[Preparation for state registration of the reagent kit for the detection and differentiation of the DNA of burkholderia «pseudomallei¼ group.]

The reagent kit designed to detect and simultaneously differentiate the DNA of three species of Burkholderia pseudomallei - causative agents of melioidosis (B. pseudomallei), glanders (B. mallei) and B. thailandensis by the set of genes of ß-lactamases with B and D molecular classes using a multiplex polymerase chain reaction with electrophoretic detection was developed for clinical laboratory diagnosis. The functional properties of the reagent kit were evaluated, tests were carried out, the stages of examination and registration in the Federal Service for Surveillance on Consumer Rights' Protection and Human Well-being were completed. During clinical testing the effectiveness of the reagent kits in the study of various samples of clinical material and isolated cultures of microorganisms was confirmed. It has been established that the indicator of diagnostic sensitivity of the reagent kit for the detection and differentiation of the glanders, melioidosis and B. thailandensis causative agents was less than 99 %, diagnostic specificity - not less than 99 % with a confidence probability of 90 % in the analysis of each of the indicators.

[The application of time-of-flight mass spectrometry with matrix activated laser desorption-ionization (MALDI-ToF) for identifying agents of glanders and melioidosis].

The article presents the results of application of developed methodological approach to identifying Burkholderia pseudomallei and Burkholderia mallei using direct mass spectrometry profiling of cellular proteins. The protocol of sampling preparation of cultures of melioidosis and glanders was optimized with taking in account characteristics of observation of requirements of biological safety for operations with pathogenic biological agents of pathogenicity group II. The dependence of quality of mass spectrums (number of individual peaks and their intensity) from medium of fermentation of microorganisms was evaluated. The characteristic mass spectrums of collection strains B.pseudomallei (5) and B.mallei (5) were obtained. The set of reference mass-spectrums was generated for identification data base S.A.R.A.M.I.S.TM (Anagnostec Gmbh.). The mentioned data base was used for identification of 43 strains of pathogenic Burkholderia. The opportunity of reliable identification of taxonomic belonging of examined microorganisms up to species' level. The cluster analysis of obtained mass-spectrums of common cellular proteins of collection strains of pathogenic Burkholderia demonstrated grouping of examined strains according to their species' belonging. The supplemented data base of mass-spectral characteristics hereinafter will permit applying express-identification of isolates suspicious for belonging to agents of melioidosis and glanders. The updated data base will become a basis for developing schemes of hemotyping of strains of Burkholderia using mass spectrometry technique.

[CONJUGATIVE INTEGRATIVE ELEMENTS (ICEs) OF MICROORGANISMS].

Integrative conjugative elements (ICEs) are an extensive group of mobile genetic elements found in the Gram-positive and Gram-negative bacteria. These genetic elements are replicated being incorporated into host chromosome, but retain the ability for excision and conjugative transfer. Given a set of the genes of the conjugative transfer, control of removal and integration, ICEs are directly involved in the processes of horizontal transfer of genetic determinants, which increase the adaptive potential of the bacterial species, as well as act as a mobilizing factor for other genetic elements.

[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.

[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.

[Cloning and expression of 7.55 kb KPNI fragment of Burkholderia pseudomallei PPM1 plasmid in Escherichia coli]. / Klonirovanie i ékspressiia 7,55 t.p.h. KPNI-fragmenta plazmidy PPM1 Burkholderia pseudomallei v Escherichia coli.

BamHI, SalI, PstI, and KpnI fragments of pPM1 (B. pseudomallei 12.95 kb plasmid) were cloned in E. coli. The recombinant clones carrying a 7.55 kb KpnI fragment of pPM1 were highly resistant to several aminoglycosides (streptomycin, kanamycin, and gentamycin) and fluoroguinolones (perfloxacin, ofloxacin). Two outer membrane proteins (23 and 27 kDa) absent in E. coli and capable to form 120 kDa oligomer complex were detected by the Western blot method in the strain carrying recombinant pS19 plasmid. The integration of a cloned 7.55 kb sequence in the chromosome was observed by the dot and Southern hybridization analysis in the clones carrying recombinant plasmids pS12 and pS14.

[Isolation and primary characteristics of a Pseudomonas (Burkholderia) pseudomallei plasmid]. / Vydelenia i pervichnaia kharakteristika plazmid Pseudomonas (Burkholderia) pseudomallei.

Plasmid screening of reference Pseudomonas pseudomallei strains isolated from patients and animals revealed cryptic plasmids with different molecular weights in 30 strains. Plasmids were investigated by restriction analysis and DNA-DNA hybridization. Cryptic plasmids were denoted as pPM1, pCM2, pCM3, and pCM4. The presence of plasmids in melioidosis agent permits their use for intraspecies typing of strains and for genetic studies.

[Endoscopy in the diagnosis and treatment of abdominal adhesions]. / Vozmozhnosti éndoskopicheskogo metoda pri spaechnoi bolezni briushnoi polosti.

Therapeutic and diagnostic laparoscopy was performed in 48 patients with chronic abdominal pain due to an adhesive process in the abdominal cavity. Laparoscopic treatment was applied only in patients who experienced pain when the adhesions were pulled with instruments. The adhesions were divided, the vessels were coagulated, and the organs were separated from the adhesions with instruments introduced into the abdominal cavity through additional punctures made in the anterior abdominal wall. The immediate results of the treatment were good in 44 out of 48 patients (91.6%). The late-term results were studied in 46 patients in follow-up periods of 12 months to 6 years. Recurrences were found in only 4 cases. Complications requiring special treatment were not encountered. Therapeutic and operative endoscopy is an effective and safe method for treating patients with adhesive disease manifested by abdominal pain.