[IDENTIFICATION OF CAUSATIVE AGENTS OF GLANDERS AND MELIOIDOSIS BASED ON PRINCIPLES OF POLYPHASE TAXONOMIC APPROACH].

AIM: Determine an optimal set of the most effective methods of identification and intraspecies typing ofcausative agents ofglanders and melioidosis. Materials andmethods. Bacteriologic, immunochemical, molecular-genetic methods were used. RESULTS: A possibility to identify collection strains of pathogenic and closely related Burkholderia in semiautomatic systems is studied. Means of detection of informative variable genome segments ofthe specified microorganisms were developed, methods of their genetic typing were selected. Effectiveness of application of precipitating mAbs for differentiation of Burkholderia was established. Data on diagnostic possibilities of immunoglobulins fluorescing based on monoclonal antibodies of various etiotropic directionality for detection and identification of B. mallei and B. pseudomallei are generalized. Experimental series of amplification test-systems for identification of glanders and melioidosis causative agents in real-time PCR format are created. CONCLUSION: A number of methods for identification and typing of glanders and melioidosis causative agents is proposed.

Use of the phytopathogenic effect for studies of Burkholderia virulence.

The phytopathogenic effect of the pseudomallei group Burkholderia is demonstrated on the Peireskia aculeata model. A method for evaluation of the effect is suggested. The effect correlates with the levels of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia thailandensis virulence for laboratory animals. P. aculeata can be used as a model for preliminary studies of the virulence of the above species.

[Bacteriocins and bacteriophages of pathogenic Burkholderia].

AIM: Detection of bacteriocins and phages in pathogenic bacteria of Burkholderia genus and study of their specificity range. MATERIALS AND METHODS: Sixty strains of B. pseudomallei, 11 strains of B. mallei, 18 strains of B. cepacia, 5 strains of B. thailandensis, and 3 strains of B. gladioli were used in the study. The agar-overlay method was used to determine bacteriocin activity. For the accumulation of bacteriocins, strains-producers were grown on nutrient broth, inactivated by chloroform and an aqueous phase was spread on the culture surface of indicator strains cultivated on semisolid agar. Phages were isolated with Gratia agar method. Microscopy of phage particles was performed using the electron microscope JEM-100 SX by instrumental magnification 50,000 - 60,000. RESULTS: It was shown that all studied clinical and collection strains of pathogenic Burkholderia--B. pseudomallei, B. cepacia, B. thailandensis, B. gladioli (total: 97 strains) produced bacteriocins and bacteriophages. The range of their inhibiting activity includes both strains of the same species and heterologous Burkholderia, including B. mallei, which does not have neither bacteriocins nor phages. For the first time presence of bacteriocins in B. pseudomallei strains were detected. Phage B. cepacia B623 effectively lysing B. mallei and not reproducing on B. pseudomallei cultures was identified which is suitable for differentiation of these two species. High sensitivity to the phages of heterologous Burkholderia has been established for B. thailandensis. Set of strains of the latter species allows to detect phagoproduction in virtually all lysogenic cultures of studied Burkholderia species. CONCLUSION: Pathogenic Burkholderia being inhabitants of the environment (B. pseudomallei, B. cepacia, B. thailandensis, B. gladioli) possess antagonistic factors that were lost in the process of evolution in strictly pathogenic B. mallei species.

[Some properties of plasmocoagulase of the causative agents of glanders and melioidosis].

Criteria for the evaluation of the plasmocoagulase activity of natural isolates and mutant strains of the causative agents of glanders and melioidosis were worked out, which made it possible to subdivide them by this sign into pathogens with high, moderate and low activity. Plasmocoagulase produced by pathogenic Burkholderia was shown to be a thermolabile enzyme, comparatively stable with respect to the action of such chemico-biological agents as hydrogen peroxide and chloramine.

[The use of the plasmid pTH10 for isolating the donor strains of Pseudomonas mallei]. / Primenenie plazmidy pTH10 dlia polucheniia donornykh shtammov Pseudomonas mallei.

The plasmid pTH10 was transfered by conjugation into the Pseudomonas mallei strains. An attempt to construct the donor strains using the widely known technique employing the homology between the plasmid and chromosome due to the transposon Tn1 carried by the plasmid was unsuccessful. Among the clones resistant to bacteriophage PRD1 the variants were selected with the supposed integration of the plasmid into the chromosome. The latter clones required the ability to transfer the auxotrophic chromosomal markers in conjugation after the repeated conjugational transfer of the plasmid pTH10 into them.