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Aim: To analyze results of conducted preclinical animal tests of ointment and gel preparations based on the somatic antigen ("Somatin") of Staphylococcus aureus., Materials and Methods: Contents of ointment and gel as well as method of "Somatin" extraction from cytosomatin ointment are presented. Standard serum was obtained by immunization of rabbits by "Somatin". Tests for acute and chronic toxicity and sensitizing effect of cytosomatin ointment were performed. 1% gentamycin ointment was used for comparison with cytosomatin ointment and gel for treatment of pyodermia. Preparations were patented., Results: Authenticity and specific activity of ointment and gel preparations were demonstrated. Animal tests for acute and chronic toxicity of cytosomatin ointment did not reveal inflammatory reaction of skin and sclera as well as influence on biochemical parameters of the blood or abnormalities of visceral morphological structure. Assessment of sensitizing effect of cytosomatin ointment did not reveal promotion of allergy. Advantages in treatment of staphylococcal skin infections and wounds with cytosomatin ointment and gel as compared with gentamycin ointment were established., Conclusion: The main immunogenic mechanism of "Somatin" substance was activation of complete phagocytosis. The character of immune response was specific. Selected optimal ratios of therapeutic doses for ointment and gel bases promote active permeation of the drug into skin that enhances its therapeutic effect. Duration of treatment with cytosomatin ointment was shortened on 5 days as compared with gentamycin ointment. Duration of treatment with cytosomatin gel was shortened on 8 days as compared with prototype.

Experimental in vitro study of influence of 2% solution of pectins (red beet, apple, citrus, manufactured by "Vitaline" company, citrus high- and low-etherified pectins, manufactured by "Hercules" company, Unipectine OB 700, and biologically active supplement "Pecto") on growth of staphylococci and production by them of type A and B enterotoxins was performed. It was shown that red beet, citrus high- and low-etherified pectins, as well as biologically active supplement "Pecto" render bactericidal effect on staphylococci and inhibit synthesis of types A and B staphylococcal enterotoxins. Citrus pectin "Vitaline" and Unipectine OB 700 don't have such influence. The most effective pectins, which were able to inhibit synthesis of types A and B staphylococcal enterotoxins, were red beet, apple, and citrus low-etherified pectins as well as biologically active supplement "Pecto".

Detection rate of enterotoxigenic Staphylococcus aureus isolated from faeces of 62 children aged from 3 months old to 7 years old with intestinal dysbacteriosis was studied by indirect hemagglutination assay and enzume immunoassay. It was shown that strains of S.aureus producing staphylococcal enterotoxin A (SEA) are prevailed (40.3%) in children with disturbances of intestinal microflora while staphylococcal enterotoxin B (SEB)-producing strains were detected in 20.9% of children. Amount of produced enterotoxin varied for SEA from 125 ng/ml to 2000 ng/ml and for SEB--from 125 ng/ml to 250 ng/ml. Inverse dependence of detection rate of enterotoxin-producing strains in faeces on age of children was established. The most number of enterotoxigenic strains of S.aureus was detected in infants. These data point to expediency of determination of enterotoxin-producing ability of S. aureus strains isolated from children with dysbacteriosis as measure of danger of this microorganism for children's health and indication for adequate actions for its elimination.

The content of methicillin resistant S. aureus (MRSA) genes, coding the synthesis of staphylococcal enterotoxins A, B, C (sea, seb, sec) and the toxin of the toxic shock syndrome (tst-H) which was classified with pyrogenic toxins of superantigens (PTSAgs), was studied with the use of PCR amplification. The study revealed the specific features of the content of genes sea and sec, detected in epidemic strains, identified earlier and found to circulate in Russian hospitals. Among the isolates, genetically related to international epidemic strain EMRSA-1, isolates containing no gene sea were detected, while among the isolates genetically related to strain EMRSA-2, isolates containing not only gene sea, but also gene sec were detected, which was indicative of the tendence of this epidemic strain in the direction of further acquisition of pathogenicity genes. As revealed in further studies, among the cultures obtained in bacteriemia, 88% contained gene sea. Two out of three isolates obtained from patients with the symptoms of toxic shock also contained this gene. The differences in the content of genes PTSAgs (sea, seb, sec and tst-H) could serve as a genetic criterium for the differention of isolates circulating in a hospital, as well as for a more complete characterization of the epidemic strains MRSA. The determination of the given genetic markers in genetic strains in circulating strains will make it possible to prognosticate the structure, severity and outcomes of hospital infections. The conditions of PCR amplification for the determination of genes sea, seb, sec and tst-H, as well as multiplex PCR for the determination of genes sea and seb, were developed.

The occurrence of Staphylococcus aureus strains producing enterotoxins of types SEA and SEB, which isolated from patients of different profile and caused the infectious process accompanied by pronounced intoxication without vomiting and enteric disturbances, was determined by means of the indirect hemagglutination test. The collection included 28 strains isolated in sepsis, 38 strains isolated in pneumonia, 57 strains isolated from patients with burns and 23, from the hands and nasopharynx of the medical staff. Among the staphylococcal strains isolated in sepsis, 75.6% synthesized SEA and 5.4%, SEB. The occurrence of SEA- and SEB-positive strains isolated in pneumonia was, respectively, 42.1% and 2.6%. From patients with burns SEA-positive staphylococci were mainly isolated (92.9%). Only 3% of the cultures isolated in wound infections produced SEA. From the medical staff, 13.4% of SEA-positive strains and 17.3% of SEB-positive strains were isolated. The data obtained from this study indicate the expediency of the determination of the enterotoxigenic properties of S. aureus clinical isolates in medical institutions for prophylactic measures with a view to the prevention of the spread of pathogenic clones.

The study of the length of the amplification products of the coagulase gene with the subsequent restriction analysis (the method of PCR--restrictive fragment length polymorphism, or RFLP) was used for typing 90 S. aureus strains. Among the strains under study, 78 were methicillin-resistant S. aureus (MRSA) strains, including 74 obtained in 1986 - 2002 in hospitals of different cities of the Russia and Belarus, as well as epidemic strains EMRSA-1, -2, -3, -12, obtained from the National Laboratory of Health, London (UK). The use of this method made it possible to type all the strains under study, which were differentiated into 9 groups by means of endonuclease Sfo1 and 7 groups by means of Alu1. Majority of clinical MRSA strains, belonged, according to the type of restriction, to groups 4 and 5. The study of the coagulase gene by the method of PCR - RFLP made it possible: to analyze the epidemic situation in hospitals for a period of several years; to compare the properties of strains isolated in different hospitals; to establish the genetic relationship of strains, isolated in 1998 - 2002, with strains, isolated in 1986 - 1990. The results of the study suggest that at least two epidemic MRSA strains, genetically similar to international strains, circulate in hospitals of Russia.

This method was used for typing of 31 Staphylococcus aureus methicillin-resistant (MRSA) strains; of these, 27 were clinical isolates obtained in hospitals of different cities of Russia and Belarus and 4 were international epidemic strains EMRSA-1, -2, -3, -12. The sequencing of the variable area, located in the middle part of the coagulase gene between nucleotides 979-1355 and detected with the use of information technologies, was carried out. The results of this sequencing were compared with those of the earlier study on the polymorphism of the area of the same gene between nucleotides 1513-2188, carried out by the method of PCR-restrictive fragment length polymorphism. The sequencing of the part of the coagulase gene made it possible to confirm the presence of essential differences in the nucleotide sequences of the coagulase gene in international strains EMRSA-1, -3, -12, grounds for classifying clinical isolates of MRSA strains with two groups (4 and 5), as well as the genetic relationship of different phage types, isolated in different clinics. The study revealed considerable similarity in the nucleotide composition of strains EMRSA-2 and EMRSA-12 despite the fact that, according to the results of Cfol restriction of the 3'-end, they were classified with different groups; the study also revealed the identity of the nucleotide sequences of the coagulase gene in the cultures of group 5, isolated in hospitals of Moscow, St. Petersburg, Orenburg, and strain EMRSA-2, as well as methicillin-sensitive S. aureus strain 8325-4; in addition, in clinical isolates of group 4 and strain EMRSA-1 a considerable degree of homology was revealed. The study of two different loci made it possible to find out the strain with the recombinant form of the coagulase gene. The approach used in this study permitted the differentiation of the international epidemic strains EMRSA-1, -2, -3 and -12 into individual groups, which coincided with the results of Enright et al. (2002) who used multilocus sequencing.

The typing of S. aureus methicillin-resistant strains, isolated in different hospitals of Moscow; was carried out with the use of three collections of phages: the International Set of Phages; the set of phages of the International Center of S. aureus phage typing in London (L); and the experimental collection of phages of the Gamaleya Institute of Epidemiology and Microbiology in Moscow (M). In this study made with the use of both the phages of the International Diagnostic Set and phages L in the standard typing dose of 1 TP about 6% of the cultures under study proved to be sensitive. When the typing dose was increased to 100 TP the phages of the international diagnostic set lyzed 75.5% of the cultures. The typed strains were found to belong to phage types 77 (71.7%), 77/84/85 (19.6%) and 94/96 (6.5%). At a concentration of 100 TP phages L lyzed 83.7% of the cultures, but the dominating phage types could not be determined due to a great variety of phage markers. In contrast to the two preceding collections, the third phage collection M was composed in such a way that in the study of the investigated culture the specificity of its restriction modification was primarily evaluated and only then the presence of antiphage immunity was determined. This latter collection was used in the evaluation of 93.1% of the cultures. By the specificity of their restriction specification system the majority of them were classified with two new groups, heretofore not described. Only this collection M made it possible to differentiate epidemic and sporadic strains and to evaluate the epidemic situation in all 6 hospitals.

An enzyme immunoassay (EIA) system for detection of staphylococcal enterotoxin, type C, has been developed. The sensitivity of the system is 1 ng/ml. The optimum EIA parameters have been worked out. The absence of false positive results with heterologous toxins confirms the specificity of the assay system. The possibility of the detection of staphylococcal enterotoxin, type C, in staphylococci isolated from different sources has been shown.

The data on a high level of interleukin production in mice, resistant to staphylococcal infection (CBA), in comparison with that in mice, sensitive to this infection (C3HA), are presented. The production of interleukin 1 in intact and infected mice of different strains exhibited no differences between these strains, but its level considerably increased after the inoculation of the animals. A the same time differences in the synthesis of IgM and IgG1 in intact and infected mice of different strains were noted.

The difference between the proliferative response of spleen cells to S. aureus cytoplasmic antigen on days 5 and 9 of the infectious process was shown in experiments of CBA and C3HA mice, opposite in their susceptibility to systemic staphylococcal infection. Hybrid mice F1 (CBA x C3HA) inherited a high level of proliferative response on day 5 (as in resistant CBA mice) and its low level on day 9 (as in susceptible C3HA mice). These data on genetic differences in proliferative response to S. aureus cytoplasmic antigen were confirmed in the test of the proliferation of lymph node lymphocytes taken from mice of these lines immunized with the above-mentioned antigen in Freund incomplete adjuvant into the pad of their paws 14 days before the test.

It was shown in experiments on a model of fatal staphylococcal sepsis in 200 mice and 15 dogs that the perfusate of a pig's spleen obtained by perfusion of the isolated organ with a dextran solution (rheopolyglucin, polyglucin) is capable of preventing the animals' death. Intravenous infusion of 400-500 ml of a xenospleen perfusate at an interval of 1-3 days, which was included in the complex of postoperative treatment of 45 patients with destructive diseases of the abdominal organs complicated by peritonitis and sepsis, contributed to reduction of lethality and the terms of recovery. It is shown that the main effect, besides detoxification, is based on immunomodulation at the cost of biologically active substances produced by the cells of a xenospleen and contained in the perfusate. The best results were produced with the use of the perfusate of a xenospleen in the early periods after radical operation and thorough cleansing of the source of the infection.

Protein antigen A was isolated from the microbial cells of Cowan I strain of Staphylococcus aureus by hot extraction method. Ion exchange chromatography on DEAE-cellulose-32 and gel-filtration on sephadex F-100 were used for purification of protein A. Microprecipitation in agar test against homologous immune rabbit serum and normal human serum showed purified protein A to be identical by serological specificity to the standard preparation obtained from Prof. Oeding's laboratory. In order to assess the immunogenic properties of protein A by various doses of the preparation (from 2 to 1000 microgram) mixed with A1 (OH)3 albino mice were immunized and then infected with the microbial culture of the homologous strain. As revealed, protein A not only failed to protect the animals from death, but even aggravated the course of infection.

Crude capsular material isolated from S. aureus strain 1193/74 was characterized chemically and subjected to purification by 4 different methods. The best results were obtained by the method including deproteinization of the material with a mixture of chloroform and butanol, fractional precipitation with ethanol and separation on SE-Sephadex. Galactose, uronic acids, and a small amount of protein served as components characteristic of the purified capsular material. Galacturonic and glucuronic acids were identified amoung the uronic acids. Besides, there were revealed other acids with a carbohydrate base, whose nature is still obscure.

The data on the stimulating action of S. aureus cells, strains B-243, 2287, Wood-46, Cowan I, as well as cell-wall peptidoglycan, on the formation of endogenous colonies in the spleen of sublethally irradiated mice are presented. Teichoic acid, S. aureus ribosomal and cytoplasmic antigens produced no such effect. Whole S. aureus cells and their components were incapable of activating transitory colonies in the spleen of sublethally irradiated mice. After immunization with cell walls, peptidoglycan and protein A the mice showed a rise in the absolute and relative content of blood-forming stem cells in the marrow and the spleen. Killed S. aureus cells increased the relative content of blood-forming stem cells in the marrow, while in the spleen a rise in both absolute and relative content of such cells occurred, which was detected in the exocolonization test.

The mitogenic effect of corpuscular antigens with respect to the splenocytes of animals was found to depend on the strain of Staphylococcus aureus. The maximum synthesis of DNA in the cells was induced by corpuscular antigen Smith and the minimum synthesis, by Wood-46. The synthesis of DNA was activated in both B- and T-splenocytes in response to corpuscular antigens Wood-46, Cowan-1 and Smith, as well as to the cell wall and protein A. Peptidoglycan produced a mitogenic effect only in B-lymphocytes, and teichoic acid showed no mitogenic activity in mouse splenocytes. The mitogenic effect of staphylococcal antigens on splenocytes depended on the dose of the antigen and the time of cultivation. After 48-hour cultivation the incorporation of 3H-thymidine into the DNA of mouse cells was 5 times higher than into the DNA of guinea-pig cells. The optimum mitogenic dose in thymectomized BALB/c mice with respect to splenocytes was higher than in normal BALB/c mice practically by one order.

The preparations of staphylococcal ribosomes, pure and homogenous in their chemical composition, have been obtained by differential centrifugation of the cytoplasm. These preparations have been found to consist mainly of RNA and protein in the ratio 1.5:1. The characteristic feature of ribosomes has proved to be the presence of one symmetrical peak in centrifugation in sucrose density gradient and the relation of the optical density values at 260 nm and 280 nm equal to 2, which is peculiar only to purified ribosomes.

Experiments were conducted on 1500 albino mice; protective activity of 7 batches of S. aureus capsular antigen isolated from 5 strains was studied. The animals were immunized subcutaneously with different doses of the preparation (from 0.001 to 10000 microgram) once, twice, and thrice, and infected intraperitoneally with 6 encapsulated cultures. Capsular antigen proved to possess weak protective properties. The following regularities were revealed: 1) high antigen doses (over 5 microgram) increased mouse sensitivity to staphylococcus infection; 2) the greatest protective activity was shown by doses within 0.01-1 microgram; 3) there was no distinct "dose-effect" dependence; 4) single immunization was less effective than double and triple; 5) antigenic specificity of the capsular material was not connected with its protective properties.

The authors elaborated suitable conditions for cultivation of freshly isolated Staph. aureus, which offered a possibility of detection among them of a high percentage (75.2) of capsular cultures. It was shown that when grown in serum semifluid agar capsular staphylococci formed diffuse colonies of three different types. No correlation between the presence of the capsule, on the one hand, and the absence of the flocculus-forming factor and resistance to the type bacteriophage, on the other hand, was revealed in the Staph. aureus strains.

Albino mice were immunized with a purified capsular antigen isolated from the S. aureus strain 1193/74. The presence of specific anticapsular antibodies in the sera of animals were determined by two methods: 1) by conversion of diffuse growth of a homologous strain into compact one, and 2) by determination of opsonic index in phagocytosis of homologous staphylococci by human neutrophils. It was revealed that antibodies converting the microbical growth were absent in the sera of normal mice and reached the highest level after the second antigen injection; opsonins were present in the sera of normal mice in widely varying quantities; their maximal level was noted after the 3rd immunization.

Immunization with purified and concentrated staphylococcal toxoid leads to the rapid 12-fold rise of the level of anti-alpha-toxin and to the 17-fold rise of the titres of antibodies to extracellular staphylococcal products. At the period of immunization phasic changes in cell-mediated and humoral immunity characteristics, indicative of the state of the nonspecific resistance system, can be observed. These changes consist in the transient suppression of phagocytosis, including a decrease in the activity of intraleukocytic bactericidal systems (myeloperoxidase and cation protein), a decrease in the activity of the complement and the bactericidal activity of the blood serum, which should be taken into account when using this immune preparation for therapy.