GYRASE MUTATIONS IN LABORATORY-SELECTED, FLUOROQUINOLONE-RESISTANT MUTANTS OF MYCOBACTERIUM-TUBERCULOSIS H37RA

To characterize mechanisms of resistance to fluoroquinolones by Mycoba cterium tuberculosis, mutants of strain H37Ra were selected in vitro w ith ofloxacin. Their quinolone resistance-determining regions of gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B, Three types of mutants were obtained: (i) one mutant (TKp1) ha d no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations r esulting in either two altered gyrase A residues or an altered residue in both gyrases A and B, The TKp1 mutant had slightly reduced levels of uptake of [C-14]norfloxacin, which was associated with two- to four fold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxac in. Gyrase mutations caused a much greater increase in the MICs of flu oroquinolones, For mutants with single gyrA mutations, the increases i n the MICs were 4 to 16-fold, and for mutants with double gyrase mutat ions, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated w ith 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxac in compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested, No sparfloxacin-resistant single-step mutants were selected at concentrations of >2.5 mu g/ml, and high-level resistance (i.e., MIC, greater than or equal to 5 mu g/ml) was associated with two gyrase mu tations. Mutations in gyrB and possibly altered levels of intracellula r accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance, B ecause sparfloxacin is more active in vitro and selection of resistanc e appears to be less likely to occur, it may have important advantages over ofloxacin or ciprofloxacin for the treatment of tuberculosis.