Sequence Analyses of Just Four Genes To Detect Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Multidrug-Resistant Tuberculosis Patients Undergoing Treatment

Abstract

The rapid detection of Mycobacterium tuberculosis isolates resistant to second-line drugs is crucial for the institution of appropriate treatment regimens as early as possible. Although molecular methods have successfully been used for the rapid detection of resistance to first-line drugs, there are limited data on mutations that confer resistance to second-line drugs. To address this question, we analyzed Mycobacterium tuberculosis strains resistant to ofloxacin (n ‫؍‬ 26) and to capreomycin and/or amikacin (n ‫؍‬ 48) from Uzbekistan for variations in target genes (gyrA, gyrB, rrs, and tlyA). Strains susceptible to ofloxacin (n ‫؍‬ 49) and capreomycin and/or amikacin (n ‫؍‬ 39) were included as controls. Mutations in gyrA or gyrB were found in 96% (25/26 strains) of the ofloxacin-resistant strains, while none of the susceptible strains displayed mutations in those two genes. The most common mutation occurred in gyrA at codon 94 (17/26 strains [65.4%]), followed by mutations at codons 90 and 91. Two strains showed a mutation in gyrB, at codons 485 and 543, respectively; both mutations have not been reported previously. The most frequent mutation in strains resistant to both amikacin and capreomycin was A1401G in rrs (34/40 strains [85.0%]). Three strains had mutations in tlyA, of which two (at codons 18 and 118) were associated with resistance to capreomycin alone. Overall, none of the 10 resistant strains (5 amikacin-resistant and capreomycin-susceptible strains) and none of the 39 susceptible control strains had mutations in the genes investigated. Our results clearly demonstrate the potential of sequence analyses of short regions of relatively few target genes for the rapid detection of resistance to second-line drugs among strains isolated from patients undergoing treatment for multidrug-resistant tuberculosis. The mechanisms that confer amikacin resistance in this setting remain unclear.

Molecular Characterization of Multidrug- and Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Jiangxi, China

“…The latter result indicates that the inclusion of gyrB may not considerably change the performance of the genotyping assay. This finding is in agreement with previous observations (21,51) that gyrB mutations are less common than gyrA mutations, and it may reflect the fact that the phenotypic resistance to FQ is mainly due to mutations in the QRDR of gyrA rather than gyrB, as suggested in a recent review and meta-analysis (12). In addition, the correlation between FQ resistance and the gyrB mutations (Asp500His and Glu540Asp), which was first reported by Duong et al in 2009 (18), has not yet been validated.…”

“…Since the C1402T mutation has not been shown to confer resistance to AMK (19), this suggests at least another unknown mechanism for AMK resistance in the isolate. Similar to other studies (9,21), no mutation was detected in the rrs gene in four resistant strains. The resistance phenotypes are probably attributed to the alterations within other loci, such as tlyA (for CAP resistance) (32) or the promoter region of eis (KAN), which confer low-level resistance to KAN (53), or an enhanced multidrug efflux pump (54).…”

Detection by GenoType MTBDR sl Test of Complex Mechanisms of Resistance to Second-Line Drugs and Ethambutol in Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates

“…Therefore, it is likely that other yet-uncharacterized molecular mechanisms are the cause of the observed monoresistance to KAN or CAP in these strains. Among other plausible causes of isolated resistance to capreomycin in M. tuberculosis, mutations inactivating the tlyA gene encoding a 2Ј-O-methyltransferase were previously reported by Feuerriegel et al (9). However, such mutations were not detected by DNA sequencing in our study, and further investigations are needed to elucidate the causes of monoresistance to KAN or CAP.…”

“…For fluoroquinolone resistance, the quinolone resistance-determining regions of the gyrA and gyrB genes were amplified and sequenced using primers PRI8 (5Ј-YGGTGGRTCR TTRCCYGGCGA-3Ј) and PRI9 (5Ј-CGCCGCGTGCTSTATGCRATG-3Ј) for gyrA and primers gyrBa (5Ј-GAGTTGGTGCGGCGTAAGAGC-3Ј) and gyrBe (5Ј-CGGCCATCAAGCACGATCTTG-3Ј) for gyrB (8). For aminoglycosides/ cyclic peptide resistance, the rrs, rpsL, and tlyA genes were amplified and sequenced using primers TB53 (5Ј-GATGACGGCCTTCGGGTTGT-3Ј) and TB54 (5Ј-TCTAGTCTGCCCGTATCGCC-3Ј) for the region from positions 513 to 516 in the rrs gene (13), primers TB55 (5Ј-GTAGTCCACGCCGTAAACG G-3Ј) and TB56 (5Ј-AGGCCACAAGGGAACGCCTA-3Ј) for the region around position 907 in the rrs gene (13), primers RRSA (5Ј-GGCGTTCCCTT GTGGCCTGTG-3Ј) (primer designed in-house) and RRS1539 (5Ј-GGGGCG TTTTGCTGGTGCTCC-3Ј) (1) for the region from positions 1401 to 1484 in the rrs gene, primers rpsl1 (5Ј-CCAACCATCCAGCAGCTGGTC-3Ј) and rpsl2 (5Ј-ATCCAGCGAACCGCGGATGA-3Ј) for the rpsL gene (primers designed inhouse), and primers tlyAs (5Ј-GCATCGCACGTCGTCTTT-3Ј) and tlyAas (5Ј-GGTCTCGGTGGCTTCGTC-3Ј) for the tlyA gene (9). With regard to ETB resistance, the embB gene in the region of codon M306 was amplified using primers embBs (5Ј-CCGACCACGCTGAAACTG-3Ј) and embBas (5Ј-GTAAT ACCAGCCGAAGGGATCCT-3Ј) (15).…”

Quinolone Resistance in Tuberculosis Treatment: A Structural Overview

“…Mutations within the QRDRs are responsible of at least 75% of fluoroquinolone resistance in M. tuberculosis, affecting most commonly the GyrA subunit (G88A, A90V, S91P, D94G, H, N, Y) but also the GyrB subunit (Aubry et al, 2006b;Veziris et al, 2007;Siddiqi et al, 2002;Feuerriegel et al, 2009). Modeling of the M. tuberculosis DNA gyrase catalytic core allowed to clearly establish that QRDR residues of both subunits are spatially close and form the quinolone-binding pocket (QBP) (Fig.…”