To develop more effective antituberculosis agents, we have been studying the fluoroquinolones. These compounds, which act by trapping gyrase on DNA, are generally used only as second-line therapeutics because resistance mutations in Mycobacterium tuberculosis
often render them ineffective. For example, in the early 1990s a strain of M. tuberculosis
resistant to isoniazid, rifampin, streptomycin, and ethambutol spread among immunocompromised persons in New York City (1
). Patients were treated with the fluoroquinolone ciprofloxacin, and within a few months some were found harboring strains of M. tuberculosis
that were resistant to fluoroquinolones as well as to the four other agents (11
). When we examined these strains, we noticed that some fluoroquinolones (e.g., sparfloxacin) were more effective than others (e.g., ciprofloxacin) at blocking the growth of resistant mutants after the data were normalized to the results obtained with wild-type cells (5
). This observation led to the idea that some quinolones might be better than others at trapping resistant gyrase in mycobacteria. Subsequent work showed that compounds with a methoxy group attached to the C-8 position (C-8-OMe) were particularly effective against resistant mutants (5
While examining the effects of C-8-OMe groups we observed a complex relationship between the recovery of resistant mutants and the fluoroquinolone concentration (6
). Increases in fluoroquinolone concentration cause the fraction of cells that form colonies on quinolone-containing agar to drop sharply to a plateau and then drop sharply a second time. We proposed that the plateau is due to the presence of first-step, resistant mutants in the population. If this were true, the second drop in mutant recovery should occur when the MIC for these mutants is approached: once the growth of first-step mutants is blocked, no mutant should be recovered at the cell amounts used because a rare, double mutation would be required to overcome the quinolone effect. The minimal quinolone concentration that allows no mutant recovery when more than 1010
cells are applied to drug-containing agar was defined as the mutant prevention concentration (MPC). If relevant concentrations of an antibiotic in tissue can be maintained above the MPC, selection of resistant mutants should be severely restricted.
MPC has been measured for only a few fluoroquinolones; consequently, structure-activity relationships are still poorly defined. In the present work we determined the MICs and MPCs for Mycobacterium smegmatis of fluoroquinolones that differed in moieties attached to the C-8 position and to the C-7 piperazinyl ring. C-8 substituents influenced the effect that C-7-ring alkyl groups have on fluoroquinolone MICs and MPCs. When resistant gyrA (gyrase) mutants were examined, fluoroquinolone susceptibility varied with drug structure in a way that allowed inferences about quinolone binding to gyrase. Data obtained with the mutants also revealed that the MPC often correlates better with the MIC for the most resistant first-step mutant than with the MIC for wild-type cells, consistent with the two-mutation explanation for MPC. Collectively, these data show that the MPCs for and the susceptibilities of resistant mutants can be used to discriminate among closely related fluoroquinolones.