Extending the Definition of the GyrB Quinolone Resistance-Determining Region in Mycobacterium tuberculosis DNA Gyrase for Assessing Fluoroquinolone Resistance in M. tuberculosis

The newer fluoroquinolones moxifloxacin (MXF) and levofloxacin (LVX) are becoming more common components of tuberculosis (TB) treatment regimens. However, the critical concentrations for testing susceptibility of Mycobacterium tuberculosis to MXF and LVX are not yet well established. Additionally, the degree of cross-resistance between ofloxacin (OFX) and these newer fluoroquinolones has not been thoroughly investigated. In this study, the MICs for MXF and LVX and susceptibility to the critical concentration of OFX were determined using the agar proportion method for 133 isolates of M. tuberculosis. Most isolates resistant to OFX had LVX MICs of >1 g/ml and MXF MICs of >0.5 g/ml. The presence of mutations within the gyrA quinolone resistance-determining regions (QRDR) correlated well with increased MICs, and the level of LVX and MXF resistance was dependent on the specific gyrA mutation present. Substitutions Ala90Val, Asp94Ala, and Asp94Tyr resulted in low-level MXF resistance (MICs were >0.5 but <2 g/ml), while other mutations led to MXF MICs of >2 g/ml. Based on these results, a critical concentration of 1 g/ml is suggested for LVX and 0.5 g/ml for MXF drug susceptibility testing by agar proportion with reflex testing for MXF at 2 g/ml.I t is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), and 5% to 10% of infected persons will become ill with active disease necessitating a complicated treatment regimen. Patients with fully susceptible TB are generally treated with a combination of first-line drugs, including rifampin, isoniazid, ethambutol, and pyrazinamide for 6 months (1). Intolerance or acquisition of resistance to one or more of the first-line drugs requires deviation from the standard treatment regimen, and second-line drugs, including the injectables amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) and one of a number of fluoroquinolone (FQ) derivatives, may be needed to successfully treat the patient.FQs are a class of broad-spectrum, synthetic antibiotics effective against many different bacterial infections such as those of the urinary and respiratory tracts, including infections by M. tuberculosis. Early FQs had a limited spectrum of activity; however, two of the newest FQ derivatives, moxifloxacin (MXF) and levofloxacin (LVX) (the levo isomer of ofloxacin [OFX]), exhibit superior bactericidal activity against a wide range of organisms, have longer half-lives, and are less toxic than older FQs (2, 3). Studies have demonstrated the effectiveness of these new FQs against M. tuberculosis (4, 5), with comparatively low MICs (MXF MIC Ͻ 0.5 g/ml) (1, 6-9). Although clinical resistance to FQs has been reported, estimating the global burden of FQ-resistant TB is difficult given that drug susceptibility testing (DST) is not performed in many areas and testing for FQ resistance is typically undertaken only when resistance to first-line drugs is detected. However, in 2013, the World Health Organization (WHO) estima...

Section: Resultsmentioning

confidence: 99%

Correlation between GyrA Substitutions and Ofloxacin, Levofloxacin, and Moxifloxacin Cross-Resistance in Mycobacterium tuberculosis

Willby

Sikes

Malik

et al. 2015

“…The construction of a database of resistance-related mutations is required for the reliable genotypic prediction of drug resistance. We demonstrate that mutations within not only QRDR-A but also QRDR-B should be considered potential determinants of quinolone resistance (14,18,19), while mutations outside the QRDRs are likely linked to the genotypic background of M. tuberculosis (31,32). Furthermore, knowing the gyrase mutations not only is relevant for the prediction of quinolone resistance but also may be useful for estimating levels of resistance to various quinolones (16,17,40), which can aid in the selection of the appropriate quinolone and its dosing.…”

Section: Discussionmentioning

confidence: 99%

“…Quinolone binds to the quinolone binding pocket (QBP) in the DNA gyrase-DNA complex. Resistance to quinolones in M. tuberculosis results primarily from mutations in gyrA and gyrB, particularly in the quinolone resistance-determining regions (QRDRs) of GyrA (QRDR-A, residues 74 to 113) and GyrB (QRDR-B, residues 500 to 540), which cause alterations in the amino acids of the QBP (14,15). A wide variety of mutations within the QRDRs have been reported; however, different amino acid substitutions in gyrase may cause different levels of resistance to quinolones (16,17).…”

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confidence: 99%

Role of gyrB Mutations in Pre-extensively and Extensively Drug-Resistant Tuberculosis in Thai Clinical Isolates

Disratthakit

Prammananan

Tribuddharat

et al. 2016

DNA gyrase mutations are a major cause of quinolone resistance in Mycobacterium tuberculosis. We therefore conducted the first comprehensive study to determine the diversity of gyrase mutations in pre-extensively drug-resistant (pre-XDR) (n ‫؍‬ 71) and extensively drug-resistant (XDR) (n ‫؍‬ 30) Thai clinical tuberculosis (TB) isolates. All pre-XDR-TB and XDR-TB isolates carried at least one mutation within the quinolone resistance-determining region of GyrA ( . MIC and DNA gyrase supercoiling inhibition assays were performed to determine the role of gyrase mutations in quinolone resistance. Compared to the MICs against M. tuberculosis H37Rv, the levels of resistance to all quinolones tested in the isolates that carried GyrA-D94G or GyrB-N538D (8-to 32-fold increase) were significantly higher than those in isolates bearing GyrA-D94A or GyrA-A90V (2-to 8-fold increase) (P < 0.01). Intriguingly, GyrB-E540D led to a dramatic resistance to later-generation quinolones, including moxifloxacin, gatifloxacin, and sparfloxacin (8-to 16-fold increases in MICs and 8.3-to 11.2-fold increases in 50% inhibitory concentrations [IC 50 s]). However, GyrB-E540D caused low-level resistance to early-generation quinolones, including ofloxacin, levofloxacin, and ciprofloxacin (2-to 4-fold increases in MICs and 1.5-to 2.0-fold increases in IC 50 s). In the present study, DC159a was the most active antituberculosis agent and was little affected by the gyrase mutations described above. Our findings suggest that although they are rare, gyrB mutations have a notable role in quinolone resistance, which may provide clues to the molecular basis of estimating quinolone resistance levels for drug and dose selection.

“…The QRDR of gyrA ranged from codons 88 to 94 (8,9); the QRDR of gyrB ranged from codons 500 to 538 (21).…”

Section: Methodsmentioning

confidence: 99%

“…The binding target of FQs in M. tuberculosis is DNA gyrase, consisting of two A and two B subunits encoded by the gyrA and gyrB genes, respectively (7,8). Missense mutations within the quinolone resistance-determining region (QRDR), including a conserved region of gyrA (codons 88 to 94) and gyrB (codons 500 to 538), have been identified as the primary mechanism conferring fluoroquinolone resistance (8,9).…”

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confidence: 99%

Prevalence and Molecular Characterization of Fluoroquinolone-Resistant Mycobacterium tuberculosis Isolates in China

Zhang

Wang

et al. 2014

China is one of the countries with the highest burdens of multidrug-resistant (MDR) and fluoroquinolone (FQ)-resistant tuberculosis (TB) globally. Nevertheless, knowledge about the prevalence and molecular characterization of FQ-resistant Mycobacterium tuberculosis isolates from this region remains scant. In this study, 138 M. tuberculosis isolates determined by the agar proportion susceptibility method to be resistant to ofloxacin (OFX) were enrolled from a national drug resistance survey of China. All these strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, and sparfloxacin using liquid Middlebrook 7H9 medium. The entire gyrA and gyrB genes conferring FQ resistance were sequenced, and spoligotyping was performed to distinguish different genotypes. Overall, the prevalence of resistance in China was highest for ofloxacin (3.76%), intermediate for levofloxacin (3.18%) and moxifloxacin (3.12%), and lowest for sparfloxacin (1.91%) and gatifloxacin (1.33%). Mutations in the gyrA gene were observed in 89 (64.5%) out of the 138 OFX-resistant M. tuberculosis strains. Positions 94 and 90 were the most frequent sites of mutation conferring FQ resistance on these strains, accounting for high-level FQ resistance. Furthermore, the Beijing genotype showed no association with high-level FQ resistance or distribution in hot spots in the quinolone resistance-determining region (QRDR) of gyrA. Our findings provide essential implications for the feasibility of genotypic tests relying on detection of mutations in the QRDR of gyrA and the shorter first-line treatment regimens based on FQs in China.