[Characterization of oligonucleotides with LNA-monomers for PCR detection of point mutations in mycobacteria tuberculosis genome].

Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mer duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.

[Vibrio cholerae strains certification].

AIM: Certification of V. cholerae strains stored at State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk. MATERIALS AND METHODS: 50 V. cholerae strains were studied. Real-time PCR with primers to genes ctxA, ctxB, ace, zot, tcpA, toxR, hlyA, rtxC, rfbO1, ompU, ompW was used. RESULTS: Membership of the studied strains in V. cholerae species was confirmed by molecular-biological methods. 46 strains belong to O1 serogroup, 42 of those - E1 Tor toxigenic, having all the virulence genes and 4 non-toxigenic strains. A strain had ace, zot, tcpA, toxR, rtxC, hlyA, ompU genes. 2 strains had ace, toxR, rtxC, hlyA genes, a strain had only toxR gene which is a global regulatory gene. 2 of the 4 serogroup O1 strains were non-toxigenic and had all the virulence genes (ctxA, ctxB, ace, zot, tcpA, toxR, rtxC, hlyA, ompU). 1 non-toxigenic strain has ace, zot, toxR, hlyA, ompUgenes, and the other - toxR, hlyAgenes. CONCLUSION: Genome certificates of all the V. cholerae strains stored in State collection of pathogenic microorganisms and cell cultures SCPM - Obolensk were created. Markers of epidemic threat - ctxA, ctxB, tcpA, toxR and additional virulence genes were determined.

[Detection of wild type isolates and point mutations in genome of isoniazid-resistant Mycobacteria tuberculosis].

The isoniazid resistance of mycobacteria tuberculosis (MBT) is associated with point mutations in the codon 315 of katG gene of MBT. The two PCR-techniques for detection of point mutations in codon 315 have been developed. A use of two sets of primers comprising the additional competitive blocking primer with 3'-terminal phosphate group (in order to eliminate unspecific amplification) allowed to identify most frequent point mutations AGC-->ACC and AGC-->AGA in the codon 315. PCR with a set of two primers one of which contained 5 LNA-monomers allowed to discriminate between wild type and isoniazid-resistant MBT isolates; any of 6 known mutations in codon 315 of kafG gene being detected. A structure and purity of the LNA-containing oligonucleotides (length of 17 nucleotides) was characterized by MALDI-TOF mass spectrometry; the duplex formed by two LNA-containing complementary oligonucleotides (length of 17 b.p.) was anlyzed by thermal denaturation.

Molecular epidemiology of tuberculosis in the Tula area, Central Russia, before the introduction of the Directly Observed Therapy Strategy.

Tuberculosis remains a major public health concern in Russia and worldwide. Given the great geographical, ethnic, and socio-economic heterogeneities between Russian regions, epidemiological data cannot be generalized from a regional to a country-wide level. We present data on the epidemiology of tuberculosis in Central Russia. We report a high level of resistance to major antitubercular drugs in both new and previously treated patients in the region. The level of drug resistance in new cases was almost twice as high as the estimated average national level. The Mycobacterium tuberculosis strains that circulated in the region were predominantly represented by LAM-RUS and Beijing genotypes. These two lineages were strongly associated with drug resistance and clustering. Using molecular epidemiology techniques, we showed a high interpenetration by M. tuberculosis strains between the prison and civilian populations. A limited number of identical strains were responsible for the majority of drug-resistant tuberculosis cases in both settings.

[Development of a comprehensive approach to detecting the antituberculous activity of low molecular-weight chemical compounds].

The optimal parameters of the Alamar Blue test have been determined to detect the antituberculous activity of the chemical compounds under study. The duration of mycobacterial cell incubation before addition of Alamar Blue is 24 hours; that is 17 hours for both H37Ra and H37Rv M. tuberculosis. A method has been devised to evaluate the bactericidal/bacteriostatic activity of the chemical compounds. A thoroughly characterized collection of clinical M. tuberculosis strains that differ in drug sensitivity has been created. A procedure has been developed to reveal the activity of the chemical compounds, by applying mono and multiresistant M. tuberculosis strains. Variability in the growth rate for the clinical strains of mycobacterial cultures is shown. A method has been devised to evaluate the toxicity of the chemical compounds for eukaryotic cells.

[Determination of critical kanamycin concentration for Mycobacterium tuberculosis in the MGIT Bactec 960 system].

The critical concentration of canamycin for the MGIT Bactec 960 system, which has been determined on a panel of the tho--roughly characterized genetically heterogeneous clinical Mycobacterium tuberculosis isolated in the Central Region of Russia, is equal to 1.25 microg/ml.

[Determining drug resistance of the clinical strains of Mycobacterium tuberculosis to pyrazinamide].

Description of the pyrazinamide-resistant clinical strains of M. tuberculosis derived from sputum of patients treated in TB clinics in Tula was made (June, 2001 - July, 2002). It was demonstrated that 30.3% (n = 91) strains were resistant to pyrazinamide. It was found out that these strains were resistant to other antituberculosis drugs in most cases. The method of PCR-sequencing was used to find the mutations in the gene pncA determining resistance to pyrazinamide. 44 different types of mutations localized in 28 codons were detected. The predominance of the mutations in 57 (13.2%), 63 (7.7%), 97 (7.7%), 12 (6.6%), 103 (6.6%) codons and in -11 (6.6%) promoter ofp ncA was observed in the pyrazinamide-resistant strains. Several new mutations determining resistance of the clinical strains of M. tuberculosis to pyrazinamide were described. A high correlation between resistance of mycobacteria to pyrazinamide and activity of pyrazinamidase was observed.

[Predominant genotypes of Mycobacterium tuberculosis strains from prisoners in Ozerky].

The correlation of the Beijing/W and LAM strains with multiple drug resistance was studied. The transmissibility of the multidrug-resistant strains of these families was demonstrated.

Association of specific mutations in katG, rpoB, rpsL and rrs genes with spoligotypes of multidrug-resistant Mycobacterium tuberculosis isolates in Russia.

Most multidrug-resistant (MDR) Mycobacterium tuberculosis isolates in Russia belong to the Beijing or Latino-American and Mediterranean (LAM) spoligotype families. The objective of this study was to investigate possible associations between genotype and the frequencies of mutations that confer drug resistance in a population that has two large families of circulating strains. Spoligotyping, IS6110 restriction fragment length polymorphism typing, and sequencing of the katG and rpoB genes, were performed for 217 consecutive MDR M. tuberculosis isolates from patients. The rpsL and rrs genes were also sequenced for selected streptomycin-resistant isolates. Of the 217 MDR isolates, 99 (46%) belonged to the LAM family, 92 (42%) to the Beijing family, 21 (10%) to the Haarlem family and four (2%) to the T family. There was one unique spoligotype. Mutations in the katG gene were identified in 207 (95%) isolates, all of which had mutations in codon 315. Mutations in the rpoB gene were identified in 200 (92%) isolates; 75% of LAM isolates carried a mutation in codon 516, whereas 71% of Beijing isolates carried a mutation in codon 531. In the 33 isolates resistant to streptomycin 50 mg/L, the 43AGG rpsL mutation was found in 27% of Haarlem, 75% of Beijing and 0% of LAM isolates, and rrs mutations were found in 17% (516C-->T) of Beijing and 100% (513A-->C) of LAM isolates. Overall, there appeared to be a correlation between the genotype and specific mutations conferring resistance to rifampicin or streptomycin in the Beijing and LAM families. The biological implications of this correlation remain to be explored.

[Structure of deletions detected in the genomes of clinical Mycobacterium tuberculosis strains].

Deletions were analyzed in the genomes of four Mycobacterium tuberculosis strains isolated from the sputum of patients living in the Central Region of Russia. The strains of the Beijing family were found to have deletions affecting 40 open reading frames (ORF) and to amount to 0.7% of the genome H37Rv. Genome deletions in a strain from the Haarlem family affected 20 ORF and accounted for 0.26% of the genome H37Rv. Six of the eight deletions were located at the site of preferred cloning on the insertion element IS6110. Prophage phiRv1-associated deletion sequence 149 is the only common to the strains of both families. The deletion ends were evenly distributed among the intergenic and coding chromosome regions with an insignificant preference of the latter. The authors revealed a new deletion in strain 1540 belonging to the Haarlem family and a two-component deletion in the region RvD2. The deletions detected in the genomes of Russian Beijing strains were typical of strains from South-East Asian countries.

[Genetic of modern mycobacteria of the tuberculosis complex].

The review of scientific periodicals relevant to research on the mycobacteria of the tuberculosis complex with the use of modern methods of molecular biology is presented. The main mechanisms of the intraspecific variability of mycobacteria and the updated view on the tuberculosis complex mycobacteria evolution are described.

Preventive and therapeutic effects of alpha-acid glycoprotein in mice infected with B. anthracis.

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

Characterization of drug-resistant isolates of Mycobacterium tuberculosis derived from Russian inmates.

SETTING: Tuberculosis ward of a prison in Russia. OBJECTIVE: Molecular characterization of drug-resistant isolates. DESIGN: Isolates were collected from all tuberculosis patients occurring in the prison over a 1-year period. RESULTS: Of 130 patients studied, 17 patients produced pan-susceptible isolates and 113 produced isolates resistant to at least one drug, including 85 multidrug-resistant isolates. Mutations at katG315 occurred in 98% of isoniazid-resistant isolates. Mutations in rpoB were found in 89% of rifampicin-resistant isolates. Mutations in pncA occurred in 13% of the 75 isolates tested. By spoligotyping, members of the Beijing (55 isolates) and LAM (31 isolates) families were identified. By IS6110 genotyping, two groups (34 and 55 isolates) of related isolates were found, including three clusters (10, 12, and 16 isolates) with identical patterns. In a study of samples collected 3 months apart from 28 patients, four patients produced isolates containing a mixture of strains and five patients produced specimens containing distinctly different isolates. Isolates of nine patients acquired additional drug resistance. CONCLUSION: Three families of strains accounted for much of the drug-resistant tuberculosis in this population. Multiple resistance, acquisition of resistance, and infection with two or more strains as well as reinfection were observed.

[PCR with subsequent sequencing of the 16S gene rRNA in species identification of mycobacteria of the non-tuberculosis complex]. / Vidovaia identifikatsiia mikobakterii netuberkuleznogo kompleksa metodom amplifikatsii i sekvenirovaniia genov 16S rRNK.

One hundred of mycobacterium cultures were assayed by the method of PCR with subsequent sequencing of the 16S rRNA region. The below mycobacterium species were identified: M. tuberculosis complex (n = 55), M. avium (n = 17), M. intracellulare (n = 4), M. scrofaleceum (n = 2), M. kansasii - M. gastri (n = 3), M. gordonae (n = 3), M. ulcerans - M. marinum (n = 1), M. smegmatis (m = 2), M. fortuitum (n = 11), M. peregrinum (n = 1) and M. chelonae - M. abscessus (n = 1). The method enabled the differentiation of species M. avium from M. intracellulare and M. peregrinum from M. fortuitum, which could not be differentiated by using the classic biochemical and bacteriological methods. Genetic heterogeneity of the mycobacterium strains of M. avium, M. fortuitum and M. gordonae was also established by PCR plus sequencing of the 16S rRNA region.

[Spoligotypes of clinical Mycobacterium tuberculosis strains isolated in patients with tuberculosis in the Central Region of Russia]. / Spoligotipy klinicheskikh shtammov Mycobacterium tuberculosis, vydelennykh u bol'nykh tuberkulezom v Tsentral'nom regione Rossii.

Spoligotyping and RFLP-IS6110 techniques were used to analyze 353 clinical Mycobacterium tuberculosis strains isolated from patients with tuberculosis in the Central Region of Russia. All spoligotypes were classified according to the international database. Most clinical M. tuberculosis strains in the collection obtained from patients living in the Central Region of Russia belong to the LAM (49.6%) and Beijing (34%) families, as shown by spoligotyping, and to A1 (51.3%) and W (34%) families, as evidenced by the RFLP-IS6110 technique.

[Study of collections of Mycobacterium non-tuberculosis by a restriction test of the amplified fragment spacer sequence 16S-23S of ribosomal DNA]. / Issledovanie kollektsii mikobakterii netuberkuleznogo kompleksa restriktsionnym analizom amplifitsirovannogo fragmenta speisernoi posledovatel'nosti 16S-23S ribosomnoi DNK.

Fifty mycobacterial cultures were studied by restriction analysis of the amplified fragment of spacer sequence 16S-23S of ribosomal DNA. The following types were identified: M. tuberculosis complex, M. avium, M. intracellulare, M. scrofulaceum, M. kansasii, M. gordonae, M. ulcerans, M. fortuitum, M. smegmatis. The genetic heterogeneity of M. fortuitum was detected. The strains under study belong to 5 patterns: M. fortuitum I, M. fortuitum II, M. fortuitum X, M. fortuitum Y, M. fortuitum Z. The pattern characteristics for M. ulcerans (the size of a CPR product was 370 pn; the characteristics of the enzyme HaeIII-induced restriction profile was 214/155 pn) were obtained.

[Characteristics of clinical isolates of Mycobacterium tuberculosis using molecular biological methods]. / Kharakteristika klinicheskikh izoliatov Mycobacterium tuberculosis s ispol'zovaniem molekuliarno-biologicheskikh metodov.

A total of 230 clinically drug-resistant and 3 drug-sensitive isolates of M. tuberculosis obtained from patients in Tula and Tula region in 1998-2001 were studied. The RFLP-IS6110 genotyping showed that 52 (30.2%) of isolates had unique patterns, and 120 (69.8%) of them were grouped to form 16 clusters. 95 (55.2%) and 53 (30.8%) of isolates were attributed to groups A1 and W (Beijing), respectively. Double mycobacterium cultures were detected in 4.1% of isolates. 2 to 4 clinical isolates were obtained from each of 55 patients during 1.5 years. A replacement of mycobacterium isolates was registered in the course of treatment in 12 (21.8%) patients. No replacement of clinical isolates occurred in 43 (78.2%) patients during the whole follow-up period. Repeatedly obtained isolates acquired the determinants of drug-resistance in 5 (11.6%) patients. Changes in the quantity of IS6110 elements were registered only in 1.05% of isolates during a 3-year follow-up.

[Biological properties of clinical isolates of Mycobacterium tuberculosis]. / Biologicheskie svoistva klinicheskikh izoliatov Mycobacterium tuberculosis.

The invasiveness of 2 grud sensitive and 8 holy-resistant M. tuberculosis clinical isolated was evaluated in experiments on BALB/c mice. Mycobacterial suspension was injected into the caudal vein of experimental animals. The results were evaluated by the degree of contamination of lungs and spleen of infected animals euthanized at different periods on time. The study revealed high variability in the degree of contamination of the organs of the animals infected with M. tuberculosis drug-resistant clinical isolates.