In this work, we studied the variation in the gyrA and gyrB genes in ofloxacin-and multidrug-resistant Mycobacterium tuberculosis strains circulating in northwest Russia. Comparison with spoligotyping data suggested that similar to the spread of multidrug-resistant tuberculosis, the spread of fluoroquinolone-resistant tuberculosis in Russia may be due, at least partly, to the prevalence of the Beijing genotype in a local population of M. tuberculosis.Fluoroquinolones (FQ) are potent second-line drugs recommended to treat multidrug-resistant tuberculosis. The main target of FQ, in Mycobacterium tuberculosis and other pathogens, is DNA gyrase, a type II topoisomerase composed of two A and two B subunits encoded by the gyrA and gyrB genes, respectively; mutations in the quinolone resistance-determining regions (QRDR) in these genes serve as a primary mechanism of the development of FQ resistance (1,7,10,20).FQ susceptibility testing is not performed in all regional laboratories in Russia, and data on FQ resistance rates are limited. In 2006, the rate of ofloxacin (OFL)-resistant M. tuberculosis strains was in the range of 1.1 to 1.6% for patients newly diagnosed with tuberculosis and 4.1 to 10.3% for previously treated patients in different regions of northwest Russia. Here, we report the results of the first population-based study of the molecular basis of FQ resistance in M. tuberculosis strains currently circulating in Russia.The bacteriology laboratory in the St. Petersburg Research Institute of Phthisiopulmonology serves as a reference center for northwest Russia. In the present study, we included all OFL-resistant strains isolated from March to November 2006 (see the table in the supplemental material); these strains were recovered from 48 patients among the total of 261 patients screened. The patients were epidemiologically unlinked and originated from different regions of the Russian Federation.Drug susceptibility testing was done by the absolute concentration method according to the guidelines of the Russian Ministry of Health (order no. 109 of 21 March 2003) by using recommended MIC breakpoints (in particular, 2 g/ml for OFL). All 48 strains were multidrug resistant (resistant to at least rifampin [RIF] and isoniazid [INH]), while 45 of them were resistant to at least INH, RIF, OFL, and kanamycin (see the table in the supplemental material) and were classified as extensively drug resistant (22).DNA extraction from Löwenstein-Jensen cultures, spoligotyping, and variable-number tandem repeat (VNTR) typing (using a 24-locus format and three hypervariable loci, QUB-3232, VNTR-3820, and VNTR-4120) were done as described previously (8,9,19). The gyrA QRDR was amplified and sequenced using the forward primer 5Ј-ACCGCAGCCACGCC AAGTCG and the reverse primer 5Ј-CCTGGCGAGCCGAA GTTGCC. The gyrB QRDR was amplified and sequenced using primers described by Takiff et al. (20). Mutations in the rpoB hot-spot region (codons 507 to 533) and katG S315T (AGCϾACC) were detected as described previously (12, 15). To minimize th...
BackgroundTuberculosis (TB) poses a worldwide threat due to advancing multidrug-resistant strains and deadly co-infections with Human immunodeficiency virus. Today large amounts of Mycobacterium tuberculosis whole genome sequencing data are being assessed broadly and yet there exists no comprehensive online resource that connects M. tuberculosis genome variants with geographic origin, with drug resistance or with clinical outcome.DescriptionHere we describe a broadly inclusive unifying Genome-wide Mycobacterium tuberculosis Variation (GMTV) database, (http://mtb.dobzhanskycenter.org) that catalogues genome variations of M. tuberculosis strains collected across Russia. GMTV contains a broad spectrum of data derived from different sources and related to M. tuberculosis molecular biology, epidemiology, TB clinical outcome, year and place of isolation, drug resistance profiles and displays the variants across the genome using a dedicated genome browser. GMTV database, which includes 1084 genomes and over 69,000 SNP or Indel variants, can be queried about M. tuberculosis genome variation and putative associations with drug resistance, geographical origin, and clinical stages and outcomes.ConclusionsImplementation of GMTV tracks the pattern of changes of M. tuberculosis strains in different geographical areas, facilitates disease gene discoveries associated with drug resistance or different clinical sequelae, and automates comparative genomic analyses among M. tuberculosis strains.
The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.
Extrapulmonary and, in particular, spinal tuberculosis (TB) constitutes a minor but significant part of the total TB incidence. In spite of this, almost no studies on the genetic diversity and drug resistance of Mycobacterium tuberculosis isolates from spinal TB patients have been published to date. Here, we report results of the first Russian and globally largest molecular study of M. tuberculosis isolates recovered from patients with tuberculous spondylitis (TBS). The majority of 107 isolates were assigned to the Beijing genotype (n ؍ 80); the other main families were T (n ؍ 11), Ural (n ؍ 7), and LAM (n ؍ 4). Multidrug resistance (MDR) was more frequently found among Beijing (90.5%) and, intriguingly, Ural (71.4%) isolates than other genotypes (5%; P < 0.001). The extremely drug-resistant (XDR) phenotype was exclusively found in the Beijing isolates (n ؍ 7). A notable prevalence of the rpoB531 and katG315 mutations in Beijing strains that were similarly high in both TBS (this study) and published pulmonary TB ( Extrapulmonary tuberculosis (EPTB) remains a major health problem due to a significantly high rate of morbidity and mortality in both developing and developed countries and constitutes a significant part of the total TB incidence (1, 2). In Russia, the rate of EPTB decreased from 9.4% in 1992 to 3.4% in 2011, and the EPTB incidence constituted 2.4/100,000 in 2011 (3, 4). At the same time, the rate of tuberculous spondylitis (TBS) among all new EPTB cases increased in Russia from 23% in 2006 to 33% in 2010, which is higher than TB rate for of all other sites of the disease (3, 5).Tuberculous spondylitis constitutes about half of bone and joint TB cases and thus represents the most severe orthopedic disease, frequently leading to irreversible neurological disorders and disability and constituting a serious social and economic problem (6-8). TBS is developed as a result of blood-born dissemination of Mycobacterium tuberculosis. Bacteriological diagnosis of bone and joint TB is a most challenging task, and findings of drug resistance in these isolates may be crucial for adequate management of such cases. Delayed initial diagnosis and confirmation (from 3 months to 10 years after onset of the disease) and the increasing incidence of the multidrug-resistant TBS with spondyloarthropathy in adults require improvement of the etiological diagnostics, taking into account biological and molecular properties of the pathogen (4, 6, 7).M. tuberculosis has a clonal population structure, and it has been demonstrated that not only its large genetic families but also their variants or subgenotypes may play a special role in the disease progression. Russian strain Beijing B0/W148, initially defined by IS6110-restriction fragment length polymorphism (RFLP) analysis (9), presents a remarkable example of a successful clone highly associated with multidrug resistance (MDR) (10). Correlating different typing schemes, 24-locus mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) type 1...
Starting from the structure of antimycobacterial screening hit OTB-021 which was devoid of activity against ESKAPE pathogens, we designed, synthesized and tested two mutually isomeric series of novel simplified analogs, 2- and 4-(3-nitro-1,2,4-triazol-1-yl)pyrimidines, bearing various amino side chains. These compounds demonstrated a reverse bioactivity profile being inactive against M. tuberculosis while inhibiting the growth of all ESKAPE pathogens (with variable potency patterns) except for Gram-negative P. aeruginosa. Reduction potentials (E1/2, V) measured for selected compounds by cyclic voltammetry were tightly grouped in the −1.3–−1.1 V range for a reversible single-electron reduction. No apparent correlation between the E1/2 values and the ESKAPE minimum inhibitory concentrations was established, suggesting possible significance of other factors, besides the compounds’ reduction potential, which determine the observed antibacterial activity. Generally, more negative E1/2 values were displayed by 2-(3-nitro-1,2,4-triazol-1-yl)pyrimidines, which is in line with the frequently observed activity loss on moving the 3-nitro-1,2,4-triazol-1-yl moiety from position 4 to position 2 of the pyrimidine nucleus.
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