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Aim: Study of bactericidal effect of phenol on Yersinia pseudotuberculosis produced in various nutrient media., Materials and Methods: Bacteria were produced in nutrient broth (NB) and NB with glucose (NB+Glu) or galactose (NB+Gal) at 8 degrees C. Effect of phenol on bacteria was evaluated by changes in optical density of suspension and quantity of viable cells, and by staining of cells with ethidium bromide. Lipids were analyzed by thin-layer and gas-liquid chromatography, gas-liquid- chromatography--mass-spectrometry, differential scanning calorimetry; lipopolysaccharides (LPS)--by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulfate (SDS-PAGE)., Results: Survival rate of bacteria is dependent on phenol concentration, biocide treatment time and parameters of cell cultivation. Addition of glucose or galactose into the nutrient medium increases the resistance of Yersinia against phenol. Bacterial cultures are heterogeneous in the resistance against phenol independently of the production parameters. Phenol causes damage in outer bacterial membrane, as evidenced by accumulation of lysophosphatidylethanolamine in the cell, the main product of enzyme activity of membrane-bound phospholipase A, and release into the cultural medium of part of LPS. Treatment by phenol in bactericidal concentration is accompanied by changes in phospholipidic and fatty acid composition of bacterial cell envelope., Conclusion: New data are obtained on environmental factors that contribute to the increase of resistance of bacteria against phenolic biocides.

Two strains of Legionella isolated from patient and hot water supply system during outbreak in Sverdlovsk region in 2007 were studied. Using genetic analysis methods (genes sequencing, VNTR-typing and PCR-based study of omp 28 gene), it was shown that tested strains are pathogenic but do not belong to one genetic group and epidemic cluster. Performed immunochemical analysis confirmed that both isolated strains belong to Legionella serogroup 1.

Technical approaches to construction of preparations for serologic diagnostics of Legionella infection were presented in the article; antigenic- and immunoglobulin-based diagnostic kits with known characteristics were developed. Immunogenic properties of protein and lypopolysaccharide antigens, which have diagnostic value, were studied; similarity of protein antigens from 7 serogroups of L. pneumophila was demonstrated. Soluble antigen with known composition was obtained and used for the development of antigen-based polymeric kit for diagnostics of Legionella infection. On the basis of hyperimmune sera, immunoglobulin-based polymeric diagnostic kit and array of coagglutinating diagnostic kits for the mentioned 7 serogroups were developed. Antigen-based polymeric diagnostic kit was recommended for licensure.

The biopolymer composition, immunotropic and immunogenic properties of the fractions of B. pseudomallei and B. mallei were under study. The first two capsular fractions of these agents were found to be similar in their biopolymer composition that was indicative of their close relations. At the same time the causative agents of glanders proved to have decreased content of high molecular glycoproteids and LPS fragments. In the causative agents of melioidosis, capsular fractions K3 and K4 were characterized by the domination of proteins with a molecular weight of 42-25 kD. Fraction K4 in B. pseudomallei and fraction K1 in B. mallei had pronounced immunosuppressing properties ensuring the protection of encapsulated microbial cells in the body. The biopolymers forming fractions K1, K2, K3 in B. pseudomallei and fraction K2 in B. mallei were characterized by immunomodulating properties.

The effect of the ingredients of a semisynthetic culture medium on the synthesis of Y. pestis antigens (F1, LPS, "mouse" toxin) under the conditions of batch cultivation at 28 degrees C was studied. The study revealed that the amount of antigens produced by bacterial cells depended on the character of the limitation of growth.

A new method for the determination of lipopolysaccharide (LPS) admixtures in protein solutions has been developed. The method includes the periodate oxidation of LPS, biotinylation with biotin hydraside, immobilization on a nitrocellulose membrane and the development of biotinylated LPS in the streptavidin--alkaline phosphatase system. Proteins are previously removed from the solution by treatment with hot phenol. Development with the use of 5-bromoinodyl phosphate and nitrotetrazolium blue makes it possible to detect about 30 pg of LPS immobilized on the nitrocellulose membrane.

K. pneumoniae lipopolysaccharide (LPS) was isolated from biomass grown under the conditions of controlled multicycle and continuous cultivation. A considerable yield of LPS containing the minimal amount of protein and nucleic acid admixtures was obtained from biomass grown by continuous cultivation with the concentration of glucose in the medium equal to 20 g/l and the content of dissolved oxygen at a level of 0% of complete saturation. Erythrocyte diagnosticum prepared on the basis of this LPS was found to have a sensitizing dose of 100 micrograms per ml of solid erythrocytic precipitate and high specificity, studied in the passive hemagglutination test with the use of rabbit sera.

In this investigation isogenic avirulent variants obtained from F. tularensis standard virulent strain 503 were used. The capsule-deficient variants (cap-) were shown to have no species-specific capsular antigens and to be capable of producing R-LPS having no the polysaccharide part of the molecules. The capsule-defective forms (cap +/- ) were found to synthesize capsular antigens and S-LPS whose polysaccharide part essentially differed from the O-lateral chains of LPS of the virulent strain. The study of bacterial dissemination revealed that virulent bacteria rapidly spread in the macroorganism, and their subsequent proliferation shortly led to the death of animals. Avirulent mutants (cap- and cap +/- ) appeared in the organs of animals later and proliferated slower, parasitizing in the macroorganism without fatal outcome. The cap- variants were not capable of inducing the synthesis of antitularemic antibodies and possessed no protective properties. The cap +/- mutants were capable of inducing the synthesis of antitularemic antibodies in mice. These antibodies facilitated the elimination of avirulent strains from the macroorganism, but did not ensure protection from infection with virulent strains.

A preparation containing P. aeruginosa cell-wall proteins with a mol. wt. of 9-55 kD has been obtained from P. aeruginosa by the method of extraction with the use of tris-EDTA buffer. In experiments on mice this protein preparation has shown pronounced protective properties, but, according to the data of Shwartzman's local reaction, proved to be toxic, perhaps due to the admixture of LPS. The purification of the protein preparation from the admixture of LPS will make it possible to obtain an effective vaccine against P. aeruginosa infection.

The preparations of lipopolysaccharide (LPS) were obtained from N.meningitidis cells grown in synthetic nutrient medium in a fermenter. Different N.meningitidis strains, groups A, B, C, were found to give different yield of the preparation of LPS: the maximum yield was obtained from N.meningitidis cells C 133 and the minimum yield, from N.meningitidis cells B 125. The comparison of group B strains 125 and 150 showed that the former gave a higher yield of LPS. The influence of the phase of growth on the yield of the preparation of LPS obtained from N.meningitidis cells 125 was shown to be manifested under the conditions of oxygen excess; in the exponential phase the highest yield of the preparation was observed. Oxygen dissolved in the medium was found to influence the yield of the preparation of LPS: with the same content of glucose in the medium the yield of LPS was higher under the conditions of the limited concentration of oxygen; with the same concentration of oxygen in the medium the yield of LPS increased with the increase of the concentration of glucose.

The comparative study of the lipopolysaccharides (LPS) of virulent and avirulent strains of S. sonnei, phase I (smooth colonies), has been made. Electrophoresis of LPS and subsequent densitometry of electrophoregrams have revealed the increase of the fraction of long 0-chains with a considerable number of recurring elements in 2 out of 3 LPS preparations obtained from avirulent shigellae. In mice immunized with these LPS preparations a considerably greater number of antibody-producing cells can be detected in Jerne's test on sheep red blood cells (SRBC) sensitized with the LPS of a virulent strain than on those sensitized with the above LPS preparations. Long 0-specific chains supposedly inhibit the fixation of individual complement components on the corresponding sensitized SRBC. The LPS of the third avirulent strain of S. sonnei, phase I, with transposon integrated into its genome, which has led to the formation of the avirulent variant of a previously virulent strain, seems to contain fine structural differences from the initial virulent strain. The immunogenicity of the LPS of this avirulent strain is greatly (3-4 times) decreased, which is manifested by the number of antibody-producing cells detected in Jerne's test on SRBC sensitized with LPS preparations obtained from these two strains.

The solid-phase liposomal immunoassay procedure for the determination of Francisella tularensis lipopolysaccharide (LPS) has been developed. This assay has been made with the use of monolayer liposomes, on the average, 360 nm in diameter with their phospholipid bilayer modified with F. tularensis LPS and their internal space filled with calcein used as fluorescent marker. The assay is based on the principle of the competitive immunosorption of liposomes and antigenic LPS on the surface of polystyrene plates sensitized with specific monoclonal antibodies. The dynamic range of the method is 50-2,500 mg/ml, the variation index being 2.3-11.5%. Optimization of this method and its comparison with the enzyme immunoassay system for the given antigen-antibody pair has been made.

The capacity of Y. pseudotuberculosis strains for disassociation with the appearance of S- and P-forms has been studied. Strains 852 and 9547 show high stability in S-forms, their conversion into R-forms occurring at 40-42 degrees C. Strain 6953 shows pronounced polymorphism and instability of its associations at different growth temperatures. Strain 9532 exists in S- and R-forms which retain their stability during numerous subculturings at different growth temperatures and prolonged storage. This strain has plasmids of 130, 72.2, 5.7 kb. All plasmids are retained in S- and R-forms, i. e. the dissociation of the strain is not accompanied by the loss of plasmids. The conversion of the strain from the S-form into the R-form leads to changes in the structure of lipopolysaccharide and the composition of low-molecular (less than 23 kD) proteins in the outer and inner membranes. In tests on guinea pigs the LD50 of the R-form of the strain is tenfold greater than that of its S-form. The dissociants of strain 9532 are transformed by plasmid DNA with equal efficiency and equally inherit them without selective pressure.

The molecular heterogeneity of S. sonnei lipopolysaccharide (LPS), reflecting the size of lateral O-specific polysaccharide chains, has been established by the method of electrophoresis in acrylamide gel in the presence of sodium dodecyl sulfate and urea. The dominating components fall into three types, viz. those with 0-3, 10-16 and 35-40 repeating structures, the remaining components being minor ones. The electrophoretic profile of S. sonnei LPS considerably differs from the profiles of Escherichia coli and S. flexneri LPS, but coincides with the LPS profiles of other strains with different virulence. The preparations of LPS obtained by extraction with trichloroacetic acid have the same electrophoretic profiles as LPS obtained by the method of aqueous phenol extraction. The domination of certain molecular variants reflects, seemingly, specific features of the biosynthesis of LPS, characteristic of a given strain. The mechanisms of the preferable synthesis of lateral O-specific chains of the definite size and the importance of the molecular parameters of lateral chains for the biological properties of LPS require further study.

Along with classical lipopolysaccharide (LPS), O-specific material not precipitated by ultracentrifugation has been isolated from the water-phenol extract of S. sonnei avirulent strain 9090 possessing complete antigenic properties. The purification of O-antigen contained in the supernatant fluid has been carried out by the gel filtration of the fluid, previously treated with ribonuclease, in a column packed with Sephadex G-100. The polysaccharide nature of O-antigen thus obtained, the absence of lipid A and KDO and the low content of hexoses, or core-specific saccharides of S. sonnei LPS, in this antigen make it possible to classify this material with O-components of microbial cells, described by different authors as "native protoplasmic polysaccharide" or "L-hapten" and formed by polymers of LPS O-side chains. The content of this component in S. sonnei strains under study is, on the average, 2.5% of the weight of dry microbial substance. L-hapten preparations obtained in the course of our investigations have been found to contain two O-specific antigens detected by immunoelectrophoresis and immunodiffusion, as well as by sedimentation in saccharose gradient, where they form peaks corresponding to 4.3 S and 10.8 S. This polysaccharide O-antigen is supposed to be capable of interaction with ribosomal particles and suitable for use as a component of ribosomal dysentery vaccines.

To determine the content of lipopolysaccharide (LPS) in P. aeruginosa aqueous extracts the ELISA was used. Membrane filtration and ultracentrifugation followed by precipitation with ammonium sulfate at 80% saturation and gel filtration on Sephadex G-100 have proved to be the most effective methods for purification of the aqueous extract from LPS.

P. aeruginosa slime has been separated into fractions XM-300 (3 X 10(5) daltons and more), XM-100 (1 X 10(5) = 3 X 10(5) daltons), PM-30 (3 X 10(4) = 1 X 10(5) daltons) and PM-10, (1 X 10(4) = 3 X 10(4) daltons) by ultrafiltration. The high-molecular slime components (3 X 10(4) daltons and more) have been found to be serologically more active than the low-molecular components (1 X 10(4) = 3 X 10(4) daltons). As shown in experiments on mice, both high-molecular toxic and low-molecular nontoxic slime components have protective activity, but the high-molecular components are more active than the low-molecular ones. The slime components stimulate the formation of immunity to homologous and partially heterologous P. aeruginosa strains in mice. Antigenic relationship between the slime components (especially the high-molecular ones) and the corresponding lipopolysaccharides has been noted.

The high-molecular fraction of substances of the cell wall of meningococci, groups A and B, isolated in free volume in gel filtration through sepharose 4B and containing both group and intergroup antigens proved to be consisting of 2 subfractions in gel-filtration through Bio-Gel A-150m. Molecular weight of the first was within the range of 100--150 million dalton, and of the second--of 3 to 100 million dalton. In dissociation in sodium deoxycholate the high molecular fraction complex compound of the cell wall of meningococcus strain, group A, isolated from the cerebrospinal fluid of a patient suffering from meningitis broke down into 5 fragments differing in chemical nature and mol wt. There were revealed protein and protein-lipopolysaccharide components with a relatively high mol wt. polypeptide components and low molecular residues of the initial lipopolysaccharide.

The comparison of the copolymer of 2-O-(alpha-abequosy)-alpha-alyl-alpha-mannopyranoside and acrylamide (AMA), used as a synthetic antigen, with its natural prototype, Salmonella typhimurium lipopolysaccharide, in the enzyme immunoassay (EIA) has revealed that the serological activity of AMA is related to the specific content of antigenic determinants in its molecule. The analysis of the serological specificity of AMA indicates that this specificity corresponds to factor 4 of Salmonella O-antigen. The high activity and monofactor specificity of the synthetic antigen AMA suggest that the use of this antigen in EIA as a diagnostic preparation holds good promise.

The antigenic composition of some cell structures of P. pseudomallei has been studied and the chemical nature of the antigens has been determined by the method of two-dimensional electrophoresis. In some cell components common antigens have been detected; at the same time these components have been found to possess their own characteristic antigenic complexes. The place of the cell structure antigens in the total antigenic structure of P. pseudomallei has been determined.

The comparative study of the chemical composition and biological properties of antigens isolated from Proteus vulgaris with the use of hydroxylamine and by two classical methods (Boivin's and Westphal's methods) has been made. As shown in this study, the treatment of bacteria with hydroxylamine makes it possible to obtain antigenic complexes with lower toxicity. At the same time hydroxylamine produces no denaturing effect on lipopolysaccharides and protein fractions of bacterial cells.

The comparative study of the chemical composition, immunobiological activity and some physico-chemical properties of lipopolysaccharide (endotoxin) of Brucella cultures isolated from myomorph rodents in the North Caucasus has been carried out. Lipopolysaccharide obtained from these cultures has been found to be most similar to B. suis lipopolysaccharide. The authors suggest that the strains isolated from myomorph rodents outside the foci of brucellosis affecting livestock should be regarded as a separate biotype of B. suis.

The method of the highly sensitive (up to 10 ng/ml) and specific determination of soluble tularemia antigen, based on the use of "sandwich" type ELISA techniques, has been developed. The dependence of the specificity and sensitivity of the method on the degree of purification of antibodies and their peroxidase conjugates used in the assay has been studied. The study has revealed that the best results can be obtained with the use of purified IgG and its conjugate free of unbound peroxidase. Both foreign peroxidase preparations and type A enzyme manufactured in the USSR can be equally used as enzymatic labels.

The work deals with the isolation of serologically active polysaccharide from S. typhimurium 415. Particular attention is paid to different conditions for the hydrolysis of lipopolysaccharide (LPS) and Boivin's antigen, permitting the authors to obtain polysaccharides with different activity of the determinant groups. The action of 1% acetic acid on LPS, lasting for several hours, did not result in the hydrolysis of the antigen, while its hydrolysis in 1.5% acetic acid led to the decomposition of the greater part of the determinant groups. Polysaccharide isolated from Boivin's antigen after hydrolysis in 1% acetic acid retained the specific activity of all antigenic determinants of the natural biopolymer.

The comparative study of the fatty acid composition of bacterial lipopolysaccharides (LPS) in the genera Escherichia, Shigella, Salmonella has been carried out under identical experimental conditions. The LPS of the bacteria under study have been found to contain a number of saturated fatty acids and oxyacids which could not be previously detected in these bacteria in other studies. In all bacterial strains under study LPS include mainly 3-oxytetradecanoic, tetradecanoic and dodecanoic fatty acids. The essential feature of the fatty acid composition of Salmonella is the presence of 2-oxytetradecanoic acid; this acid is absent in Escherichia and Shigella, which can thus be used as a differentiating criterion. The content of other fatty acids in Salmonella is similar to that in Escherichia and Shigella. These data confirm that the genera Escherichia, Shigella and Salmonella are phylogenetically related, the relationship between Escherichia and Shigella being more close.

The authors studied antigens obtained by Grasset's method from 13 strains of Citrobacter of the International collection. The strains possessed O- and H-antigens whose behaviur in the electric field differed. All the strains under study were divided into two groups (by the number of serologically-active components of their O-antigens); representatives of the second group had no cathode O-antigen component. Chemical composition of specific lipopolysaccharides (LPS) obtained by Westphal's method was determined. Fourteen different sugars were revealed. The strains under study were referred to the known chemotypes. Strain 16/52 (8a, 8c) was for the first time studied in respect to the monosaccharide composition of specific LPS, and was referred to chemotype designated as CC-L.

The structure of O-specific polysaccharides and the protective activity of lipopolysaccharides (LPS) obtained from seven P. aeruginosa immunotypes (according to Fisher's classification) have been studied. The structure of O-specific polysaccharides, immunotypes 2, 3, 4, 5 and 6, is identical to that of polysaccharides of serotypes 011; 0(2a), 2c; 01; 010a, 10b; 07a, 7d respectively. No structural analogs of O-polysaccharide characteristic of immunotypes 1 and 7 have been detected among serotypes classified according to the scheme of Lányi-Bergan-Akatova-Smirnova. The specific character of O-polysaccharides is confirmed by the results of the passive hemagglutination inhibition test, but the data of the passive hemagglutination test indicate that LPS of different immunotypes are antigenically related. As revealed in experiments on the active immunization of mice, LPS of seven immunotypes possess more or less pronounced cross protective properties. The causes of the cross protective activity observed in experiments with these LPS are discussed.

The results of the comparative analysis of LPS isolated by different methods of extraction from the cultures of several Brucella species differing in their virulence are presented. Purified LPS preparations have been obtained from Brucella virulent and vaccine strains by using such methods as water-phenol extraction, Boivin's method, mild alkaline hydrolysis of the antigen according to Boivin's procedure. The presence of certain relationship between the method used for the extraction of Brucella LPS to be compared and their chemical composition, immunological characteristics and serological activity has been established. As shown in this investigation, in the process of the preparation of a highly sensitive diagnosticum for the passive hemagglutination test the use of LPS obtained from Brucella virulent strains, but not from the vaccine strain, by the method of mild alkaline hydrolysis according to Boivin's procedure is expedient. The data presented in this work indicate that the soluble complex of lipid A obtained from Brucella LPS has been found to possess serological activity. The results of the study of the serological properties of lipid A indicate that the lipid component may also play a certain role in the manifestation of the serological activity of Brucella LPS.

It was shown that Y. pseudotuberculosis strains causing the Far-Eastern scarlatina-like fever in the Primorsk region belonged to subtype IB-ipopolysaccharides of the standard strain of subtype IB and of the local strain were closely affiliated by the analytic data and monosaccharide composition, but differed from the lipopolysaccharide of strains belonging to subtype IA. Living vaccines should be used to obtain the sera against the subtypes IA and IB of the causative agent.