[Application of multiplex-PCR for bifidobacteria and propionibacteria genus identification].

AIM: Creation of a PCR test-system for determination of Actinobacteria class bacteria belonging to 2 genera that are the most widely represented among obligate anaerobic microbiota of human intestine: Bifidobacterium and Propionibacterium. MATERIALS AND METHODS: 8 strains of Bifidobacterium spp. and 6 strains of Propionibacterium genus were identified by morphologic, cultural and biochemical properties. Isolation of matrix DNA of the strains for PCR was carried out by "DNA-Express" kit (SPF "Lytech", Russia). Primers for determination of genus membership for obligate anaerobes were developed based on variability of 16S RNA gene by using "Lasergene 7.1" ("DNASTAR, Inc.", USA) program. PCR screening of the isolated DNA was carried out based on "Syntol" LLC primers and reagents. Amplicon detection was carried out by agarose gel electrophoresis. RESULTS: Multiplexing of 2 different primer pairs in a single probe at.68 degrees C annealing temperature for 30 cycles showed the presence of non-specific amplicons that form in samples with bifidobacteria DNA-matrix. The increase of annealing temperature to 70 degrees C and reduction of the number of PCR cycles to 25 resulted in the exclusion of formation of non-specific amplicons. Because the annealing temperature reached the level of values optimal for Taq-polymerase, a 2-phase PCR algorithm could be implemented. This solution allowed reducing the overall time of reaction to 45 minutes. Further increase of annealing temperature to 72 degrees C and reduction of elongation phase up to 15 seconds at 30 PCR cycles did not result in a visible reduction of reaction effectiveness: CONCLUSION: A rapid system for identification of Bifidobacterium and Pronionibacterium genera using a system of 2-phase multiple PCR was developed. The system is part of a screening system for identification of major genera and species of cultured obligate anaerobic bacteria isolated from human intestine biotopes.

[Genetic determinants of secreted lysozyme inhibitors and enterobacteria anti-lysozyme phenotype].

AIM: Determination of interconnection of genetic determinants of secreted lysozyme inhibitors with enterobacteria anti-lysozyme phenotype. MATERIALS AND METHODS: 50 cultures of non-hemolytic Escherichia coli, Klebsiella pneumoniae isolated from feces of patients with stage I - II intestine dysbiosis and Salmonella enterica isolated from patients with acute intestinal infections (AII) and reconvalescent salmonella carriers served as material for the study. Isolation of matrix DNA of the studied strains for PCR was carried out by DNA-Express kit (Lytex Co., Russia). PCR screening of secreted lysozyme inhibitors genes ivyC and pli was performed based on Syntol Co. (Russia) primers and reagents. Amplicon detection was carried out by agarose gel electrophoresis method. Detection of general anti-lysozyme activity (ALA) was performed by plate method, secreted and sorption ALA components - photometrically by O.V. Bukharin et al. (1999) and V.Yu.Sokolov and A.P.Luda (1987) methods, respectively. RESULTS: Screening of chromosome localization genes - ivyC in escherichiae and klebsiellae and pliC in salmonellae by PCR method showed their ubiquitous spread among the studied cultures. The search for homologues of pliC gene that are characteristic for K. pneumoniae virulence plasmid revealed positive result in 19 +/- 5.5% of cases. The lowest level of ALA wa noted in carriers of only ivyC gene accounting for an average of 2.8 +/- 0.6 microg/mlxOD, while all enterobacteria with pliC gene had average ALA value of 4 +/- 0.3 microg/mlxOD. CONCLUSION: Anti-lysozyme activity of enterobacteria is part of the general mechanism of microorganism lysozyme resistance and is determined by the presence of secreted lysozyme inhibitor genes. Application of molecular-genetic approach in the form of the developed experimental test-system based on PCR method allowed to detect secreted lysozyme inhibitor genes in screening mode. High level of secretory ALA is coupled with the presence of pliC gene, while in ivyC gene carriers lower values of the attribute were detected.

[The increase of plasmid DNA copy number is a possible mechanism of the amplification of bacteria anti-lysozyme activity under the effect of chloramphenicol].

AIM: Determination of copy number dynamics of plasmid DNA and anti-lysozyme activityunderthe influence of chloramphenicol in cells of Escherichia coli 252 and 242 strains (ICIS), that have identical biochemical profile and differ by antibiotic resistance, plasmid profile and level of anti-lysozyme activity. MATERIALS AND METHODS: E.coli strains were isolated during screening of antibiotic resistant enteric bacteria of human intestinal biotope. The selected strains were cultivated in medium with 0.17; 0.5; 1 and 1.5 MIC of chloramphenicol by nonspecific amplification ofplasmid DNA in medium with chloramphenicol (technique by T. Maniatis et al.). Plasmid DNA was recovered by alkaline lysis method with subsequent electrophoresis in agarose gel. The quantity ofplasmid DNA was evaluated by comparison of luminescence intensity of marker and experimental DNA sample bands. Anti-lysozyme activity was measured photometrically (O.V.Bukharin et al.). RESULTS: E. coli 252 strain had higher direct correlation of bacteria anti-lysozyme activity and increase of plasmid copy number per cell (r > or = 0.9) during increase of chloramphenicol concentration in sub-bacteriostatic range. Dynamics of these parameters in E. coli 242 control strain were not detected. CONCLUSION: The data obtained give evidence in favor of the action of plasmid localized "gene dose" effect as a mechanism of anti-lysozyme activity increase of bacteria under the influence of chloramphenicol.