Gatifloxacin activity against quinolone-resistant gyrase: allele-specific enhancement of bacteriostatic and bactericidal activities by the C-8-methoxy group

Abstract

Antibacterial activities of gatifloxacin (AM1155), a new C-8-methoxy fluoroquinolone, and two structurally related compounds, AM1121 and ciprofloxacin, were studied with an isogenic set of ten quinolone-resistant, gyrA (gyrase) mutants of Escherichia coli. To compare the effect of each mutation on resistance, the mutant responses were normalized to those of wild-type cells. Alleles exhibiting the most resistance to growth inhibition mapped in alpha-helix 4, which is thought to lie on a GyrA dimer surface that interacts with DNA. The C-8-methoxy group lowered the resistance due to these mutations more than it lowered resistance arising from several gyrA alleles located outside alpha-helix 4. These data are consistent with alpha-helix 4 being a distinct portion of the quinolone-binding site of GyrA. A helix change to proline behaved more like nonhelix alleles, indicating that helix perturbation differs from the other changes at helix residues. Addition of a parC (topoisomerase IV) resistance allele revealed that the C-8-methoxy group also facilitated attack of topoisomerase IV. When lethal effects were measured at a constant multiple of the minimum inhibitory concentration for each fluoroquinolone to normalize for differences in bacteriostatic action, gatifloxacin was more potent than the C-8-H compounds, both in the presence and absence of protein synthesis (an exception was observed when alanine was substituted for aspartic acid at position 82). Collectively, these data show that the C-8-methoxy group contributes to the enhanced activity of gatifloxacin against resistant gyrase and wild-type topoisomerase IV.

FIG. 1

Fluoroquinolone structures.

FIG. 2

Bacteriostatic action of fluoroquinolones against resistant mutants. Isogenic strains of E. coli containing the indicated alleles of gyrA (A) or gyrA parC (B) were applied to LB agar plates containing various concentrations of the fluoroquinolones for determination of the MIC₉₉s. The MIC₉₉ for each mutant was then divided by the MIC₉₉ of wild-type or parC (S80L) cells to generate the normalized MIC₉₉ (the MIC₉₉s with wild-type cells were 0.038, 0.013, and 0.011 μg/ml for gatifloxacin [C-8-methoxy; solid bars], AM1121 [C-8-H; shaded bars], and ciprofloxacin [C-8-H; open bars], respectively. The data shown are the averages of two determinations.

FIG. 3

Relationship between bacteriostatic activities of fluoroquinolones against gyrA mutants and gyrA parC mutants. The MIC₉₉s for gyrA (Fig. 2A) and gyrA parC (Fig. 2B) mutants, normalized to the MIC₉₉s of wild-type and parC mutant cells, respectively, as described in the legend for Fig. 2, were plotted such that each data point represents one gyrA allele with wild-type and mutant parC alleles. Symbols: ■, gatifloxacin; □, AM1121; and ○, ciprofloxacin. Arrows indicate values for the D82A gyrA allele.

FIG. 4

Effect of fluoroquinolone structure on bacteriostatic activity against resistant gyrase. Normalized MIC₉₉s of gyrA parC mutants (Fig. 2B) are expressed as gatifloxacin/AM1121 MIC₉₉ ratios. Filled bars represent alleles located in α-helix 4 according to reference ; open bars represent alleles outside α-helix 4. The values shown are the averages of two independent determinations. Error bars indicate the upper ranges of the determinations.

FIG. 5

Bactericidal action of fluoroquinolones against resistant mutants. The survival rates of the indicated mutant strains of E. coli were determined following a 2-h incubation in gatifloxacin (filled bars), AM1121 (shaded bars), or ciprofloxacin (open bars) at 10 times the MIC₉₉ for each compound. (A) gyrA mutant strains; (B) gyrA parC double mutants. The data shown are the averages of two determinations.

FIG. 6

Bactericidal action of fluoroquinolones against resistant gyrA and gyrA parC mutants in the presence of chloramphenicol. The survival rates of mutants were determined following a 2-h incubation in chloramphenicol plus gatifloxacin (filled bars), AM1121 (shaded bars), or ciprofloxacin (open bars) at 10 times the respective MIC₉₉s (A) gyrA mutant strains; (B) gyrA parC double mutant strains. The data shown are the averages of two determinations.

FIG. 7

Relative positions of resistance alleles in GyrA dimer. The numbers indicate amino acid positions in the E. coli GyrA protein for resistance mutations and for the active center tyrosine (solid circles). The resistance alleles that reside within α-helix 4 are shown as open circles; residues outside the helix that confer resistance are shown as open squares. The dashed line approximates the interface of the two GyrA subunits. DNA is predicted to lay across the protein at an angle from upper left to lower right such that the two helices fit in the major groove of DNA (14).