Abstract
Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.
Highlights
Mycobacterium tuberculosis is the etiologic agent of tuberculosis (TB), a potentially fatal illness which results in approximately 2 million deaths worldwide each year [1]
We introduced several mutations identified within gyrA and gyrB into M. tuberculosis laboratory strains and assessed their true significance in FQ resistance
The Significance of gyrA Mutations and FQ Resistance In order to ensure that the Minimum Inhibitory Concentrations (MICs) observed with clinical isolates harboring mutations in gyrA or gyrB is directly related to those specific mutations, we introduced single or double mutations into the well-characterized M. tuberculosis strains H37Rv and Erdman using the mycobacteriophage allelic exchange system and determined the MIC for CIP, LVX, MXF and OFX
Summary
Mycobacterium tuberculosis is the etiologic agent of tuberculosis (TB), a potentially fatal illness which results in approximately 2 million deaths worldwide each year [1]. TB treatment requires a lengthy multi-drug regimen, and TB control efforts have been hampered by the emergence of resistance to the first-line drugs. Treatment of patients infected with a drug-resistant strain requires the use of more toxic and less efficient drugs with a longer treatment period as compared to drug-susceptible strains [3]. FQs have excellent in vitro and in vivo activity against M. tuberculosis and have proven to be among the most effective secondline antimicrobial drugs used for the treatment of individuals infected with MDR TB and patients experiencing severe adverse effects due to first-line drugs [5,6]. Range of MIC (mg/mL) CIP OFX LVX MXF. 2, no mutation, CIP, ciprofloxacin, OFX, ofloxacin, LVX, levofloxacin, MXF, moxifloxacin. Resistance defined as; CIP (.2 mg/mL), OFX (.2 mg/mL), LVX (.1 mg/mL) and MXF (.0.5 mg/mL). M. tuberculosis lacks parC and parE homologs, and DNA gyrase appears to be the sole target for FQ antibiotics [14]
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