[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.

[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.

[Molecular typing of the Burkholderia pseudomallei strains with different resistance to antibiotics].

Wild type strains of Burkholderia pseudomallei, spontaneous mutants with high resistance to fluoroquinolones and ceftazidime, and Tn5-induced mutants with reduced resistance level were studied using polymorphic and gene-specific DNA fingerprinting. Cluster analysis of genomic DNA patterns obtained using PCR with arbitrary primer (5'-GTTTCGCTCC-3') and primer specific to the class I integrase intll gene (5'-CCTCCCGCACGATGATC-3') was performed. According to the DNA pattern conformity, the distinct groups submitted by high-level resistant B. pseudomallei derivatives were revealed by both typing approaches. The obtained results may be useful in searching for molecular markers associated with different types of antimicrobial resistance among pathogenic and related burkholderiae.

[Properties of the Burkholderia pseudomallei insertional mutants deficient in membrane proteins production].

Transposon-induced B. pseudomallei mutants deficient in membrane proteins production were obtained for evaluation of the functional role of these cell components. In comparison with the wild type strain B. pseudomallei 57576, mutant clones TTM6, TTM7 and TTM9 carrying Tn5 chromosome insertions were characterized by lost or decreased production of outer membrane proteins 27, 48, 52, 150, 200 kDa. Alterations in outer membrane protein spectra were accompanied by twofold increase in susceptibility of bacteria to fluoroquinolones (pefloxacin, ofloxacin) and cephalosporins (ceftazidime) and noticeable reduction of virulence for white mice and guinea pigs in contrast to the initial strain, the obtained mutants were also less resistant in in vitro phagocyte killing.

[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.

[Mobilization of a cryptic plasmid from the melioidosis pathogen in heterologous species of microorganisms]. / Mobilizatsiia kripticheskikh plazmid vozbuditelia melioidosis v geterologichnye vidy mikroorganizmov.

Cryptic plasmids with different molecular weights have been detected in B. pseudomallei strains. Mobilization of B. pseudomallei plasmid DNA in heterologous B. mallei species was performed by the conjugate plasmid RP1::Tn10. Possibility of detecting phenotypical characteristics of plasmids and behavior of B. pseudomallei non-chromosomal replicons in B. mallei have been determined.

[The efficacy and outlook for the study of live vaccines for the prevention of melioidosis]. / Effektivnost' i perspektivy issledovaniia zhivykh vaktsin dlia profilaktiki melioidoza.

The effect of immunization with Burkholderia pseudomallei, (Pur- and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2, carrying a plasmid with fragments of B. pseudomallei chromosome, was studied on four species of experimental animals, essentially differing by their sensitivity to melioidosis. B. pseudomallei mutants formed the statistically significant level of protection in subcutaneously challenged animals, moderately sensitive to melioidosis, but were not effective when tested, under the same conditions, in animals, highly sensitive to melioidosis. The effect produced by the experimental vaccines under study in animals of all species, subjected to aerogenic challenge, was leveled. The study showed good prospects for the use of tularemia vaccine with a view to create heterologous immunity to melioidosis and the possibility of its use as the basis of bivalent gene engineering vaccine.

[The outlook for the development of live vaccines for the prevention of melioidosis]. / Perspektivy razrabotki zhivykh vaktsin dlia profilaktiki melioidoza.

The effectiveness of immunization with Burkholderia pseudomallei attenuated strains (Pur and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2 carrying a plasmid with fragments of B. pseudomallei chromosome was studied in four species of experimental animals, essentially differing in their sensitivity to melioidosis. The most immunogenic B. pseudomallei mutants, introduced subcutaneously, created a statistically significant level of protection in animals, moderately sensitive to melioidosis, but proved to be ineffective in highly sensitive animal models when tested under the same conditions. In aerogenic infection the effectiveness of the experimental vaccines under study in all species of the animals was on the same level. The study showed good prospects of using tularemia vaccine for inducing heterologous immunity to melioidosis, as well as the possibility of its use as the basis of a bivalent gene-engineering vaccine.

[Insertion mutations in Pseudomonas pseudomallei genome induced by transposons Tn10. Tn9, and Tn5]. / Insertsionnye mutatsii v genome Pseudomonas pseudomallei, indutsirovannye transpozonami Tn10, Tn9, Tn5.

Tn10 and Tn9 from temperature-sensitive replicons RPI::Tn10 Rep is and RP1::Tn9 Rep ts and Tn5 from the "suicidal" vector pSUP5011 may integrate in the P. pseudomallei chromosome. Transposons induced auxotrophic mutations of different spectrum, depending on the strain, Tn element, and defects in the genes responsible for the mobility sign, function of some extracellular elements (lecithinase and lipase), hemolytic activity, and antigen 8 synthesis, which are considered as the potential factors of the pathogenicity of melioidosis agent. No secondary restructuring induced by Tn elements were detected in the domains adjacent to insertion sites. Plasmids RP1::Tn10 Rep ts and RP1::Tn9 Rep ts were stable in insertion mutant cells, mediating the variable TR-TS phenotype probably indicating that plasmid integration with the chromosome is unstable.

[Spontaneous phage production in Pseudomonas pseudomallei and in a range of hosts of melioidosis phages among representatives in the genus Pseudomonas]. / Izuchenie spontannoi fagoproduktsii Pseudomonas pseudomallei i kruga khoziaev melioidoznykh fagov sredi predstavitelei roda Pseudomonas.

A capacity to spontaneous phage production has been studied in 57 melioidosis museum strains. The strains were comparatively analyzed by the levels of frequencies of spontaneous phage production. It is established that 40 of 57 studied strains of Pseudomonas pseudomallei are capable to spontaneously produce phages with frequency from 10(7) to 1. On the indicator lawn of P. mallei phages formed negative colonies of different morphology transparent, turbid and turbid with secondary growth in the centre. No distinct correlation between the frequency of spontaneous phage production and morphology of negative colonies was revealed. The range of hosts of melioidosis phages was determined among 88 representatives of Pseudomonas genus. All melioidosis strains were resistant to their own phages. Phages, identical by the spectrum of lithic effect, were not found even in the case of quantitative coincidence of sensitive strains. Maximum activity was observed in 8 lysates which were lysed 42 to 53 strains of 88 under study.

[Mobilization using incompatibility group P1 plasmids in strains of Pseudomonas pseudomallei and Pseudomonas mallei as potential vectors for DNA cloning]. / Mobilizatsiia s pomoshch'iu plazmid gruppy nesovmestimosti P1 v shtammyPseudomonas pseudomallei i Pseudomonas mallei potentsial'nykh vektorov dlia klonirovaniia DNK.

The cells of Pseudomonas pseudomallei and Pseudomonas mallei have been shown to serve as recipients for the plasmid RSF1010 and its recombinant derivatives pVA1 and pVA4. The conjugative plasmids RP1 and pTH10 of the incompatibility group P1 are able to mobilize the nontransmissive vector plasmids for conjugation transfer into Pseudomonas pseudomallei and Pseudomonas mallei strains. The SmR determinant of the plasmid RSF1010 is expressed in the latter strains. These data makes the mentioned vector plasmids the candidates for DNA cloning in these strains.

[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.

[Use of transformation for the chromosome mapping of Pseudomonas pseudomallei]. / Ispol'zovanie transformatsii dlia kartirovaniia khromosomy Pseudomonas pseudomallei.

The mapping of the chromosome sections in P. pseudomallei, responsible for the synthesis of histidine, has been carried out with the use of two- and three-factor crossing. Two new groups of marker linkage have been found: his 522 with ilv 658 and his 37 with asp 37 glu 37. The linkage of some of the existing histidine mutations with locus his 37 has been revealed and the relative position of the markers in this linkage group has been shown.