Fluoroquinolone action against mycobacteria: effects of C-8 substituents on growth, survival, and resistance

Abstract

Fluoroquinolones trap gyrase on DNA as bacteriostatic complexes from which lethal DNA breaks are released. Substituents at the C-8 position increase activities of N-1-cyclopropyl fluoroquinolones against several bacterial species. In the present study, a C-8-methoxyl group improved bacteriostatic action against gyrA (gyrase-resistant) strains of Mycobacterium tuberculosis and M. bovis BCG. It also enhanced lethal action against gyrase mutants of M. bovis BCG. When cultures of M. smegmatis, M. bovis BCG, and M. tuberculosis were challenged with a C-8-methoxyl fluoroquinolone, no resistant mutant was recovered under conditions in which more than 1, 000 mutants were obtained with a C-8-H control. A C-8-bromo substituent also increased bacteriostatic and lethal activities against a gyrA mutant of M. bovis BCG. When lethal activity was normalized to bacteriostatic activity, the C-8-methoxyl compound was more bactericidal than its C-8-H control, while the C-8-bromo fluoroquinolone was not. The C-8-methoxyl compound was also found to be more effective than the C-8-bromo fluoroquinolone at reducing selection of resistant mutants when each was compared to a C-8-H control over a broad concentration range. These data indicate that a C-8-methoxyl substituent, which facilitates attack of first-step gyrase mutants, may help make fluoroquinolones effective antituberculosis agents.

FIG. 1

Structures of fluoroquinolones examined. The structure common to the compounds is shown, with positions of variable groups indicated by C5, C8, X, Y, or Z. Specific groups that distinguish the compounds are shown at the bottom of the figure. Abbreviations: H, hydrogen; Br, bromo; OMe, methoxyl; Me, methyl; Et, ethyl; NH₂, amino.

FIG. 2

Effect of a C-8-OMe group on bactericidal action of fluoroquinolones. (A) Wild-type M. bovis BCG (strain KD1295) was incubated at the indicated concentrations of fluoroquinolone for 48 h. Cultures were then diluted in drug-free medium, and the number of viable colonies was determined by growth on agar. Survival was calculated as the number of colonies recovered for each fluoroquinolone concentration normalized to the number observed in samples taken at the time of drug addition. (B) M. bovis BCG strain CX1 (first-step gyrA mutant) was treated as described for panel A. (C) M. bovis BCG strain CX2 (second-step gyrA mutant) was treated as described for panel A. Arrows indicate concentrations that are 4 times the ID₅₀ for each compound. Symbols: circles, PD161148 (C-8-OMe); squares, PD160793 (C-8-H).

FIG. 3

Effect of a C-8-Br group on bactericidal action of fluoroquinolones. M. bovis BCG strain KD1295 (wild type; open symbols) or strain CX1 (first-step gyrA mutant; filled symbols) was treated as in Fig. 2. Arrows indicate concentrations that are 4 times the ID₅₀ for each compound. Symbols: circles, PD163753 (C-8-Br); squares, PD138032 (C-8-H).

FIG. 4

Effect of chloramphenicol on bactericidal action of C-8-OMe and C-8-H fluoroquinolones. (A) Wild-type M. bovis BCG was treated with the indicated concentrations of PD161148 (C-8-OMe) in the presence (filled circles) or absence (open circles) of 20 μg of chloramphenicol/ml added 60 min prior to the fluoroquinolone. Incubation was continued for 48 h, after which percent survival was determined as described in the legend for Fig. 2. (B) Conditions were as described for panel A except that cells were treated with PD160793 (C-8-H), in the presence (filled squares) or absence (open squares) of chloramphenicol.

FIG. 5

Effect of fluoroquinolone concentration on selection of resistant mutants of M. bovis BCG. Wild-type cells were spread on agar plates containing the indicated concentrations of PD161148 (C-8-OMe; filled circles), PD160793 (C-8-H; filled squares), PD163753 (C-8-Br; open circles), or PD138032 (C-8-H; open squares). The plates were then incubated for 28 days at 37°C, and the number of fluoroquinolone-resistant colonies was recorded.