Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

Abstract

The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 ؋ 10 ؊8 and 5.7 ؋ 10 ؊9 , respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 ؋ 10 ؊10 ).Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16-to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 g/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 g/ml) resulted in 4-to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.Bacterial infections caused by multidrug-resistant pathogens are a major global problem, especially for nosocomial infections (6). Methicillin-resistant Staphylococcus aureus (MRSA) is one such pathogen against which options for effective antibacterial therapies are already limited (2). While certain newly developed drugs have promising activity against MRSA (1, 9), their relatively narrow spectra of activity could limit their clinical use in empirical therapy.Recently, a series of 8-methoxy, nonfluorinated quinolones (NFQs) (Fig. 1) F-1509, p. 210, 2000). On the basis of in vitro potency data, these compounds are more potent against MRSA and coagulase-negative staphylococci than several fluoroquinolones and have activities comparable to that of clinafloxacin (10). An apparent advantage of the NFQs against S. aureus lies in their ability to (i) better utilize both DNA gyrase and topisomerase IV as dual targets than certain quinolones, such as ciprofloxacin and trovafloxacin, and (ii) largely circumvent existing mutations in serine and glutamate "hot spots" of the target genes, gyrA and grlA, commonly associated with quinolone resistance (10, 11). However, it is imperative to ascertain the potential for development of de novo resistance to the NFQs in these pathogens. This report describes the in vitro isolation of S. aureus mutants with reduced susceptibilities to the NFQs and other quinolones by two approaches: stepwise isolation of mutants and spiral plater-based serial passage. MATERIALS AND METHODSMaterial...

Topoisomerase Targeting with and Resistance to Gemifloxacin in Staphylococcus aureus

“…Recently, we and others have identified many novel mutations in both subunits of topoisomerase IV outside the QRDRs in S. aureus and S. pneumoniae and have proved the role of some of these mutations in resistance to quinolones (24,25,27,42,48). In contrast, the range of mutations reported for DNA gyrase appears to be less, with only a few novel mutations in or outside the QRDR isolated, and proved to be responsible for quinolone resistance in S. aureus (26,42), S. pneumoniae (38,48), and E. coli (13). These findings suggest that a smaller range of tolerated mutations occur on DNA gyrase and that the study of various topoisomerase IV mutants may provide additional insights into enzyme-DNA-quinolone interactions.…”

Molecular Characterization of 8-Methoxyfluoroquinolone Resistance in a Clinical Isolate of Methicillin-Resistant <i>Staphylococcus aureus</i>

“…The grlA mutations were associated with both high-and low-level resistance to ciprofl oxacin in clinical isolates of S. aureus, while the gyrA mutations were responsible for an increase in resistance in the grlA mutant [11] . Previous in vitro studies have shown that multiple mutations in the QRDRs of both grlA and gyrA genes appeared to confer high-level resistance to fl uoroquinolones, including an 8-methoxyfl uoroquinolone [11,12,[26][27][28][29][30][31][32][33] . However, few previous reports have characterized clinical S. aureus isolates highly resistant to 8-methoxyfl uoroquinolones, including gatifl oxacin [18] .…”

“…Although the MIC breakpoint of si- tafl oxacin has not been defi ned by the CLSI/NCCLS, some resistant bacteria to older fl uoroquinolones with alterations in the QRDRs may remain susceptible to a newer fl uoroquinolone with increasing potency [19] . Investigations in vitro have shown that some mutants selected by fl uoroquinolone treatment acquired resistance via mutations in GrlA, GrlB, GyrA, GyrB and/or the norA promoter and coding regions [12,17,26,27,35] . In S. aureus, fl uoroquinolone resistance that emerged only shortly after these drugs were introduced has become recognized as a clinical problem [1,36] .…”

Unique Biological Properties and Molecular Mechanism of 5,6-Bridged Quinolones

“…5,6-Bridged dioxinoquinolones, unsubstituted (5-H, 6-H) quinolones, ciprofloxacin, and clinafloxacin (see Table 3) were synthesized at Procter & Gamble Pharmaceuticals as described previously (2,24). All other antibacterials were purchased from Sigma (St. Louis, Mo.).…”

“…For example, resistance to the nonfluorinated quinolones and other 8-methoxy quinolones can arise from mutations at a variety of previously unreported topoisomerase residues (16,24). In this study, we have reinvestigated an early series of 5,6-bridged dioxinoquinolones originally reported to exhibit structure-activity relationships (SARs) that diverged from those of other quinolones (2).…”