entire contents of the tubes are mixed, first placed in a thermostat for 15 minutes, then in the cold for 5 minutes, and 2-3 drops of 15% acetic acid are added. The presence of hyaluronidase in bacteria is recorded by the absence of a clot in the test sample. A good clot should appear in the control tube due to the presence of intact hyaluronic acid.
For a more accurate quantitative determination of the hyaluronidase activity of the test culture, the sample is placed according to the following detailed scheme.
| Scheme for determining the hyaluronidase activity of cultures | ||
| Components | Test tubes | |
| 1 | 2 | |
| (main sample) | control of hyaluronidase acid | |
| Hyaluronic acid extract in a working dose (in ml) | 0.2 ml | 0.2 ml |
| Filtrate - an enzyme or 2 billion suspension of microbes in saline (in ml.) | 0.3 ml | - |
| Distilled. water or nat. solution (in ml.) | 0,2 | 0.5 ml |
| In a thermostat at +37 ° C for 15-30 minutes, then in the cold for 5 minutes to stop the enzyme action | ||
| 15% acetic acid solution | 2 drops | 2 drops |
| Scheme for the quantitative determination of hyaluronidase activity | ||||||
| Components in ml | Test tubes | |||||
| 1 | 2 | 3 | 4 | 5 | 6 | |
| control of hyaluronic acid | ||||||
| Hyaluronic acid extract in the slave. dose (in ml) | 0,2 | 0,2 | 0,2 | 0,2 | 0,2 | 0,2 |
| Suspension of bacteria 2 billion (in ml) | 0,5 | 0,4 | 0,3 | 0,2 | 0,1 | - |
| Distilled. water or nat. solution in ml | 0,2 | 0,3 | 0,4 | 0,5 | 0,6 | 0,7 |
| Keep in a thermostat, add indicator 15% acetic acid in the cold | ||||||
| Result (clot formation) | - | - | - | + | + | + |
The titer of hyaluronidase activity of the culture in this example is 0.3 ml.
DETERMINATION OF FIBRINOKINASE is based on the ability of staphylococcus to lyse fibrin clots of fresh blood. For testing, take 0.5 ml of a daily broth culture of staphylococci, add to 0.2 ml of fresh human plasma or blood with 0.8 ml of saline. Then gently mix, after adding 0.5 ml of 0.25% solution of calcium chloride. The control in this reaction is a test tube with all the same components, but without bacteria. The tubes are placed in a thermostat at 37 ° C. Coagulation occurs within the first 15 minutes. From this moment, the progress of dissolution of the formed clot is monitored. Typically, pathogenic cultures provide complete fibrinolysis within 24 hours.
DIFFERENTIATION OF CROPS ON THE ENVIRONMENT IS CHEPMEN. Preparation of the medium: 3.3 ml of 0.1% aqueous solution of gentian violet or crystal violet are added to 1 liter of 3.5% MPA. The prepared medium is poured into cups. After hardening, it has a grayish-lilac color. Pathogenic staphylococci give purple or orange colonies on it, non-pathogenic ones - white or lilac.
DIFFERENTIATION OF PATHOGENIC STAPHYLOCOCCUS WITH REGARDING TO MANNITOL is carried out on a liquid Gyos medium containing 0.5% polyhydric alcohol - mannitol. Mannitol destroys pathogenic staphylococci after 36 hours, non-pathogenic ones much later. This sign is very unstable and is not an independent indicator of pathogenicity.
ACCELERATED BACTERIOLOGICAL ISOLATION METHOD
AND IDENTIFICATION OF PATHOGENIC STAPHYLOCOCCUS ACCORDING TO PETER DANIL
The method is based on the combined use of conventional blood MPA with lamb erythrocytes and a medium with mannitol, sodium chloride and potassium tellurite, the recipe for which was developed by Petru Danila.
THE MEDIUM TO PETRO DANIL HAS THE FOLLOWING COMPOSITION: distilled water - 100 ml; peptone - 0.5 g; sodium chloride -10 g; mannitol or lactose, 0.5 g; potassium tellurite - 0.5 g; bromtimoblau - 0.004 g.
Preparation of the medium: dissolve peptone and salt in warm water, add 2 ml of bromothymolbau solution (0.1 g per 3.2 ml of N / 20 sodium hydroxide solution and 50 ml of distilled water), sterilize at +120 ° C for 15 minutes. After cooling, add 5 ml of a 10% aqueous solution of mannitol or lactose, sterilized by boiling and 5 ml of a 1% aqueous solution of sodium tellurite, sterilized in an autoclave. The medium is dark green, pH -7.6. When the color is blue, a few drops of 10% hydrochloric acid are added to it. The medium is poured into test tubes, 1-2 ml.
Yellowing of the medium to Petru Danil 24 hours after sowing the culture and hemolysis around the colonies of staphylococcus indicate the release of pathogenic bacteria.
ACCELERATED COMPREHENSIVE IDENTIFICATION OF PATHOGENIC STAPHYLOCOCCUS ACCORDING TO PETER DANIL
Differentiation of pathogenic staphylococci is carried out in one test tube. The method is based on the ability of pathogenic staphylococci to agglutinate in the presence of human plasma, coagulate it, and also cause agglutination of homologous erythrocytes. To perform the test, 10 ml of physiological solution and 0.05 ml of erythrocyte mass settled under the plasma are added to 1 ml of nitrate human plasma. The tube is shaken and 0.5 ml of the mixture is poured into another tube. As much as possible of the culture of staphylococcus is introduced here, which is carefully rubbed on the wall of the test tube close to the liquid substrate, slightly touching its surface. Rubbing is continued until a homogeneous suspension is obtained. Pathogenic staphylococcus aureus is agglutinated by plasma, and homogenization does not occur, non-pathogenic - forms a uniform homogeneous mass.
Pathogenic staphylococci, when the culture is rubbed in a plasma substrate, form a granular inhomogeneous suspension, and after incubation in a thermostat, they cause hemagglutination of erythrocytes and plasma coagulation. In this case, non-pathogenic stamps form a uniform turbid suspension, and plasma coagulation and erythrocyte adhesion do not occur.
| Tab. 1. Types of phages | |
| Group | Bacteriophages |
| I | 29, 52, 52A, 79, 80 |
| II | 3A, 3B, 36, 55, 71 |
| III | 6, 7, 42E, 47, 53, 54, 75, 77, 83A |
| IV | 42D |
| V | 81 and 187 |
PHAGOTYPING OF STAPHYLOCOCCUS
Different phage types of staphylococci circulate in different localities. Therefore, their determination in staphylococcal cultures is of great importance for clarifying the possible source of infection and the ways of its spread.
Among plasma-coagulating staphylococci, according to the nomenclature of the International Committee for Phageotyping, 22 types of phages are distinguished (Table 1):
Determination of lysizability of staphylococci by the named bacteriophages is performed as follows.
PREPARATION OF CROPS FOR PHAGOTYPING
PHAGOTYPING
A positive result is considered if the degree of lysis is determined by at least 2 plus points. In this case, the lysis zone is about 50% of the area at the site of application of the bacteriophage.
In many cases, staphylococcal cultures are lysed not by one, but by several phages, forming a kind of phagomosaic from sterile spots. Staphylococci showing the same mosaic or differing by 1 phage are considered identical.
BIOLOGICAL METHOD
The biological diagnostic method is used only if there is a suspicion of food intoxication caused by enterotropic staphylococci that release enterotoxin. Its principle is reduced to feeding the remains of food suspicious of infection with staphylococcus, isolated culture, wash water. The best biological model is 1.5-2 months old kittens (for culture research) and adult cats (if enterotoxin is detected). With drip staphylococcal infections, this method is not used.
A source: Motavkina N.S., Pyanova R.E. Microbiological diagnostics of some droplet infections and toxoplasmosis. Methodical development for students. VSMU, 1973
Staphylococcus aureus was discovered in 1878 by R. Koch and in 1880 by L. Pasteur in purulent material. L. Pasteur, having infected a rabbit, finally proved the role of staphylococcus as a causative agent of purulent inflammation. The name "staphylococcus" was given in 1881 by A. Ogston (because of the characteristic arrangement of cells), and in 1884 F. Rosenbach described its properties in detail. Staphylococci are gram-positive, regular geometric spherical cells with a diameter of 0.5 - 1.5 microns, usually located in the form of clusters (see color incl., Fig. 92), catalase-positive, reduce nitrates to nitrites, actively hydrolyze proteins and fats, ferment glucose under anaerobic conditions to form acid without gas. They can usually grow in the presence of 15% NaCl and at 45 ° C. The content of G + C in DNA is 30 - 39 mol%. Staphylococci do not have flagella, do not form spores. They are widespread in nature. Their main reservoir is the skin of humans and animals and their mucous membranes, communicating with the external environment. Staphylococci are facultative anaerobes, only one species ( Staphylococcus saccharolyticus) Is a strict anaerobic. Staphylococci are not demanding on nutrient media, grow well on ordinary media, the optimum temperature for growth is 35 - 37 ° C, pH 6.2 - 8.4. Colonies are round, 2 - 4 mm in diameter, with smooth edges, convex, opaque, colored in the color of the formed pigment. Growth in a liquid culture is accompanied by uniform turbidity, and a loose precipitate forms over time. When growing on conventional media, staphylococci do not form capsules, however, when injected into a semi-liquid agar with plasma or serum, most strains S. aureus forms a capsule. Capsule-free strains in semi-liquid agar grow in the form of compact colonies, capsule strains - form diffuse colonies.
Staphylococci have high biochemical activity: they ferment with the release of acid (without gas) glycerin, glucose, maltose, lactose, sucrose, mannitol; form various enzymes (plasma coagulase, fibrinolysin, lecithinase, lysozyme, alkaline phosphatase, DNase, hyaluronidase, tellurite reductase, proteinase, gelatinase, etc.). These enzymes play an important role in the metabolism of staphylococci and largely determine their pathogenicity. Enzymes such as fibrinolysin and hyaluronidase are responsible for the high invasiveness of staphylococci. Plasma coagulase is the main factor in their pathogenicity: it protects against phagocytosis and converts prothrombin into thrombin, which causes fibrinogen to coagulate, as a result of which each cell is covered with a protein film that protects against phagocytes.
Classification. Genus Staphylococcus includes more than 20 species, which are divided into two groups - coagulase-positive and coagulase-negative staphylococci. Various characters are used to differentiate species (Table 22).
Table 22
Differential signs of the main types of staphylococci
Note. (+) - positive sign; (-) - the sign is negative; + (-) - is a fickle sign ;? - unknown.
I. Coagulase-positive staphylococci:
1.S. aureus***.
2.S. intermedius**.
3.S. hyicusa.
II. Coagulase-negative staphylococci:
* Pathogenic for humans only.
** Pathogenic for animals only.
*** Pathogenic for humans and animals.
a Not all strains S. hyicus have coagulase.
Pathogenic for humans are mainly coagulase-positive staphylococci, but many coagulase-negative staphylococci are also capable of causing diseases, especially in newborns (neonatal conjunctivitis, endocarditis, sepsis, urinary tract diseases, acute gastroenteritis, etc.). S. aureus depending on who is its main bearer, it is divided into 10 ekovars ( hominis, bovis, ovis and etc.).
More than 50 types of antigens have been found in staphylococci, antibodies to each of them are formed in the body, many of the antigens have allergenic properties. By specificity, antigens are subdivided into generic (common to the entire genus Staphylococcus); cross-reacting - antigens common with isoantigens of human erythrocytes, skin and kidneys (autoimmune diseases are associated with them); species and type-specific antigens. By type-specific antigens detected in the agglutination reaction, staphylococci are divided into more than 30 serovariants. However, the serological method of typing staphylococci has not yet received widespread use. Species-specific include protein A, which forms S. aureus... This protein is located superficially, it is covalently bound to peptidoglycan, its molecular weight is about 42 kDa. Protein A is especially actively synthesized in the logarithmic growth phase at 41 ° C, is thermolabile, and is not destroyed by trypsin; its unique property is the ability to bind to the Fc-fragment of IgG immunoglobulins (IgG 1, IgG 2, IgG 4), to a lesser extent to IgM and IgA. On the surface of protein A, several regions have been identified that can bind to a region of the immunoglobulin polypeptide chain located at the border of the CH 2 and CH 3 domains. This property has found wide application in the coagglutination reaction: staphylococci, loaded with specific antibodies, in which active centers remain free, when interacting with the antigen, give a rapid agglutination reaction.
The interaction of protein A with immunoglobulins leads to dysfunctions of the complement systems and phagocytes in the patient's body. It has antigenic properties, is a strong allergen and induces the multiplication of T and B lymphocytes. Its role in the pathogenesis of staphylococcal diseases has not yet been fully elucidated.
Strains S. aureus differ in sensitivity to staphylococcal phages. For typing S. aureus use an international set of 23 temperate phages, which are divided into four groups:
1st group - phages 29, 52, 52A, 79, 80;
2nd group - phages 3A, 3C, 55, 71;
3rd group - phages 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85;
4th group - phages 94, 95, 96;
outside the groups - phage 81.
The relation of staphylococci to phages is peculiar: one and the same strain can be lysed either by one phage, or simultaneously by several. But since their sensitivity to phages is a relatively stable sign, phage typing of staphylococci is of great epidemiological significance. The disadvantage of this method is that no more than 65 - 70% can be typed. S. aureus... In recent years, sets of specific phages have been obtained for typing S. epidermidis.
Staphylococcus pathogenicity factors. Staphylococcus aureus is a unique microorganism. It can cause more than 100 different diseases belonging to eleven classes according to the International Classification of 1968. Staphylococci can affect any tissue, any organ. This property of staphylococci is due to the presence of a large complex of pathogenic factors in them.
1. Adhesion factors - the attachment of staphylococci to tissue cells is due to their hydrophobicity (the higher it is, the stronger the adhesive properties are), as well as the adhesive properties of polysaccharides, possibly also protein A, and the ability to bind fibronectin (the receptor of some cells).
2. Various enzymes that play the role of factors of "aggression and defense": plasma coagulase (the main factor of pathogenicity), hyaluronidase, fibrinolysin, DNase, lysozyme-like enzyme, lecithinase, phosphatase, proteinase, etc.
3. Complex of secreted exotoxins:
erythrocytes, necrosis when administered intradermally to a rabbit, destruction of leukocytes, death of a rabbit when administered intravenously. However, it turned out that this effect is caused by the same factor - a membrane-damaging toxin. It has a cytolytic effect on various types of cells, which is manifested as follows. Molecules of this toxin first bind to unknown receptors of the target cell membrane or are nonspecifically absorbed by lipids contained in the membrane, and then form a mushroom heptamer of 7 molecules, consisting of 3 domains. The domains that form the “cap” and “edge” are located on the outer surface of the membranes, and the domain of the “stem” serves as a transmembrane pore channel. Through it, the entry and exit of small molecules and ions occurs, which leads to swelling and death of cells with a nucleus, and osmotic lysis of erythrocytes. Found several types of membrane damaged from humans, it lyses the erythrocytes of humans, rabbits and rams. The lethal effect in rabbits is caused by intravenous administration after 3 - 5 minutes. Hemolysates erythrocytes of humans and many animal species. A lethal effect on a rabbit when administered intravenously causes after 16 - 24 - 48 hours. Very often, in staphylococci, ob
b) exfoliative toxins A and B are distinguished by antigenic properties, relation to temperature (A - thermostable, B - thermolabile), localization of genes that control their synthesis (A is controlled by a chromosomal gene, B - by a plasmid gene). Often in the same strain S. aureus both exfoliatins are synthesized. Associated with these toxins is the ability of staphylococci to cause pemphigus in newborns, bullous impetigo, scarlet rash;
c) true leukocidin - a toxin that differs from hemolysins in antigenic properties, selectively acts on leukocytes, destroying them;
d) exotoxin causing toxic shock syndrome (TSS). It has the properties of a superantigen. TSS is characterized by an increase in temperature, a decrease in blood pressure, skin rashes with subsequent peeling on the hands and feet, lymphocytopenia, sometimes diarrhea, kidney damage, etc. More than 50% of strains are capable of producing and secreting this toxin S. aureus.
4. Strong allergenic properties, which are possessed by both components of the cell structure and exotoxins and other waste products secreted by bacteria. Staphylococcal allergens are capable of causing both delayed-type hypersensitivity (HPR) and immediate-type hypersensitivity (HPN) reactions. Staphylococci are the main culprits for skin and respiratory allergies (dermatitis, bronchial asthma, etc.). The peculiarity of the pathogenesis of staphylococcal infection and its tendency to transition into a chronic form are rooted in the effect of the GHZ.
5. Cross-reacting antigens (with isoantigens of erythrocytes A and B, kidneys and skin - induction of autoantibodies, development of autoimmune diseases).
6. Factors inhibiting phagocytosis. Their presence can be manifested in the suppression of chemotaxis, protection of cells from absorption by phagocytes, in providing staphylococci with the ability to multiply in phagocytes and blocking the "oxidative burst". Phagocytosis is inhibited by the capsule, protein A, peptidoglycan, teichoic acids, toxins. In addition, staphylococci induce the synthesis of suppressors of phagocytic activity by some cells of the body (for example, splenocytes). Inhibition of phagocytosis not only prevents the body from cleansing staphylococci, but also disrupts the processing and presentation of antigens to T- and B-lymphocytes, which leads to a decrease in the strength of the immune response.
The presence of a capsule in staphylococci increases their virulence for white mice, makes them resistant to the action of phages, does not allow typing with agglutinating sera, and masks protein A.
Teichoic acids not only protect staphylococci from phagocytosis, but, obviously, play an essential role in the pathogenesis of staphylococcal infections. It has been established that in children with endocarditis, antibodies to teichoic acids are found in 100% of cases.
7. Mitogenic effect of staphylococci in relation to lymphocytes (protein A, enterotoxins and other products secreted by staphylococci have this effect).
8. Enterotoxins A, B, C1, C2, C3, D, E. They are characterized by antigenic specificity, thermal stability, resistance to the action of formalin (do not convert to toxoid) and digestive enzymes (trypsin and pepsin), are stable in the pH range from 4, 5 to 10.0. Enterotoxins are low molecular weight proteins with a molecular weight of 26 to 34 kDa with properties of superantigens.
It has also been established that there are genetically determined differences in sensitivity to staphylococcal infection and the nature of its course in humans. In particular, severe staphylococcal purulent-septic diseases are more often found in people with blood groups A and AB, less often in people of 0 and B groups.
The ability of staphylococci to cause food poisoning, such as intoxication, is associated with the synthesis of enterotoxins. Most often they are caused by enterotoxins A and D. The mechanism of action of these enterotoxins is poorly understood, but it differs from the action of other bacterial enterotoxins, which disrupt the function of the adenylate cyclase system. All types of staphylococcal enterotoxins cause a similar pattern of poisoning: nausea, vomiting, pain in the pancreas, diarrhea, sometimes headache, fever, muscle spasm. These features of staphylococcal enterotoxins are due to their superantigenic properties: they induce excessive synthesis of interleukin-2, which causes intoxication. Enterotoxins stimulate intestinal smooth muscles and increase gastrointestinal motility. Poisoning is most often associated with the use of dairy products infected with staphylococcus (ice cream, pastries, cakes, cheese, cottage cheese, etc.) and canned food with butter. Infection of dairy products can be associated with mastitis in cows or with pyoinflammatory diseases in humans related to food production.
Thus, the abundance of various pathogenic factors in staphylococci and their high allergenic properties determine the peculiarities of the pathogenesis of staphylococcal diseases, their nature, localization, severity of the course and clinical manifestations. Avitaminosis, diabetes, decreased immunity contribute to the development of staphylococcal diseases.
Staphylococcus resistance. Among bacteria that do not form spores, staphylococci, like mycobacteria, have the greatest resistance to external factors. They tolerate drying well and remain viable and virulent for weeks and months in dry fine dust, a source of dust infection. Direct sunlight kills them only for many hours, while diffused light acts very weakly. They are also resistant to high temperatures: heating up to 80 ° C withstand about 30 minutes, dry heat (110 ° C) kills them within 2 hours; they tolerate low temperatures well. Sensitivity to chemical disinfectants varies greatly, for example, a 3% solution of phenol kills them within 15 - 30 minutes, and a 1% aqueous solution of chloramine - in 2 - 5 minutes.
Features of epidemiology. Since staphylococci are permanent inhabitants of the skin and mucous membranes, the diseases caused by them can be either autoinfection (with various injuries of the skin and mucous membranes, including microtraumas), or an exogenous infection caused by contact-household, airborne droplets, airborne dust or alimentary (for food poisoning) methods of infection.
Carriage of pathogenic staphylococci is of particular importance, since carriers, especially in medical institutions (various surgical clinics, maternity hospitals, etc.) and in closed groups, can cause staphylococcal infections. The carriage of pathogenic staphylococci can be temporary or intermittent, but persons in whom it is permanent (resident carriers) pose a particular danger to others. In such people, staphylococci persist for a long time and in large numbers on the mucous membranes of the nose and throat. The reason for the long-term carriage is not entirely clear. It may be a consequence of a weakening of local immunity (lack of secretory IgA), dysfunction of the mucous membrane, an increase in the adhesive properties of staphylococcus, or due to any of its other properties.
Features of pathogenesis and clinical picture. Staphylococci easily enter the body through the smallest damage to the skin and mucous membranes and can cause a variety of diseases - from juvenile acne (acne) to severe peritonitis, endocarditis, sepsis or septicopyemia, in which the mortality rate reaches 80%. Staphylococci cause boils, hydradenitis, abscesses, phlegmon, osteomyelitis; in wartime - frequent culprits of purulent complications of wounds; staphylococci play a leading role in purulent surgery. Possessing allergenic properties, they can cause psoriasis, hemorrhagic vasculitis, erysipelas, nonspecific polyarthritis. Staphylococcal infection of food is a common cause of food poisoning. Staphylococci are the main culprits of sepsis, including in newborns. Unlike bacteremia (bacteria in the blood), which is a symptom of the disease and is observed in many bacterial infections, sepsis (septicemia - putrefaction) is an independent disease with a definite clinical picture, which is based on damage to the organs of the reticuloendothelial system (system of mononuclear phagocytes - SMF ). With sepsis, there is a purulent focus, from which the pathogen periodically enters the bloodstream, spreads throughout the body and affects the reticuloendothelial system (SMF), in the cells of which it multiplies, releasing toxins and allergens. In this case, the clinical picture of sepsis weakly depends on the type of pathogen, but is determined by the defeat of certain organs.
Septicopyemia is a form of sepsis, in which the pathogen causes purulent foci in various organs and tissues, that is, it is sepsis complicated by purulent metastases.
Bacteremia in sepsis and septicopyemia can be short-term and long-term.
Post-infectious immunity exists, it is due to both humoral and cellular factors. An important role in it is played by antitoxins, antimicrobial antibodies, antibodies against enzymes, as well as T-lymphocytes and phagocytes. The intensity and duration of immunity against staphylococci have not been sufficiently studied, since their antigenic structure is too diverse, and there is no cross-immunity.
Laboratory diagnostics. The main method is bacteriological; developed and implemented serological tests. If necessary (in case of intoxication), a biological sample is used. The material for bacteriological research is blood, pus, mucus from the pharynx, nose, discharge from wounds, sputum (with staphylococcal pneumonia), feces (with staphylococcal colitis), in case of food intoxication - vomit, feces, gastric washings, suspicious foods. The material is inoculated on blood agar (hemolysis), on milk-salt (milk-yolk-salt) agar (the growth of foreign bacteria is inhibited due to NaCl, pigment and lecithinase are better detected). The isolated culture is identified by species characteristics, the presence of the main signs and factors of pathogenicity (golden pigment, mannitol fermentation, hemolysis, plasma coagulase) is determined, the sensitivity to antibiotics is checked, if necessary, phage typing is carried out. Among serological reactions, RPHA and IFM are used to diagnose purulent-septic diseases, in particular, to determine antibodies to teichoic acid or to species-specific antigens.
To determine the enterotoxigenicity of staphylococci, three methods are used:
1) serological - with the help of specific antitoxic sera in the precipitation reaction in the gel, enterotoxin is detected and its type is established;
2) biological - intravenous administration of staphylococcus broth culture filtrate to cats in a dose of 2 - 3 ml per 1 kg of body weight. Toxins cause vomiting and diarrhea in cats;
3) an indirect bacteriological method - isolation of a pure culture of staphylococcus from a suspicious product and determination of its pathogenicity factors (the formation of enterotoxin correlates with the presence of other pathogenic factors, in particular RNAase).
The simplest and most sensitive is the serological method for the detection of enterotoxin.
Treatment. For the treatment of staphylococcal diseases, beta-lactam antibiotics are mainly used, to which the sensitivity should first of all be determined. In severe and chronic staphylococcal infections, specific therapy gives a positive effect - the use of an autovaccine, toxoid, anti-staphylococcal immunoglobulin (human), antistaphylococcal plasma.
Specific prophylaxis. To create artificial immunity against staphylococcal infection, staphylococcal toxoid (liquid and tableted) is used, but it creates antitoxic immunity only against staphylococci, lysed mainly by group I phages. The use of vaccines from killed staphylococci or their antigens, although it leads to the emergence of antimicrobial antibodies, but only against those serovariants from which the vaccine is made. The problem of finding a highly immunogenic vaccine effective against many types of pathogenic staphylococci is one of the most important problems of modern microbiology.
STYLAB offers test systems for analyzing the content of Staphylococcus aureus in food and the environment by microbiological methods, as well as for determining the DNA of this bacterium using PCR.
Staphylococcus aureus ( Staphylococcusaureus) is a ubiquitous gram-positive immobile facultatively anaerobic non-spore-forming bacterium belonging to cocci - globular bacteria. This microorganism is part of the normal microflora of the skin and mucous membranes in 15-50% of healthy people and animals.
Some strains of this bacterium are resistant to. The most famous of these is methicillin-resistant Staphylococcus aureus (MRSA). For a long time, it was considered the causative agent of nosocomial infections, but since the mid-1990s, it has been known about diseases in people who were not in hospitals. Most often, these were purulent skin lesions, however, when scratching the lesions, MRSA entered the bloodstream and affected other organs. Methicillin-resistant Staphylococcus aureus was susceptible to vancomycin, a toxic antibiotic that nevertheless eradicates the organism.
Another antibiotic-resistant bacterium is vancomycin-resistant Staphylococcus aureus (VRSA). Doctors and scientists have been expecting this organism ever since they learned about the existence of MRSA and vancomycin-resistant enterococcus (VRE), a non-pathogenic organism that lives in the intestines, since horizontal transfer allowed the exchange of genes between these bacteria. VRSA was first discovered in 2002 and indeed was resistant to all powerful antibiotics that existed at that time. However, his weak point was his sensitivity to the old sulfonamide - bactrim.
Staphylococcus aureus is found in soil and water, often contaminates food and can infect all tissues and organs: skin, subcutaneous tissue, lungs, central nervous system, bones and joints, etc. This bacterium can cause sepsis, purulent skin lesions and wound infections.
The optimum temperature for Staphylococcus aureus is 30-37 ° C. It can withstand heating up to 70-80 ° C for 20-30 minutes, dry heat - up to 2 hours. This bacterium is resistant to drying out and salinization and is able to grow on media with 5-10% table salt content, including fish and meat balyk and other products. Most disinfectants kill Staphylococcus aureus.
Staphylococcus aureus secretes a wide variety of toxins. Membranotoxins (hemolysins) of four types provide hemolysis; in addition, membranotoxin α in experiments causes skin necrosis, and when administered intravenously, death of animals. Exfoliatins of two types damage skin cells. Leukocidin (Panton-Valentine's toxin) causes disturbances in the water-electrolyte balance in the cells of leukocytes, especially macrophages, neutrophils and monocytes, which leads to their death.
In accordance with TR CU 021/2011 and other documents, the content of coagulase-positive staphylococci is also limited in food. These are bacteria that produce coagulase, an enzyme that causes blood plasma to clot. In addition to S. aureus These include S. delphini, S. hyicus, S. intermedius, S. lutrae, S. pseudintermedius and S. schleiferi subspecies. coagulans... According to some reports, S. leei is also coagusal positive.
To determine Staphylococcus aureus in samples, both microbiological methods, including selective media, and DNA analysis using the PCR method are used.
