title: 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.
authors.affiliation: Central Research Division, Pfizer, Inc., Groton, Connecticut 06340, USA. thomas_d_gootz@groton.pfizer.com
authors.forename: T D
authors.initials: TD
authors.lastname: Gootz
authors.forename: R P
authors.initials: RP
authors.lastname: Zaniewski
authors.forename: S L
authors.initials: SL
authors.lastname: Haskell
authors.forename: F S
authors.initials: FS
authors.lastname: Kaczmarek
authors.forename: A E
authors.initials: AE
authors.lastname: Maurice
mesh_terms: D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000890:Anti-Infective Agents / Q000494:pharmacology*; D000995:Antitubercular Agents / Q000494:pharmacology; D002939:Ciprofloxacin / Q000494:pharmacology; D027081:DNA Gyrase; D027101:DNA Topoisomerase IV; D004250:DNA Topoisomerases, Type II / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004347:Drug Interactions; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D024841:Fluoroquinolones; D064704:Levofloxacin; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009287:Naphthyridines / Q000494:pharmacology*; D015242:Ofloxacin / Q000494:pharmacology; D012110:Reserpine / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D059005:Topoisomerase II Inhibitors
chemical_list: D018759:Adrenergic Uptake Inhibitors; D000890:Anti-Infective Agents; D000995:Antitubercular Agents; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D004791:Enzyme Inhibitors; D024841:Fluoroquinolones; D009287:Naphthyridines; D059005:Topoisomerase II Inhibitors; D002939:Ciprofloxacin; D064704:Levofloxacin; D012110:Reserpine; C080163:trovafloxacin; D015242:Ofloxacin; D027101:DNA Topoisomerase IV; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II; C061363:sparfloxacin