[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.

[The comparative evalution of informativeness of immunologic and molecular genetic methods and means during stages of specific indication of melioidosis agent].

The reference-center of monitoring of agents of glanders and melioidosis carried out testing of reagents kits for diagnostic of agent of melioidosis and other close-related species of Burkholderiae in vitro. At the stage of specific identification of pathogenic Burkholderiae the diagnostic possibilities of commercial and experimental kits of reagents for express- and rapid analysis were evaluated. The criteria of evaluation of diagnostic value of kits of reagents were sensitivity, specificity and time of implementation of studies. The analysis with application of mono- and multi-locus amplification systems, including real-time polymerase chain reaction permitted during 5-6 hours to implement identification and differentiation of Burkholderia pseufomallei, B. thailandensis and B. cepacia.

[Comparative characteristic of Burkholderiae pseudomallei group].

AIM: Comparative characteristic of diagnostic value of main cultural-biological characteristics of Burkholderiae pseudomallei group. MATERIALS AND METHODS: 59 strains of B. pseudomallei, 14 --B. mallei and 5--B. thailandensis were used in the study. Biochemical characteristics were studied by generally accepted methods, antigenic properties were evaluated in agglutination reaction and immunoelectrophoresis, virulence was determined by Dlm for laboratory animals, antibiotic sensitivity was verified by disc-diffusion method. RESULTS: Passaging of B. pseudomallei and B. mallei in mice results in increase of virulence, preservation of initial sensitivity to antibiotics, contraction of precipitogen specter. During therapy of experimental melioidosis in guinea pigs resistance to chemopreparations of various groups is formed. Varying degree of virulence and sensitivity to antibiotics of various B. thailandensis strains was established. Dependence of sensitivity on in vitro cultivation was not detected. CONCLUSION: Stability of diagnostically significant tests used for identification of Burkholderiae pseudomallei group was established. Relevance of attribute set expansion that facilitates their differentiation is justified.

[Experimental study on chemotherapy of acute glanders].

Glanders is a zoonotic infection inducing acute forms of the disease (pneumonia, sepsis) in humans and animals under certain conditions, which even with the use of modern chemotherapy have unfavourable prognosis. Insufficient of efficacy of antibiotics with in vitro low MIC for planktonic bacterial suspension of Burkholderia mallei in chemotherapy of acute forms of glanders was due to the capacity of the pathogen for intracellular survival and formation of biofilms. Under such conditions the susceptibility of B. mallei to antibiotics lowered by several orders of magnitude. Chemotherapy of the glanders acute forms in animals usually provided only an increase of the lifespan, while among the survivors there was recorded a high relapse rate. More favourable outcomes were observed with the use of in vitro effective antibiotics in the form of clathrate compounds or especially liposomal forms. In the experiments with golden hamsters the survival rate reached 100% in 1000 Dlm infection even with the treatment onset by meropenem liposomal form 48 hours after the infection. Chemotherapeutics in the liposomal form significantly lowered resistance of B. mallei in both the experiments with a suspension of planktonic organisms and the use of bacteria interned in eukaryotic cells (Tetrahymena pyriformis).

[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.

[In vitro antibiotic susceptibility compliance with efficacy of chemotherapy in infections due to pathogenic Burkholderias].

Among the known species of Burkholderia only two are obligate pathogens, i.e., B. mallei and B. pseudomallei, causative agents of glanders and melioidosis respectively. The other species are saprophytes as natural inhabitants of water reservoirs and soil, still capable of causing opportunistic infections in humans and animals under definite conditions. All the species of Burkholderia are characterized by high resistance to antibacterials, including antibiotics. By the MICs, the most efficient chemotherapeutics against pathogenic burkholderias are tetracyclines, fluoroquinolones, penems and combined sulfanilamides. In the treatment of experimental glanders and melioidosis the set of the effective drugs had the inverse variation dependence on the infection severity and the desease process rate. Co-trimoxasole showed the best results, then followed doxicycline, ciprofioxacin and ceftazidime in the diminishing succession. The modification of the method for determination of antibiotic susceptibility with addition of native blood to the medium and the subculture under the atmosphere of 5% CO2 was shown useful in estimation of the prospects of the use of chemotherapeutics for the treatment of Burkholderia infections.

[Phenotypic and genotypic identification of bacteria from the Burkholderia cepacia complex].

AIM: Evaluation of the diagnostic value of pheno- and genotypic characteristics of B. cepacia strains collection. MATERIALS AND METHODS: Phenotypic and genetic methods of identification and differentiation of 25 strains of the B. cepacia complex. RESULTS: Polyphasic taxonomic approach utilizing multiple diagnostic tests was used for accurate identification of Burkholderia species. Algorithm for identification of microorganisms from the B. cepacia complex was developed. CONCLUSION: Combined use of phenotypic and molecular genetic tests, such as recA gene PCR, is recommended for differentiation of the B. cepacia complex genomovars.

[Procedure for preliminary rapid estimation of bacteria susceptibility to chemotherapeutics].

Principles and procedure for rapid estimation of bacteria susceptibility to antibiotics on a glucose-tryptone medium with an indicator are described. The results of the tests with 50 microbial strains of 17 species showed practically complete identity to the results of the antibiograms when the data were estimated in 3-6 hours according to the described procedure in comparison to the findings estimated in 18-24 hours on the standard media.

[Experimental study on the possibility of using live tularemia vaccine to increase resistance to heterologous infection disease]. / Eksperimental'noe obosnovanie vozmozhnosti primeneniia tuliaremiinoi zhivoi vaktsiny glia povysheniia rezistentnosti k geterologichnym infektsionnym zabolevaniiam.

In experiments on guinea pigs immunized with Francisella tularensis 15, or live tularemia vaccine (LTV), the level of heterologous protective effect to dangerous infectious diseases caused by Yersinia pestis, Burkholderia pseudomallei, B. mallei, Mycobacterium tuberculosis was studied. The study revealed that during the first 4 weeks after the subcutaneous immunization with LTV the level of resistance of the immunized animals to heterologous infective agent reliably increased as indicated by the survival rate of the animals, as well as by the survival time of those killed by infection, in comparison with the controls. Later (on day 150 after immunization) differences in death rate between the groups perceptibly decreased. Nevertheless, the 1 1/2-fold increase of the survival time of the challenged immunized animals in comparison with the controls proved the possibility of using immunization with LTV for the urgent prophylaxis and treatment not only of tularemia, but also of plague, glanders, melioidosis and tuberculosis.

[Burkholderia thailandensis: biological properties, identification and taxonomy]. / Burkholderia thailandensis: biologicheskie svoistva, identifikatsiia i taksonomicheskaia pozitsiia.

Burkholderia pseudomallei-like microorganisms have been isolated from soil and water in regions with endemic melioidosis. These strains have biochemical and antigenic profiles identical to melioidosis agents, except that they differ by virulence and L-arabinose (vir-, ara+). There are minor differences between these species by rRNA sequence. DNA hybridization and, more so, positive transformation of DNA auxotrophic mutants of B. pseudomallei by cell lysates of B. thailandensis and B. mallei confirmed the homology of these species' genomes. These members of the Burkholderia genus (pseudomallei, mallei, and thailandensis) can be regarded as a supraspecies taxon: pseudomallei group. B. thailandensis strains are not virulent for guinea pigs and slightly virulent for golden hamsters. Immunization with live cultures of B. thailandensis protected more than 50% guinea pigs challenged with 200 LD50 B. pseudomallei 100. B. thailandensis is suggested as a potential melioidosis vaccine.