Development of an immunoassay test system based on monoclonal antybodies and immunomagnetic particles for the detection of F. tularensis cells.

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

The use of loop-mediated isothermal DNA amplification for the detection and identification of the anthrax pathogen.

The results of detection and identification of Bacillus anthracis strains in loop-mediated isothermal DNA amplification (LAMP) reaction performed under optimized conditions with original primers and thermostable DNA polymerase are presented. Reproducible LAMP-based detection of chromosomal and plasmid DNA targets specific for B. anthracis strains has been demonstrated. No cross reactions with DNA from bacterial strains of other species of the B. cereus group were detected. The development of tests for anthrax-pathogen detection based on the optimized reaction of loop isothermal DNA amplification is planned. These tests will be convenient for clinical studies and field diagnostics due to the absence of requirements for sophisticated equipment.

[CONSTRUCTION AND PROPERTIES OF THE FRANCISELLA TULARENSIS VACCINE STRAIN WITHOUT ONE COPY OF THE IGLC GENE AND WITHOUT RECA GENE].

The live vaccine based on the Francisella tularensis subsp. holarctica vaccine strain 15 NIIEG line is used in Russia against tularemia. This vaccine is highly effective, but fairly unstable. Therefore, development of stable live tularemia vaccine with minimal side effect is rather urgent. The method of allel removal in the F. tularensis vaccine strain was used to remove one copy of the iglC gene, which is required to provide intracellular production of the vaccine strain, as well as removal of the recA gene. The latter is crucial for homological recombination. pGM5 suicide vector based on pHV33 bireplicon plasmid was constructed to provide replacement of intact F. tularensis chromosome segments by modified segments. Modified chromosome segments contain F. Tularensis DNA fragment without iglC structural gene segment 545 p. b. (in pGMΔiglC plasmid), as well as DNA fragment containing no recA structural gene segment 1060 p.b. (pGMΔrecA plasmid). The constructed 15/23-1ΔrecA mutant, in contrast to the vaccine strain 15, was capable of reproducing in the macrophage-like cells J774A.1 line, whereas the efficiency of the reproduction was 8-10 times less. BALB/c mouse responded to immunization by the 15/23-1ΔrecA strain by smaller weight decrease (-2%) as compared to the strain 15 (-14%). Bacteria of the 15/23-1ΔrecA strain were virtually incapable of germinating from the BALB/c murine spleen 14 days after invasion, whereas bacteria of the strain 15 were found in the murine organs even after 21 days. The F. tularensis 15/23-1ΔrecA strain having smaller reaction ability can be used as a basis for construction of stable live safe tularemia vaccine.

[Vibrio cholerae strains certification].

AIM: Certification of V. cholerae strains stored at State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk. MATERIALS AND METHODS: 50 V. cholerae strains were studied. Real-time PCR with primers to genes ctxA, ctxB, ace, zot, tcpA, toxR, hlyA, rtxC, rfbO1, ompU, ompW was used. RESULTS: Membership of the studied strains in V. cholerae species was confirmed by molecular-biological methods. 46 strains belong to O1 serogroup, 42 of those - E1 Tor toxigenic, having all the virulence genes and 4 non-toxigenic strains. A strain had ace, zot, tcpA, toxR, rtxC, hlyA, ompU genes. 2 strains had ace, toxR, rtxC, hlyA genes, a strain had only toxR gene which is a global regulatory gene. 2 of the 4 serogroup O1 strains were non-toxigenic and had all the virulence genes (ctxA, ctxB, ace, zot, tcpA, toxR, rtxC, hlyA, ompU). 1 non-toxigenic strain has ace, zot, toxR, hlyA, ompUgenes, and the other - toxR, hlyAgenes. CONCLUSION: Genome certificates of all the V. cholerae strains stored in State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk were created. Markers of epidemic threat - ctxA, ctxB, tcpA, toxR and additional virulence genes were determined.

Biological properties and structure of the lipopolysaccharide of a vaccine strain of Francisella tularensis generated by inactivation of a quorum sensing system gene qseC.

A knockout mutant with a deletion in a quorum sensing system gene qseC was generated from the vaccine strain Francisella tularensis 15 by site-directed mutagenesis. The variant with the inactivated gene qseC differed from the parental strain in growth rate on solid nutrient medium but had the same growth dynamics in liquid nutrient medium. The mutation abolished almost completely the resistance of the vaccine strain to normal rabbit serum and its ability to survive in macrophages; in addition, the strain lost the residual virulence. A significant phenotypic alteration was observed in the lipopolysaccharide of F. tularensis. Particularly, the mutant strain synthesized no noticeable amount of the lipopolysaccharide with the high-molecular-mass O-polysaccharide, presumably as a result of impairing biosynthesis of the repeating unit, namely, a loss of the ability to incorporate a formyl group, an N-acyl substituent of 4-amino-4,6-dideoxy-D-glucose.

[Development of a comprehensive approach to detecting the antituberculous activity of low molecular-weight chemical compounds].

The optimal parameters of the Alamar Blue test have been determined to detect the antituberculous activity of the chemical compounds under study. The duration of mycobacterial cell incubation before addition of Alamar Blue is 24 hours; that is 17 hours for both H37Ra and H37Rv M. tuberculosis. A method has been devised to evaluate the bactericidal/bacteriostatic activity of the chemical compounds. A thoroughly characterized collection of clinical M. tuberculosis strains that differ in drug sensitivity has been created. A procedure has been developed to reveal the activity of the chemical compounds, by applying mono and multiresistant M. tuberculosis strains. Variability in the growth rate for the clinical strains of mycobacterial cultures is shown. A method has been devised to evaluate the toxicity of the chemical compounds for eukaryotic cells.

[Determination of critical kanamycin concentration for Mycobacterium tuberculosis in the MGIT Bactec 960 system].

The critical concentration of canamycin for the MGIT Bactec 960 system, which has been determined on a panel of the tho--roughly characterized genetically heterogeneous clinical Mycobacterium tuberculosis isolated in the Central Region of Russia, is equal to 1.25 microg/ml.

[Determining drug resistance of the clinical strains of Mycobacterium tuberculosis to pyrazinamide].

Description of the pyrazinamide-resistant clinical strains of M. tuberculosis derived from sputum of patients treated in TB clinics in Tula was made (June, 2001 - July, 2002). It was demonstrated that 30.3% (n = 91) strains were resistant to pyrazinamide. It was found out that these strains were resistant to other antituberculosis drugs in most cases. The method of PCR-sequencing was used to find the mutations in the gene pncA determining resistance to pyrazinamide. 44 different types of mutations localized in 28 codons were detected. The predominance of the mutations in 57 (13.2%), 63 (7.7%), 97 (7.7%), 12 (6.6%), 103 (6.6%) codons and in -11 (6.6%) promoter ofp ncA was observed in the pyrazinamide-resistant strains. Several new mutations determining resistance of the clinical strains of M. tuberculosis to pyrazinamide were described. A high correlation between resistance of mycobacteria to pyrazinamide and activity of pyrazinamidase was observed.

[Anthrax: early steps of the intracellular stage of infection development].

It was shown that spore germination of different Bacillus anthracis strains in macrophage-like cells J774A.1 depended on the genotype of the strains. The virulent B. anthracis strains contain plasmids pXO1 and pX02 responsible for the synthesis of a toxin and a capsule, respectively. The loss of one of the plasmids results in the reduction of strain virulence. It was shown that effective survival of germinating spores in macrophages occurred in the presence of plasmid pXO1 only. The spores of the B. anthracis strains ?Ames and STI-Rif deprived of plasmid pXO1 were least adapted to passing through the intracellular stage. The B. anthracis strains 81/1 and 71/12 (carrying plasmids pXO1 and pXO2 and synthesizing the toxin and capsule) less effectively survived in the cytoplasm of macrophages than the strain STI-1 which has only the plasmid pXO1. It was found that the rate of synthesis of the capsule consisting of polymer gamma-D-glutamic acid depended on the ability of bacterial cells to escape from macrophages. In the B. anthracis strains carrying plasmid pXO2, capsule synthesis by vegetative cells was activated within macrophages that promoted a rapid escape of the vegetative cells from the macrophages. On the contrary, most of capsule-free cells of the vaccine strain STI-1 remained inside macrophages during the whole period of observation. Thus, integrated regulation of two processes, namely synthesis of the toxin components participating in the transition of the germinating cell from phagosome into cytoplasm, and synthesis of the capsule whose presence promotes rapid escape of bacterial cells from macrophages by presently unknown mechanism play the key role in anthrax development at early stages.

[Microbiological degradation of asymmetrical dimethylhydrazine--a toxic component of rocket fuel]. / Mikdrobiologicheskaia degradatsiia nesimmetrichnogo dimetilgidrazina--toksichnogo komponenta raketnogo topliva.

A possibility of microbiological cleaning of water and soil polluted with asymmetric dimethylhydrazine (ADMH), a highly toxic rocket fuel ingredient (RFI), was studied. Several isolates (bacteria, yeast, and micromycetes) capable of utilizing ADMH as the only source of nitrogen, carbon, and energy were isolated from RFI-polluted tundra soil. Acceleration of RFI biodegradation was achieved using a biosorbent that involved cells of the degrader strain immobilized on granulated activated carbon. Biological testing in Escherichia coli and cereals (wheat and barley) demonstrated that biodegradation significantly decreased the integral toxicity of solutions containing ADMH, suggesting its utility for microbiological cleaning of polluted territories.

[Increase in the ecological danger upon the use of biocides for fighting corrosion induced by microorganisms]. / Povyshenie ékologicheskoi bezopastnosti pri ispol'zovanii biotsidov dlia bor'by s korroziei, indutsiruemoi mikroorganizmami.

Five synergistic combinations of biocides were found, among which the combination of kathon + copper sulfate was the most efficient against Serratia marcescens. Depending on the ratio of these biocides, the synergistic effect of this pair allowed 4-20-fold decreases in the effective concentrations. Combinations of biocides with salts (carbonates and phosphates) that facilitate passivation of steel were found, which considerably decreased the corrosion losses of mild steel in comparison to isolated treatment with biocides or salts. The data showed that biocides must be added to corrosion-prone systems simultaneously with the beginning of their exploitation. Otherwise, considerably excessive amounts of biocides or their synergistic compositions are needed.

[Creation and use of a liquid substance based on association of petroleum-oxidizing bacteria]. / Sozdanie i primenenie zhidkogo preparata na osnove assotsiatsii nefteokisliaiushchikh bakterii.

An association of four bacterial strains with high oil-oxidizing and bioemulsifying activities, psychrophilicity, resistance to chemical pollutants, and lack of pathogenicity was selected from a collection of natural oil-oxidizing microorganisms. A new liquid preparation containing stabilizers and preservatives that maintain the cell viability and oil-oxidizing activity during long-term storage was developed. A field experiment in oil-polluted sod-podzol and clay sand soils demonstrated that this preparation accelerated the biodegradation of oil and its individual fractions, especially in the presence of mineral and organic fertilizers. Treatment of oil-polluted soil with this preparation and additives decreased the oil-induced suppression of certain groups of soil microflora.