Quinolone resistance in Staphylococci: activities of new nonfluorinated quinolones against molecular targets in whole cells and clinical isolates

Abstract

The activity of three new, 8-methoxy-nonfluorinated quinolones (NFQs) against multiple-drug-resistant staphylococci was investigated. First, using Staphylococcus aureus strains containing point mutations in the serine 84-80 hot spots of the target genes (gyrA and grlA), cell growth inhibition potencies of the NFQs as a result of DNA gyrase and topoisomerase IV inhibition were estimated and compared with those of known fluoroquinolones. The NFQs and clinafloxacin showed higher affinities toward both the targets than ciprofloxacin, trovafloxacin and gatifloxacin. Furthermore, the ratio of the calculated affinity parameter for DNA gyrase to that for topoisomerase IV was lower in the case of the NFQs, clinafloxacin, and gatifloxacin than in the case of ciprofloxacin and trovafloxacin. These results suggest that the former group of quinolones is better able to exploit both the targets. Next, using clinical isolates of methicillin-resistant S. aureus (MRSA; n = 34) and coagulase-negative staphylococci (CoNS; n = 24), the NFQs and clinafloxacin were shown to be more potent (MIC at which 90% of the isolates are inhibited [MIC90] = 2 microg/ml for MRSA and 0.5 microg/ml for CoNS) than ciprofloxacin, trovafloxacin, and gatifloxacin (MIC90 = 16 to >64 microg/ml for MRSA and 4 to >32 microg/ml for CoNS). Bactericidal kinetics experiments, using two MRSA isolates, showed that exposure to the NFQs at four times the MIC reduced the bacterial counts (measured in CFU per milliliter) by > or =3 log units in 2 to 4 h. Overall, the NFQs and clinafloxacin were less susceptible than the other quinolones to existing mechanisms of quinolone resistance in staphylococci.

FIG. 1

Chemical structures of the NFQs.

FIG. 2

Effects of gyrA and grlA mutations on S. aureus cell growth in the presence of ciprofloxacin. With OD₆₀₀ after a 10-h incubation (described under Materials and Methods), cell growth of strains SS1 (gyrA; Ser84-Leu) (A), MT5224c4 (grlA; Ser80-Phe) (B), and EN1252a (gyrA, Ser84-Leu; grlA, Ser80-Phe) (C) were compared with that of the parent strain ISP794. Error bars represent standard deviations due to well-to-well variations in results. These and similar data with all the quinolones included in this study were used to generate the ETI₅₀ values.

FIG. 3

Correlation between the S. aureus topo IV ETI₅₀ values estimated during this study and previously reported IC₅₀ values measured using purified topo IV by Takei et al. (18).

FIG. 4

Cumulative susceptibility of the MRSA isolates to the NFQs and other quinolones. Percent susceptibility through the whole range of concentrations (A) and at 1- and 2-μg/ml concentrations (B) is shown.

FIG. 5

Correlation of relative susceptibilities of MRSA isolates with high levels of quinolone resistance. These isolates were chosen based on the MIC of trovafloxacin (>2 μg/ml, i.e., in the 4- to 32-μg/ml range). Linear regression analyses were performed with data points correlating the relative MICs of PGE9509924 with those of PGE9262932, PGE4175997, and trovafloxacin.

FIG. 6

Bactericidal kinetics of the NFQs, trovafloxacin, and clinafloxacin at four times the MIC. Results using two isolates of MRSA, Mi407 (A) and Mi414 (B), are shown. PGE9509924 was used as a racemic mixture in this experiment.