Activities of trovafloxacin compared with those of other fluoroquinolones against purified topoisomerases and gyrA and grlA mutants of Staphylococcus aureus

Abstract

Frequencies of mutation to resistance with trovafloxacin and four other quinolones were determined with quinolone-susceptible Staphylococcus aureus RN4220 by a direct plating method. First-step mutants were selected less frequently with trovafloxacin (1.1 x 10(-10) at 2 to 4x the MIC) than with levofloxacin or ciprofloxacin (3.0 x 10(-7) to 3.0 x 10(-8) at 2 to 4x the MIC). Mutants with a change in GrlA (Ser80-->Phe or Tyr) were most commonly selected with trovafloxacin, ciprofloxacin, levofloxacin, or pefloxacin. First-step mutants were difficult to select with sparfloxacin; however, second-step mutants with mutations in gyrA were easily selected when a preexisting mutation in grlA was present. Against 29 S. aureus clinical isolates with known mutations in gyrA and/or grlA, trovafloxacin was the most active quinolone tested (MIC at which 50% of isolates are inhibited [MIC(50)] and MIC(90), 1 and 4 microg/ml, respectively); in comparison, MIC(50)s and MIC(90)s were 32 and 128, 16 and 32, 8 and 32, and 128 and 256 microg/ml for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin, respectively. Strains with a mutation in grlA only were generally susceptible to all of the quinolones tested. For mutants with changes in both grlA and gyrA MICs were higher and were generally above the susceptibility breakpoint for ciprofloxacin, sparfloxacin, levofloxacin, and pefloxacin. Addition of reserpine (20 microg/ml) lowered the MICs only of ciprofloxacin fourfold or more for 18 of 29 clinical strains. Topoisomerase IV and DNA gyrase genes were cloned from S. aureus RN4220 and from two mutants with changes in GrlA (Ser80-->Phe and Glu84-->Lys). The enzymes were overexpressed in Escherichia coli GI724, purified, and used in DNA catalytic and cleavage assays that measured the relative potency of each quinolone. Trovafloxacin was at least five times more potent than ciprofloxacin, sparfloxacin, levofloxacin, or pefloxacin in stimulating topoisomerase IV-mediated DNA cleavage. While all of the quinolones were less potent in cleavage assays with the altered topoisomerase IV, trovafloxacin retained its greater potency relative to those of the other quinolones tested. The greater intrinsic potency of trovafloxacin against the lethal topoisomerase IV target in S. aureus contributes to its improved potency against clinical strains of S. aureus that are resistant to other quinolones.

FIG. 1

Inhibition of [³H]thymidine incorporation (Incorp.) into whole cells of the S. aureus RN4220 parent strain (○), the first-step mutant (mutant strain RN4220-20 [●]), and the second-step mutant (mutant strain RN4220-20-48 [□]) by trovafloxacin.

FIG. 2

Killing curves for the S. aureus RN4220-20-48 double mutant (GrlA, Ser80→Phe; GyrA, Ser84→Leu) by trovafloxacin (Trova), ciprofloxacin (Cipro), levofloxacin (Levo), and sparfloxacin (Spar) at 2 or 4× the MIC of each drug.

FIG. 3

Stimulation of topoisomerase IV (TOPO IV)-mediated DNA cleavage by trovafloxacin (A). Trovafloxacin concentrations of 0.0195, 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0, and 10 μg/ml were tested. (B) Relative potency (that of ciprofloxacin is given as a value of 1) of trovafloxacin for stimulation of topoisomerase IV-mediated DNA cleavage. The lowest concentration of drug required to stimulate enzyme-mediated DNA cleavage was determined by densitometric scanning of photographs of ethidium bromide-stained agarose gels. The numbers above the bars are MICs for S. aureus RN4220.

FIG. 4

Pattern of cleavage of ³²P-end-labeled pBR322 DNA by S. aureus topoisomerase IV in the presence of trovafloxacin or sparfloxacin. Enz, enzyme.