Molecular Characterization of Ofloxacin-Resistant Mycobacterium tuberculosis Strains from Russia

Abstract

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...

Evaluation of Two Molecular Assays for Rapid Detection of Mycobacterium tuberculosis Resistance to Fluoroquinolones in High-Tuberculosis and -Multidrug-Resistance Settings

“…Our data are in agreement with previous studies where FQ resistance was reported to be associated largely with mutations in codons 90 and 94 of gyrA (A90V, D94G, and D94Y [20,35]) and, more rarely, with mutations D94A, D94N, T80A, G88C, and D89 N gyrA (3,26,46). The distribution of mutations in the analyzed strains was in agreement with that reported in a recent study from St. Petersburg (26), suggesting a similar mechanism of drug resistance development in areas where the Beijing strains family dominates.…”

“…The distribution of mutations in the analyzed strains was in agreement with that reported in a recent study from St. Petersburg (26), suggesting a similar mechanism of drug resistance development in areas where the Beijing strains family dominates. Mutations in gyrB are less common (3), and their association with resistance to FQ is not known.…”

A Systematic Review Of Gyrase Mutations Associated With Fluoroquinolone-Resistant Mycobacterium Tuberculosis And A Proposed Gyrase Numbering System

“…19,21,42,46 Heteropeaks in fluoroquinolone-resistant M. tuberculosis have been described in detail previously. 48 In contrast to most bacteria, M. tuberculosis acquisition of drug resistance does not occur as a result of horizontal transfer of resistance-bearing genetic elements.…”

“…Substitutions Gly470Ala, Asn499Lys and D644+645 were never observed independently. 21,26,42 Systematic review…”

“…c Substitutions GyrA Ala74Ser and Ala90Gly, and GyrB Gly470Ala, Asn499Lys and D644-645 were never observed independently. 18,21,26,33,42,46,47 Systematic review 827 JAC …”