Search

Aim: Determine frequency of diseases caused by group A streptococci (GAS) among invasive infections of soft tissues; identify emm-types of the isolated streptococci, determine the presence of bacteriophage integrases and toxin genes in their genomes., Materials and Methods: 4750 case histories of patients with soft tissue infections of the purulent-surgical department of the 23rd City Clinical Hospital.of Moscow "Medsantrud" in 2008 - 2011 were analyzed. 46 strains of GAS isolated from patients with invasive streptococcus infection (ISI) were studied. GAS identification was carried out by latex-agglutination method. GAS emm-type was determined by molecular-genetic methods, as well as the presence of bacteriophage integrases int2, int3, int4, int5, int6, int7, int49, bacteriophage toxins speA, speI, sla, speC/J, speL, speH, speC, ssa and speB gene present on the chromosomal DNA., Results: 132 cases (2.8%) were attributed to invasive infections. In 46 cases of invasive infections (35%) GAS were isolated. 22 different emm-types of invasive GAS strains were detected. Only speB gene among all the toxin genes (as well as the expression of the gene--SpeB toxin) was detected in all the strains, whereas sla and speI genes were not detected in any of the strains. Genes of the other toxins (ssa, speL, speC, speA, speH, speC/J) occurred in a number of strains. Genes of phage integrases were detected among all the strains however in varying combinations (from 1 to 4 genes)., Conclusion: Invasive infections caused by GAS are more frequently spread than had been previously assumed and a high degree of genetic heterogeneity of invasive GAS strains was detected.

Testing the supernatants of ctx(+) strains of V. cholerae eltor and V. cholerae O139 on cell subcultures confirmed the possibility of the synthesis of hemolysin by V. cholerae under the condition of growing them in tripton medium lacking FeCl3. At the same time ctx(+) strains of V. cholerae of both serogroups retained, simultaneously with hemolysin production, their capacity for the synthesis of cholera toxin.

The immunomodulating action of genetically related pairs of strains of S. dysenteriae 1, differing by the presence of high molecular plasmid with a mol. wt. of 140 mD and transposon-mediated mutation in the tox-gene chromosome (4 phenotypes: Inv+ Tox+; Inv+ Tox-; Inv- Tox+; Inv- Tox-), was studied. The study was carried out with the use of the experimental intraperitoneal infection of CBA mice with live Shigella cells in a dose of 5x10(7) microbial cells. For the first time the role of Shiga toxin synthesis at different stages of the formation of cell-mediated immune response was demonstrated in vivo. This response was manifested by the stimulation of hematopoiesis, pronounced sensitization of effectors of delayed hypersensitivity and an immunomodulating effect on immunocompetent B lymphocytes, synthesizing antibodies, specific to S. dysenteriae 1 LPS and non-specific to heterologous antigen (sheep red blood cells).

The growth of Bacillus thuringiensis subsp. thuringiensis strains (BT) 202, 1140, 98 producing bitoxybacillin in the presence of novobiocin, mitomycin C and at high temperature 43 degrees C resulted in obtaining of the mutants for the synthesis of crystalline protein (Cry) and exotoxin (Exo). Analysis of the plasmid content of the mutants has shown the Cry Exo phenotyre to correlate with the loss of 55 MD plasmid in the strain BT202 and the loss of 60MD plasmid in BT1140. The transfer of these plasmids into Bacillus cereus leads to the transfer of endo- and exotoxin production properties. The synthesis of Cry in BT98 is controlled by the 56MD plasmid, while the synthesis of Exo is encoded by the chromosome or plasmid that cannot be eliminated or transferred into other strains. Localization of Cry on the 55 and 56 MD plasmids in BT202 and BT98 is confirmed by the hybridization of the plasmid DNA with the DNA-probe.

The experimental study of conditions for the optimization of the batch cultivation of P. aeruginosa has been made. As revealed in this study the aim of this cultivation can be achieved by using exponentially growing culture in a dose of 1.10(9) cells/ml as seed material and by ensuring the conditions of rational air supply during the main cultivation process.

The genes coding for Pseudomonas aeruginosa exotoxin-A and Staphylococcus aureus A-protein have been cloned. Different fragments of the genes were subcloned on the plasmid vestors. The plasmids pAPA4 and pAPA42 have been constructed. The plasmids are coding for the hybrid protein which consists of the immunoglobulin binding A-protein domain at its NH2-terminus and the catalytically active fragment of exotoxin-A at its COON-terminus. The hybrid gene is expressed in Escherichia coli cells under the control of lambda cro-gene expression elements (pAPA42) or lac-operon expression elements (pAPA4). The latter proved to be most productive.

The effect of ten mineral salts on the productivity and toxin synthesis was studied in Bacillus thuringiensis IPM-1140. Exotoxin synthesis was stimulated by Zn2+, Mn2+ and NH4+ ions as well as by potassium phosphates. The direct correlation between the number of viable spores and the exotoxin accumulation was disordered at extreme salt concentrations. Optimal salt concentrations in the yeast-polysaccharide medium were found using the method of a fractional factor experiment, which made it possible to increase the productivity of the culture to 5 X 10(9) spores/ml and the yield of the exotoxin to 730 micrograms/ml. The thermoresistance and the entomopathogenic activity of crystals increased when B. thuringiensis IPM-1140 was grown in this medium.

Various procedures were studied for obtaining Bacillus thuringiensis strains of serotype I which synthesized exotoxins. Mutant clones with elevated exotoxin synthesis could be selected by treating the cells with N-methyl-N-nitro-N-nitrosoguanidine. The frequency of (+) variant selection was from 17 to 12 X 10(-2). The clones of S and R types differed in the insecticide activity of the exotoxin. Its yield could be increased by optimizing the composition of growth media. The strain specificity of B. thuringiensis producing the exotoxin was assayed in terms of carbon and nitrogen requirements.

The activity of exotoxin A in culture filtrates prepared from cultures obtained by growing P. aeruginosa strains PA-7 and PA-103 in Martin's broth containing iron at a concentration of 0.08 microgram/ml, 0,05 M sodium glutamate and 1% of glycerin has been shown to be 1.5 times higher than that in filtrates prepared from cultures obtained by growing the above strains in a medium containing soybean tryptic digestion (USA). The optimun conditions for the production of exotoxin A by these strains are achieved during their cultivation in a fermenter at a temperature of 32 degrees C for 18 hours with simultaneous stirring (800 r. p. m.) and oxygenation (450 m3/h). Under these conditions the biological activity of the filtrates is 200 LD50/ml, their ADP-ribosyltransferase activity is 9500 c. p. m. and a sharply defined precipitation line appears in the double diffusion test in gel with monospecific antiserum to purified toxin, used in a dilution of 1:8.

Plasmid DNA was isolated from Yersinia pestis strains containing pesticin I or fraction I antigen and "mouse" toxin determinants. Specificity of DNA preparations was studied by using them for transformation of plague agent strains carrying no plasmids. pPstI plasmid (molecular weight 7,0-7,8 MD) encoded pesticin I, fibrinolysin and plasmacoagulase synthesis. Fraction I antigen and "mouse" toxin production determinants were borne on pFraI/Tox plasmid (molecular weight about 50 MD). The observation that some Y. pestis cultures, having lost the ability to synthesize one of pFraI/Tox products, still retained this plasmid in their cells, is regarded as an evidence for a complicated regulation of pFraI/Tox function.

The article specifies the mechanisms of toxigenicity in clinical streptococci strains. The production of type A erythrogenic toxin is found to be related to the toxigenicity gene expression localized in moderate bacteriophage genome. The possibility of using DNA probes to assess the degree of toxigenicity is discussed together with the relationship between the toxigenicity and the gene dose.