Staph infections diagnostics

In spite of the fact that staphilococcuses belong to number of most easily found and distinguished microorganisms which are not demanding complex diagnostic receptions for their revealing, microbiologists have some difficulties till now with practical work at an establishment of their causal role in a number of various diseases. On the one hand, the presence of pathogenic staphilococcuses found in the investigated stuff, not always is the convincing proof of their etiological value. With another, considering the big diversity of implications of a biological potency of these microbes, depending from different, not always factors giving in to the account, their wide variability under the influence of medicinal materials and the macroorganism, enough happens to define their potential pathogenicity. At last, the third cause is bound by that staphilococcuses are representatives of a normal microflora from bunch of conditional pathogenic microorganism and, along with nonpathogenic, pathogenic representatives dwell in an organism of humans, extending thus rather irregularly on different fields of a human body.

If abjection of a pathogenic staphilococcus, from blood of sick humans and various lumens which it is considered to be sterile, in overwhelming majority of cases can serve as the proof of its role as disease-producing factor, presence of staphilococcuses on a mucous fauces, a nose etc. Even at their obvious disease state demands the big guard of the explorer in conclusions about an aetiology of the given diseases.

Therefore, naturally, there can not be general references, both at diagnostics of various staph infections, and at microbialogic researches of fields of a body of the human and different objects of an external environment. The wide exchange of opinions between microbiologists of scientific institutions and the practical laboratories, carried out at congresses and the conferences devoted to problems of staph infections, and also at personal contacts, apparently, should lead to certain views on the various methods of research offered for abjection and identification of staphilococcuses. However the big number of the inquiries arriving from laboratories SES and medical institutions, testifies to obvious difficulties which arise at explorers at the decision of different questions of microbialogic diagnostics of staphilococcuses, and in a number of microbialogic establishments there are still some out-of-date methods and the receptions leading to irregular assessments and even to annoying misunderstanding.

The important condition of efficacyy of laboratory diagnostics is associated definition of qualitative and quantitative criteria of the maintenance of pathogenic staphilococcuses in an investigated stuff. Proceeding from it, I consider necessary to state a number of methods of the basic microbialogic researches, the most simple and accessible for each practical and scientific laboratory which we use for many years.

Main principles of detection and identification of pathogenic staphilococcuses

Preliminary diagnostics of staphilococcuses can be based on bacterioscopic studying of film preparations, gramme-stained. Pathogenic staphilococcuses, besides a uviform locating, are characterised by the correct orbicular form of cells. Detection of the staphilococcuses which are in cells, allows to answer about staph infection presence. In the majority of cases for an exact establishment of pathogenicity of the found staphilococcuses it is required to secure these microorganisms in pure culture by sowing of an investigated stuff on dense nutrient mediums.

Now the assortment of the differential, elective and memory mediums offered for abjection of pathogenic staphilococcuses, is rather appreciable and continues to extend. Many explorers on the basis of knowledge of the basic biochemical characteristics and nutrient requirements of pathogenic staphilococcuses at designing of mediums aspire to raise their drillability and to provide possibility to carry out their quantitative account, to receive a reliable mean to distinguish potentially disease-producing staphilococcuses from saprophytes.

The use of liquid memory mediums for primary abjection of staphilococcuses is inexpedient, as deprives of the explorer of possibility of a quantitative assessment of the maintenance of microbes in surveyed objects, and at casual contaminations promotes errors. Especially it concerns such researches, as detection of a carriage of pathogenic staphilococcuses where the proof is only massive growth, or definition of an etiological role of these microorganisms "at food poisonings or outside inflammatory processes. If it is a question of a blood analysis, and also about those few cases when happens it is necessary to tap even individual teleorganic individuals of these microbes, for example at the control of efficacyy of disinfection, check on sterility or titration sowings in similar cases use of memory mediums is quite justified.

From the big number of the various nutrient mediums tested for primary abjection of staphilococcuses, in practice have justified itself the few. Usual nutrient agar (pH 7,2 7,4), 2 % prepared by addition an agar-agar to a beef-extract broth or to tryptic overcook Hottinger, can be used at research of exsudates from the occluded lumens. Staphilococcuses grow in 18-24 hours of an incubation at 37 C° in the form of smooth opaque with equal edges opaque and slightly convex colonys shining with equal edges. Character of a pigment is taped better after an additional daily withstanding of cups at a room temperature (20 C°) on light. If at an investigated stuff there is an extraneous flora on outward of colony of staphilococcuses can be difficultly distinguishable.

The blood agar use gives the chance, except pigment revealing, to define and hemolitic activity of staphilococcuses. Thus it is necessary to remember, that the hemolitic ability defined on cups with a blood agar, depends on many factors - a kind of blood, its concentration, presence in it of antitoxins, thickness of a layer of medium, the carbon dioxide maintenance in atmosphere of cultivation, density of sowing, presence and character of concomitant flora etc.

At selection of colonys from a blood agar it is necessary both hemolitic, and not giving a hemolysis, especially if research is spent on the medium with human or horse blood, and absence of a hemolysis on such mediums does not allow to conclude about absence in general hemolitic activity. Therefore use of a blood agar for primary sowing expediently only at inspection of the objects which are not containing an extraneous microflora, also is desirable to add blood of a rabbit or the ram in a nutrient medium.

If research of pus of open wounds or abjointed ulcers or fistulas it is necessary to use elective-differential medium for staphilococcuses - an is vitelline-salt agar (VSA) is carried out. The selectivity of this medium is provided with the high maintenance of chloride sodium, and the added egg yolk allows to tap lecitovitellase activity which is one of indicators of pathogenicity of staphilococcuses and owing to it VSA differentiates pathogenic representatives, than a blood agar more accurately. Preparation VSA is simple.

Make for the future and sterilise in an autoclave at 120 C° within 20 minutes nutrient agar (MPA or Hottinger) pH 7,2-7,4, keeping 10 % of table salt. Before the use saline nutrient agar kindle, cool till 45-50 C° and add to it of 20 % on volume of a sterile vitelline suspension (1 yolk of an egg on 200 ml of a normal saline solution). For the best emulsification it is desirable to have flasks with a glass beads. After careful hashing medium spill on 20-25 ml in cups which can be stored in a refrigeration cabinet within several days.

It is necessary to effect massive sowing of an investigated stuff on cups C VSA, and an incubation to conduct within 2 days at 35-37 C°. The extraneous flora is sharply depressed, staphilococcuses on VSA grow practically in pure culture. Immediately at viewing of cups round colonys formation of iridescent crowns and cloudy region - lecitovitellase reaction becomes perceptible. Such colonys, not less two of each sowing, twist on the agar cut meat-peptonic for the subsequent check of evolved cultures in plazmo-coagulation reaction.

To avoid errors in definition of vitelline reaction, it is necessary to mean, that turbidity of medium without an iridescent fillet is sometimes observed, in this case assay on lecitovitellase is considered negative. If the colonys which have grown on VSA, do not give lecitovitellase reaction, i.e. Are not surrounded by an iridescent crown, but there is a growth of staphilococcuses they are considered too suspicious and also twist not less two of each such sowing on cups with simple nutrient agar maculae in diameter by of 5-10 mm. After a daily hatching at 37 C° the received macrocolonys check in plasma-agglutination reaction for what microbic mass stir a loop on glass in drops of citrate rabbity or human plasma, divorced 1:4. Formation of flakes consider during 30 seconds - 1 minutes In the presence of a plasma-agglutination consider such strains suspicious and twist on a slant for the further studying in coagulase reaction. At such selection the number of passed strains of pathogenic staphilococcuses of a human parentage is insignificant, the cultures secured from animals, it is required to check in plasma-coagulation reaction.

Coagulates assay is the basic sign defining pathogenesity of staphilococcuses, therefore its statement demands to itself the maximum attention. For revealing of coagulase activity at the staphilococcuses secured from humans, it is possible to use both a whole blood, and plasma of rabbits or the human. It is possible to use also blood or the plasma taken immediately from the human. Tinned plasma or the blood used for transfusion, are unsuitable for reaction as to them preservatives which vital activities of staphilococcuses interfere, to implication of coagulase activity are often added. It is not necessary to apply blood of animals or the humans bearing staphylococcal diseases, in particular fixing as it can appear unsterile and contain an anticoagulasa. At work with the staphilococcuses secured from horned livestock, it is possible to use the bull blood.

Aseptic the taken blood stabilise by means of 1-4 % of Citras or 0,1 % of Sodium oxalatum. For plasma reception nitrate blood centrifuging or defend on a cold. But, as it was already spoken, it is possible to use and a whole blood. Then blood plant with a sterile normal saline solution in the ratio 1: 3 or 1: 5 and spill on 0,2-0,3 ml in sterile tubes which before sterilisation should be washed carefully up as the residual of chemical materials can influence result of a plasma-coagulation.

Examinees lead agarinic cultures of staphilococcuses a loop in plasma and well stir. Bouillon cultures lead the pipet in volume of 0,1 ml In each experience it is necessary to put the control with cultures of obviously pathogenic staphilococcuses giving coagulase reaction, and strains a coagulase of negative microorganisms. Besides, the part of tubes with diffuse plasma should be abandoned not sowed by microbes and to maintain in a thermostat throughout all term of experience. In case of coagulation offensive at least in one of them all experience needs to be rearranged with new portion of blood. Tubes with investigated cultures maintain in a thermostat at 37 C° within days. The account of results is spent necessarily twice: in 2-3 hours and 24 hours. Consider coagulation of plasma and formation of clots. Usually the cultures activly producing a coagulase, give positive reaction in 2-3 hours and if they possess and the expressed fibrinolitic activity pristinely formed clots can undergo to fusion to the extremity of days. Weakly coagulabling strains can give positive reaction in serotinal terms - to the extremity of days. The experiences which have yielded doubtful result, it is necessary to repeat.

Some explorers, having received negative or doubtful result, abandon tubes on a table. It to do it is impossible, as plasma coagulation in more serotinal terms can have not specific character and it should not be taken into consideration.

The staphilococcuses giving positive coagulase assay, should be surveyed as potentially pathogenic, irrespective of presence or absence at them hemolitic properties and character of a pigment, them carry to kind St. aureus and further subject phagotypisation and check on sensitivity to antibiotics.

Profound studying of staphilococcuses has shown, that the basic sign of pathogenicity - coagulase reaction can be exposed to variability at influence of various factors, therefore in some cases, at abjection a coagulase of negative staphilococcuses in a monoculture from the pathological locuses, it is expedient to study at them other signs of potential pathogenesity and first of all - ability to ferment Mannitum in anaerobic conditions. It is known, that among a coagulase negative the part of cultures possesses ability to disjoin Mannitum in anaerobic conditions.

Definition of a fermentation of Mannitum in anaerobic conditions technically very simply also is carried out by sowing by a nyxis of investigated cultures in tubes with a high column of a semifluid Hottinger's agar. This medium before sowing "regenerate", i.e. Boil in the water bath, quickly cool, and after sowing fill in with a layer of a sterile liquid paraffin. Sowings incubate at 37 C° within 5 days. However in appreciable number of cases results can be considered already in a day.

Along with plasma-coagulation reaction, great value one more important ability of staphilococcuses characterising them potential pathogenicity, - deoxyribonucleic has got activity. Many explorers reporting about high correlation of this sign with coagulase activity and a toxigenicity of studied cultures; notice, that the staphilococcuses secured from a pathological stuff, as a rule, possess DNA-ASE. A coagulase the positive strains received from carriers, can not have some this enzyme, and usually absence deoxyribonucleic activity is combined with low biochemical ability and atoxygen such cultures of staphilococcuses.

On observations of some explorers, among a coagulase of negative strains of staphilococcuses, but possessing other signs of the pathogenicity, secured in some cases from patients with various purulent infection contaminations, deoxyribonucleic activity showed from 10 to 29 % of such cultures.

Now this test can be a reliable differential sign between pathogenic and nonpathogenic staphilococcuses and at the same time the coagulase of positive strains and, certainly can help with differentiation of degree of pathogenicity, the attention to a coagulase negative will draw, but possessing this enzyme to cultures of staphilococcuses and in some cases will allow to carry the last to disease-producing forms with larger confidence.

The definition procedure deoxyribonucleic activity is simple, method Jeffries is put in its basis. Prepare for the future 2 % MPA (pH-8,6), keeping DNA (2 mg/ml of medium), spill on vials and sterilise a fluid steam within 30 minutes Before flood in cups by medium add CaCIa (0,8 mg/ml). On the dried medium sow daily cultures of staphilococcuses. After an incubation within 18-20 hours at 37 °, on a surface of medium pour IN solution НС1 and in 7-10 minutes consider regions clarifications which estimate crosses.

For preparation of working solution of DNA, shot nucleic acid from calculation of 10 mg/ml place in the distilled water, add 0,5 ml of 0,2 % (or 0,8 %) solution NaOH on each 10 ml of water. For the best dissolution of DNA an admixture or heat up to boil in the water bath at constant hashing by a glass rod, or abandon for the night at 37 C° in a thermostat. To fused and cooled to 50 C° MPA add working solution of DNA from calculation of 1 mg/ml (on 9 ml МПА-1 ml of solution of DNA), stir, spill по10л (l in tubes, sterilise a fluid steam during 30 mines or in an autoclave at 0,5 atmospheres within 20 minutes Term of storage - 1-2 months

At statement of experience columns of an agar from DNA kindle, in the water bath add 10 % of solution CaCl, stir and pour out on a layer of well dried nutrient agar in Petri dishes and again dry. The daily agarinic culture is sowed with small plaques (diameter is peer 2-2,5 mm) or a die-replicator no more than 20-25 plaques, in order to avoid coalescence of regions with DNA depolymerizations. After 18-20-часовой incubations an agar surface fill in 5 ml IN of solution НС1 and through 3-5 mines consider regions clarifications. Reactions estimate in crosses.

Thus it is necessary to consider, that intensity of region of a depolymerization on dense medium not always coincides with quantity of production of this enzyme at investigated staphilococcuses in fluid mediums.

For Fibrinolysinum detection use Christie's a little advanced method.

The medium of Christie-Chepmena has the following structure: a meat extract-0,45 of, pepton-0,75, chloride sodium-1,2 of, an agar-2,75 of, ml water-130, pH mediums == 7,0. It is sterilised in an autoclave and remains for the future.

Before experience medium kindle, cool to 50 C° and add 15-20 % of sterile citrate human plasma. The admixture gets warm in the water bath at 65-70C° during 2 5 mines before appearance of a clear turbidity, and then spreads in cups. Examinees of culture sow with "maculae" till 15-20 on a cup. Sowings incubate at 37 C°. Formation of regions clarifications round colony pristinely in 14 hours is considered, then in 24 and 48 hours as reaction at various cultures streams unequally quickly and at a number of strains it educes in more serotinal terms.

We consider the fibrinolitic test rather suitable for definition of potential pathogenesity of staphilococcuses, but we estimate only in a complex with other signs of activity.

Hyaluronidase activity is defined on the Poppy-wedge method.

Hyalrunate solution giving in the presence of 2 N acetic acid a typical clot, spreads on 0,5 ml in sterile tubes. 0,5 ml of daily culture of a staphilococcus in peptonic water are added. Controls serve: hyaluronate + the distilled water and hyaluronate + not sowed medium. Admixtures are stirred up and maintained at 37 C° 15-20 minutes Then in each tube is added on two drops 2 N solution of acetic acid and stirred up. In monitorings the mucous lump should be formed. In experience in the presence of Hyaluronidasum it is absent, that testifies to a depolymerization of hyaluronic acid microbic enzyme.

For studying of a toxigenicity of staphilococcuses it is convenient to use a method offered Lewi, allowing to define types of hemolysins.

With that end in view cups with the nutrient agar keeping 5 % of erythrocytes washed by a normal saline solution of the ram, a rabbit and the human prepare.

On a nutrient medium surface on diameter place the sterile stria of a filter paper moistened with alpha antitoxic Serum. Having retreated 1-2 ml from stria edge, it is perpendicular to it sow strokes investigated cultures. Sowings incubate at 37 C° within days then consider results.

The alpha hemolysis shows in the form of a full clarification of medium round a stroke of culture and will be neutralised by alpha antitoxic Serum. It is taped on mediums with the rabbity and mutton erythrocytes

The beta hemolysis on an agar with the mutton blood gives the particulate clarification which region has a burovato-purple shade. It "warmly-cold" toxin also is better taped after an additional daily withstanding of cups in a refrigeration cabinet at temperature +4 C° on a characteristic level-by-level hemolysis of the mutton erythrocytes and not centralised by alpha antitoxic Serum. On the rabbity erythrocytes it is not active. The Delta-gemolizin is defined on a narrow stria of a hemolysis of the mutton and rabbity erythrocytes, will not be neutralised by alpha antitoxic Serum. However formation delta-gemoli-zina on these cups can be disguised alpha toxin action and consequently it should be checked on mediums with erythrocytes of the human or a horse. Thus, for definition of all 3 types of hemolysins are enough to use erythrocytes of the ram and the human or a horse.

For definition of virulence of staphilococcuses there are some various methods of infestation of white mice.

The most simple is introduction of 0,1 ml daily bouillon cultures of the examinee of a staphilococcus in a caudal vein. The account of  destruction of animals is carried out within 10 days, register presence of abscesses in nephroses.

It is convenient to use mean Badenski, Daily bouillon culture centrifuge at 3000 rpm - 30 minutes the Received deposit resuspend in half volume of a decantate and in number of 0,05 ml is introduced to 6 mice behind an eyeball into a periorbital fat. The culture causing  destruction of half and more infested mice within 6 days, consider virulent.

At these methods of infestation by virulent culture at animals septic process with a primary lesion of nephroses educes communities. The basic  destruction of mice descends for 3-5th day after infestation.

Other means of infestation of white mice (intraperitoneal and intranasal) demand in tens

Time of a larger infesting dose, a maximum of  destruction of mice is necessary for 1-2 days that characterises mainly toxic component of process.

At the same time a number of explorers prefer more simple technically intraperitoneal mean of infestation of mice which simultaneously allows to study cellular and humoral mechanisms of development of an infection contamination.

Comparison of virulence of staphilococcuses for white mice with separate factors of their pathogenicity has shown, that virulence of strains, according to the big number of explorers, correlates with alpha hemotoxin level. As to other signs of pathogenicity and their correlation with virulence varied data are received. So, virulence of strains correlated with production of a beta hemolysin, a lethal factor, a lecitovitellase, Hyaluronidasum and a coagulase.

For comparison of virulence of staphylococcal cultures it is possible to use their ability to cause dermonecrotic reaction at rabbits.

Prepare a 4-milliard suspension of daily agarinic culture of a staphilococcus in a normal saline solution, and of the received density do delutions to receive 2 and 1-milliard suspensions. From each delution in volume of 0,1 ml that compounds 100, 200, 400 million microbic bodies, introduce to a rabbit in cut or depilate the day before a skin. On one rabbit it is possible to put simultaneously to 8 intracutaneous tests. Daily note implications of dermonecrotic reaction, and it is definitive for 4 days. For the minimum dermonecrotic dose accept that least quantity of culture which gives a necrosis for 4 days.