ABSTRACT
We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of Streptococcus pneumoniaeto gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against S. pneumoniae 7785 (MIC, 0.06 μg/ml) compared with ciprofloxacin (MIC, 1 to 2 μg/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either parC orgyrA quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 μg/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 μg/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-stepgyrA mutants of S. pneumoniae 7785 (gemifloxacin MICs, 0.25 μg/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 μg/ml) exhibited an alteration in parC resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10- to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either S. pneumoniaegyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in S. pneumoniae, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.
Gemifloxacin (SB-265805) is a new fluoroquinolone which displays impressive activity againstStreptococcus pneumoniae (5, 24), the principal cause of community-acquired pneumonia and a major player in meningitis, otitis, sinusitis, and exacerbations of chronic bronchitis (3). The drug is effective in vitro, not only against penicillin-susceptible isolates of S. pneumoniae but also against penicillin-resistant strains, which are now commonly encountered in the clinic (5, 9, 24). A tentative breakpoint for S. pneumoniae of 0.5 μg/ml has been proposed (35). Recent work has shown that gemifloxacin also retains activity against multidrug-resistant S. pneumoniae, including strains resistant to ciprofloxacin (16), a fluoroquinolone widely used in treating gram-negative infections but which has borderline activity against pneumococci (31). Although the incidence of quinolone-resistant pneumococci is presently low (7), such strains could become important with increased quinolone usage. The mechanism underlying the susceptibility of ciprofloxacin-resistant strains to gemifloxacin is not known.
Fluoroquinolones act by inhibiting DNA gyrase and topoisomerase IV, two enzymes that operate by a double-strand DNA break mechanism (17,20) and that collaborate in ensuring DNA unwinding and strand separation during DNA replication (1, 8, 10, 12, 17, 34,36). Gyrase, an A2B2 tetramer encoded by the gyrA and gyrB genes, catalyzes negative DNA supercoiling during the initiation and elongation phases of DNA replication (13). Topoisomerase IV is made up of two C and two E subunits specified by the parC and parEgenes and is responsible for the segregation of daughter chromosomes at cell division (19). Both gyrase and topoisomerase IV form a ternary complex with quinolones and DNA (termed the “cleavable complex”), which is converted into a lethal double-strand DNA break by collision with replication forks or other mechanisms (8). Resistance to quinolones occurs through stepwise acquisition of chromosomal mutations in defined segments of the gyrase and topoisomerase IV genes, termed the quinolone resistance-determining regions (QRDRs) (23). Within the QRDRs, nucleotide hot spots are mutated in quinolone-resistant strains leading to particular changes at the protein level (6, 23), e.g., for S. pneumoniae, S81F or S81Y in GyrA; S79F or S79Y in ParC; E474K in GyrB; and D435V in ParE (11, 15, 16, 18, 22, 25-28, 30,33). Many of these mutations have been shown to confer quinolone resistance when mutant QRDRs are used to transform susceptible S. pneumoniae strains (18, 22).
By establishing the order of topoisomerase QRDR mutations in consecutive stepwise-selected drug-resistant mutants of S. pneumoniae, we were able to show that quinolones can be grouped into three archetypal mechanistic classes (27, 28). The first group, initially identified with ciprofloxacin and now including levofloxacin, norfloxacin, pefloxacin, and trovafloxacin, selects QRDR mutations in topoisomerase IV before those in gyrase, suggesting that the drugs act preferentially through topoisomerase IV in vivo (11,15, 18, 22, 25, 26, 30, 33). By contrast, a second group of drugs comprising sparfloxacin, gatifloxacin, and NSFQ-105 [a ciprofloxacin homologue bearing a 4-(4-aminophenylsulfonyl)-1-piperazinyl group at C-7] select gyrase mutations before those in topoisomerase IV, indicating that this class of quinolones acts through gyrase (2,11, 27). Finally, clinafloxacin acts through both gyrase and topoisomerase IV (28). Thus, although clinafloxacin selectsgyrA or gyrB QRDR mutations in the first step, these single-step mutants occur at low frequency and display only a small (<twofold) increase in MIC over wild type, suggesting that both gyrase and topoisomerase IV contribute substantially to drug action (28). It appears that the structure of the quinolone defines its mode of action in S. pneumoniae (2, 27).
In recent work, we and a colleague reported that gemifloxacin retained activity against S. pneumoniae clinical isolates that were highly resistant to ciprofloxacin and carried mutations inparC, gyrB, and parE genes (16). Given the absence of gyrA mutations in these strains, we speculated that gemifloxacin may act preferentially through gyrase in S. pneumoniae (16). To test this idea, we have examined the response of an isogenic panel ofS. pneumoniae strains bearing defined quinolone resistance mutations in topoisomerase genes. In complementary experiments, we have also determined the order of acquisition of QRDR resistance mutations in the topoisomerase genes of S. pneumoniae mutants selected by stepwise challenge with gemifloxacin. Finally, we have investigated the interaction of recombinant S. pneumoniae gyrase and topoisomerase IV enzymes with gemifloxacin in vitro.
MATERIALS AND METHODS
Bacterial strains. S. pneumoniae 7785 is a quinolone-susceptible clinical isolate (26) from which drug-resistant mutant strains were derived by stepwise challenge. Mutant strains developed by selection with ciprofloxacin (1C1, 2C6, 2C7, and 3C4) or sparfloxacin (1S1, 1S4, 2S1, and 2S4) have been described previously (27).
Drug susceptibilities.Gemifloxacin mesylate was supplied by SmithKline Beecham Pharmaceuticals, Harlow, Essex, United Kingdom. Gemifloxacin was dissolved in water at the appropriate concentration immediately before use. Ciprofloxacin and sparfloxacin were kindly made available by Bayer U.K., Newbury, United Kingdom, and Dainippon Pharmaceutical Co., Suita, Japan, respectively. MICs were determined by twofold agar dilution on brain heart infusion (BHI) plates containing 10% horse blood. Bacteria (104 to 105 CFU) were spotted onto plates containing the appropriate concentration of drug. Plates were incubated in air at 37°C for 18 to 24 h and were examined for growth.
Stepwise selection of gemifloxacin-resistant mutants.Approximately 109 to 1010 CFU of S. pneumoniae strain 7785 (or its drug-resistant mutants) were plated onto BHI plates containing 10% horse blood and various concentrations of gemifloxacin. Plates were incubated aerobically for 24 to 48 h. Colonies were restreaked on plates containing the same drug concentration. Frequencies of mutant selection were calculated from the ratio of colonies obtained on drug plates to the number obtained on drug-free plates. All procedures were as described previously (25,27).
PCR amplification of QRDRs and RFLP analysis.Genomic DNA was isolated from bacterial strains and used as a template in PCR amplification of the QRDRs of the parC, parE,gyrA, and gyrB genes as previously described (25). PCR primers used to amplify QRDRs for HinfI analysis were VGA3 and VGA4 (for gyrA) and M0363 and M4721 (for parC). Primers used for PCR and for sequence analysis were VGA4 and VGA9 (gyrA), M5884 and M4721 (parC), XS01 and M0361 (parE), and M4025 and M4026 (gyrB). Primer sequences and PCR conditions have been reported previously (16). Restriction fragment length polymorphism (RFLP) analysis was performed as previously described (27).
Asymmetric PCR and DNA sequencing.Single-stranded DNA was generated from QRDR PCR products by asymmetric PCR using the reported primers and reaction conditions (16). The single-stranded DNA was sequenced directly by the chain termination method using Sequenase version 2.0 and appropriate primers (32).
DNA cleavage by S. pneumoniae topoisomerases.Conditions for the purification and assay of recombinant S. pneumoniae gyrase and topoisomerase IV have been described previously (29). DNA cleavage assays were carried out as described earlier (29). The extent of DNA cleavage was determined from photographic negatives using a Molecular Dynamics personal densitometer SI and ImageQuant software.
RESULTS
Activity of gemifloxacin against isogenic S. pneumoniaestrains bearing defined quinolone-resistance mutations.To gain an understanding of which quinolone resistance mutations affect susceptibility to gemifloxacin, we made use of quinolone-susceptible clinical isolate 7785 and its characterized mutants obtained by stepwise challenge with ciprofloxacin (strains 1C1, 2C6, 2C7, and 3C4) or sparfloxacin (1S1, 1S4, 2S1, and 2S4) (Table1). The susceptibilities of the parent and mutant strains were determined for ciprofloxacin, sparfloxacin, and gemifloxacin by twofold agar dilution; the MIC values determined here for ciprofloxacin and sparfloxacin were identical to, or within one dilution of, those reported previously (27). The parent strain 7785 required MICs for ciprofloxacin and sparfloxacin of 1 to 2 and 0.5 μg/ml, respectively (Table 1). However, gemifloxacin was much more potent, requiring a MIC of only 0.06 μg/ml. This value is in agreement with previous results obtained for other clinical strains and underlines the much greater activity of gemifloxacin against S. pneumoniae (16).
Fluoroquinolone susceptibilities and QRDR statuses of isogenic mutants of S. pneumoniae strain 7785 selected with ciprofloxacin, sparfloxacin, or gemifloxacin
The responses of the defined mutants to ciprofloxacin and sparfloxacin were described earlier and are consistent with the drugs acting through topoisomerase IV and gyrase, respectively (27). Briefly, in the case of ciprofloxacin, strain 1C1 (possibly an efflux mutant; see below) showed a small increase in MIC as reported previously (25). Strains 2C6 and 2C7 (derived from 1C1) withparC changes coding S79Y or S79F alterations exhibited a two- to fourfold increase in MIC over 1C1. Strains 1S1 and 1S4 expressing S81F or S81Y GyrA proteins required MICs for ciprofloxacin similar to that of wild-type strain 7785. The parC-gyrAdouble mutants 3C4, 2S1, and 2S4 were highly resistant to ciprofloxacin with MICs of 16 to 64 μg/ml (Table 1). By contrast, gyrAchanges in strains 1S1 and 1S4 increased the sparfloxacin MIC, whereasparC changes in 2C6 and 2C7 had no effect (Table 1). The double mutants were highly resistant to sparfloxacin with a MIC of 16 μg/ml.
Results for gemifloxacin followed neither paradigm. First, the presence of a mutation either in gyrA or in parC resulted in a MIC of 0.12 to 0.25 μg/ml, i.e., a two- to fourfold increase over that of the parent (Table 1). Second, the presence of mutations in both parC and gyrA in strains 3C4, 2S1, and 2S4 increased the gemifloxacin MIC to 0.5 to 1.0 μg/ml, an 8- to 16-fold increase over that of the wild-type strain. These observations are consistent with the idea that both gyrase and topoisomerase IV contribute in setting the gemifloxacin susceptibility of strain 7785. Moreover, gemifloxacin was some 32- to 128-fold more active against the double mutants than the other quinolones tested (Table 1).
Gemifloxacin-resistant S. pneumoniae mutants from stepwise challenge.As susceptibility studies on defined mutants proved uninformative about the primary intracellular target of gemifloxacin, we decided to generate and analyze S. pneumoniae strains selected by stepwise drug challenge. Identification of QRDR changes in first-step mutants indicates the preferred in vivo target. The choice of initial drug concentration was guided by the findings in Table 1.
Approximately 109 to 1010 CFU of S. pneumoniae strain 7785 was plated on BHI agar containing 10% horse blood and gemifloxacin at either 0.06 μg/ml (the MIC) or 0.09 μg/ml (1.5 × MIC). After 48 h of incubation in air at 37°C, mutant colonies appeared at each drug concentration. Colonies were restreaked on fresh plates containing the same (selecting) concentration of gemifloxacin. The frequency of first-step mutants selected at either drug concentration was approximately 10−9. Six of the first-step gemifloxacin mutants—1GM4, 1GM5, 1GM7, and 1GM9 (selected at 0.06 μg of drug/ml) and 1GM10 and 1GM11 (selected at 0.09 μg of drug/ml)—were chosen for further characterization (Fig. 1). For the second-step selection, strain 1GM5 (gemifloxacin MIC, 0.25 μg/ml) was challenged with drug at 0.25 μg/ml (1 × MIC) and 0.5 μg/ml (2 × MIC), producing mutants 2GM1-16 and 2GM17-20, respectively (Fig. 1). The mutant frequencies in both second-step selections were approximately 10−9. Mutants 2GM1, 2GM2, 2GM17, and 2GM18 were characterized further.
Selection of S. pneumoniae mutants resistant to gemifloxacin. Mutants were selected stepwise on agar plates as described in Materials and Methods. The relationship between the parent strain 7785 and its first-step (prefix 1) and second-step (prefix 2) gemifloxacin-resistant mutants is shown. Concentrations of gemifloxacin (μg/ml) used for selection are indicated outside the boxes.
First-step mutants carry gyrA QRDR changes; second-step mutants have an additional parC QRDR mutation.All first-step mutants required MICs for gemifloxacin of 0.12 to 0.25 μg/ml, which represents a two- to fourfold increase over that of the wild-type strain 7785 (Table 1). Interestingly, with the exception of strain 1GM4, the other five first-step mutants showed only a small (<twofold) increase in MIC for ciprofloxacin compared with 7785, whereas their sparfloxacin MICs were 4- to 16-fold higher than that of strain 7785. By analogy with the data for ciprofloxacin- and sparfloxacin-selected mutants in Table 1, this result suggested that first-step mutants could have acquired changes in gyrA. Accordingly, the gyrA, parC, gyrB, andparE QRDRs were amplified from each strain by PCR prior to DNA sequence analysis (28). Strain 1GM4 required a ciprofloxacin MIC of 2 to 4 μg/ml and, like 1C1 (ciprofloxacin MIC, 3 μg/ml), did not have any QRDR mutations. To determine whether resistance in these mutants was due to an efflux mechanism, e.g., involving PmrA, their ciprofloxacin MICs and that of parental strain 7785 were determined in the presence of reserpine (7.5 μg/ml), a known efflux pump inhibitor (4, 14). Inclusion of reserpine reduced all the MICs to 0.5 to 1 μg/ml, which is consistent with the operation of an efflux mechanism in both 1C1 and 1GM4. The other five first-step mutants that were examined had acquired single-point changes in the gyrA QRDR specifying S81F, S81Y, or E85K, all of which are known quinolone resistance mutations. None of the first-step mutants had mutations in the parC, gyrB, orparE QRDRs (Table 1).
Second-step mutants 2GM1 to 2GM20 exhibited MICs of ciprofloxacin, sparfloxacin, and gemifloxacin of 64, 32, and 1 μg/ml, respectively. Based on results in Table 1, this phenotype is consistent with the presence of mutations in both gyrA and parC. RFLP analysis by HinfI digestion (28) was performed to detect mutations affecting codon 79 in parC. A 366-bpparC QRDR product was amplified by PCR from all 20 second-step gemifloxacin mutants. In each case, digestion withHinfI produced a 183-bp doublet, indicating mutational loss of a HinfI site encompassing codon 79 (data not shown). DNA sequence analysis of the four topoisomerase QRDRs of selected second-step mutants 2GM1, 2GM2, 2GM17, and 2GM18 (Table 1) confirmed the presence in all the strains of the gyrA (S81F) mutation derived from parent 1GM5. In addition, and in agreement withHinfI RFLP analysis, the strains had acquired aparC mutation encoding S79F or S79Y changes at the protein level. Thus, gemifloxacin selects gyrA and thenparC mutations in S. pneumoniae.
Gemifloxacin is more potent than ciprofloxacin in stabilizing cleavable complexes of S. pneumoniae gyrase and topoisomerase IV.In principle, the increased antipneumococcal activity of gemifloxacin could ensue from enhanced stabilization of type II topoisomerase complexes on DNA. To test this possibility, we compared the abilities of gemifloxacin and ciprofloxacin to stabilize complexes of recombinant S. pneumoniae gyrase or topoisomerase IV with DNA. Supercoiled plasmid pBR322 was incubated with enzyme in the absence or presence of drug, and DNA cleavage was effected by the addition of detergent. Samples were digested with proteinase K, and DNA was analyzed by electrophoresis in 1% agarose gels. The results are presented for gyrase and for topoisomerase IV in Fig. 2A and B, respectively. For gyrase, inclusion of either ciprofloxacin or gemifloxacin resulted in a dose-dependent increase in the production of linear DNA generated by disruption of the cleavable complex. There was no drug-dependent increase in nicked DNA. Quantitative analysis of the bands allowed the determination of CC25 values (the concentration of drug that results in conversion of 25% of the substrate DNA to the linear form under these experimental conditions). The CC25 values for ciprofloxacin and gemifloxacin were 80 and 5 μM, respectively (Fig. 2A).
Comparison of fluoroquinolone-promoted DNA cleavage byS. pneumoniae gyrase and topoisomerase IV. (A) DNA cleavage mediated by DNA gyrase in the presence of ciprofloxacin (CIP) and gemifloxacin (GEMI). Supercoiled plasmid pBR322 DNA (0.4 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) with ciprofloxacin or gemifloxacin at the concentrations indicated. After the addition of sodium dodecyl sulfate and proteinase K, samples were analyzed by electrophoresis in 1% agarose gels. (B) DNA breakage by topoisomerase IV. ParC (0.45 μg) and ParE (1.7 μg) proteins were incubated with drugs prior to induction of DNA breakage and sample processing as described above. (A and B) Lanes A and B, supercoiled and linear pBR322 DNA, respectively; N, nicked DNA; L, linear DNA; S, supercoiled DNA.
A similar experiment is shown in Fig. 2B using topoisomerase IV at levels equimolar to those of gyrase in Fig. 2A. Inclusion of either ciprofloxacin or gemifloxacin resulted in dose-dependent increases in both linear and nicked DNA species (Fig. 2B). The CC25values for ciprofloxacin and gemifloxacin (based on production of linear DNA) were 2.5 and 0.1 μM, respectively. Thus, gemifloxacin was some 25-fold more efficient than ciprofloxacin in promoting DNA cleavage by topoisomerase IV. (A similar relative difference in drug efficiency [20-fold] was found when the production of both linear and nicked species in Fig. 2B was taken into account [data not shown]). Interestingly, topoisomerase IV was much more efficient than gyrase in promoting DNA cleavage by either of the drugs (Fig. 2A and B). Table2 presents the various CC25values obtained in vitro in comparison with the ciprofloxacin and gemifloxacin MICs required for strain 7785. It can be seen that the 20-fold lower MIC of gemifloxacin compared to ciprofloxacin correlates with the enhanced stabilization of cleavable complexes by both gyrase and topoisomerase IV.
Fluoroquinolone growth inhibition of S. pneumoniae strain 7785 and drug-stimulated DNA cleavage efficiencies of DNA gyrase and topoisomerase IV
DISCUSSION
Gemifloxacin is a novel antibacterial fluoroquinolone with potent activity against S. pneumoniae. We examined its mechanism of action both in vivo, by using defined S. pneumoniae mutants and stepwise mutant selection, and in vitro, by using purified recombinant S. pneumoniae gyrase and topoisomerase IV proteins. Gemifloxacin selected gyrA QRDR mutants in the first step followed by parC changes in the second step, suggesting that gyrase is the preferred drug target in vivo (Table 1). However, given that S. pneumoniae mutants with defined mutations in parC showed a low-level increment in MIC similar to that of the first-step gyrA mutants (Table 1), it appears that the drug acts substantially through both gyrase and topoisomerase IV, i.e., shows some of the properties expected of a dual-targeting fluoroquinolone. Interestingly, topoisomerase IV was more effective than gyrase in inducing DNA cleavage mediated by gemifloxacin in vitro (Fig. 2). This is the first genetic and enzymatic analysis of gemifloxacin action in S. pneumoniae and has important implications.
One striking feature of the data presented here is the markedly greater activity of gemifloxacin compared with ciprofloxacin and sparfloxacin against wild-type S. pneumoniae and its quinolone-resistant mutants (Table 1). Thus, wild-type strain 7785 was at least 10- to 20-fold more susceptible to gemifloxacin than to the other two agents. The MIC of 0.06 μg/ml is similar to that reported (0.03 to 0.12 μg/ml) for a range of clinical isolates (16). Moreover, the gemifloxacin MICs for single gyrA or parCmutants were elevated only some two- to fourfold, to 0.12 to 0.25 μg/ml (Table 1). Even strains harboring mutations in bothparC and gyrA (3C4, 2S1, and 2S4) showed MICs of gemifloxacin of 0.5 to 1 μg/ml (Table 1), some 32- to 64-fold lower than those of ciprofloxacin and sparfloxacin (Table 1).
The factors underlying the enhanced activity of gemifloxacin againstS. pneumoniae are not fully understood. However, the DNA cleavage data suggest that the drug exhibits greater affinity for both gyrase and topoisomerase IV than ciprofloxacin in vitro. At least 10- to 20-fold higher levels of ciprofloxacin over gemifloxacin were required to produce comparable levels of DNA cleavage by either enzyme in vitro (Fig. 2). These results are consistent with the idea that enhanced cleavable complex formation is responsible for greater potency in vivo. Interestingly, similar to previous studies with other quinolones (21, 29), we have shown that gemifloxacin stimulates DNA cleavage by topoisomerase IV much more efficiently than by gyrase. Although this observation contrasts with the genetic work showing that both enzymes are targeted in vivo with a preference for gyrase, we assume that inside the bacterium, drug-enzyme affinity is only one aspect determining the killing pathway (29). Moreover, it is conceivable that the relative proficiencies of cleavable complex formation measured in vitro do not properly reflect those that hold under intracellular conditions.
Gemifloxacin is a new addition to those quinolones that selectgyrA QRDR changes in first-step challenge of S. pneumoniae. These agents include sparfloxacin, gatifloxacin, and NSFQ-105 (2, 11, 27). Identification of gemifloxacin with these agents may aid structure-activity analysis of how quinolone structure influences in vivo targeting. In fact, the response of the panel of defined gyrA and/or parC mutants to gemifloxacin and the results of enzyme studies (Table 1 and Fig. 2) both show close similarity to data previously obtained for the dual-targeting agent clinafloxacin (28, 29). Thus, similar to gemifloxacin, neither gyrA nor parC mutations alone had much effect on clinafloxacin susceptibility: both mutations were needed to give significant resistance. Moreover, gemifloxacin and clinafloxacin were comparably active against parC-gyrAdouble mutants, with MICs of 0.5 to 1 μg/ml (28). In enzyme studies, the drugs show very similar CC25 values against gyrase and topoisomerase IV. Such highly potent agents may be especially valuable in attacking mutants selected by previous clinical use of other quinolones, e.g., ciprofloxacin or norfloxacin.
As an example of this approach, we and a colleague recently described two multidrug-resistant S. pneumoniae clinical isolates that were highly resistant to ciprofloxacin (MICs, 64 μg/ml) but remained susceptible to gemifloxacin (MICs, 0.12 μg/ml) (16). Both strains expressed S79F ParC, E474K GyrB, and D435V ParE proteins. Ciprofloxacin preferentially targets topoisomerase IV in S. pneumoniae, and the high-level resistance of these strains presumably arises from the S79F change in ParC, a known quinolone resistance mutation, and D435V, which affects a hot spot for quinolone resistance in the conserved EGDSA motif of ParE. The E474K mutation in GyrB was reported previously in a first-step S. pneumoniaemutant selected with clinafloxacin (28). The mutation lies outside the classical GyrB QRDR and had at most a twofold effect on susceptibility to ciprofloxacin. We hypothesized that the markedly superior activity of gemifloxacin over ciprofloxacin against these strains could be explained if gemifloxacin acts preferentially through gyrase in S. pneumoniae (16). The data presented here support this hypothesis (Table 1).
In summary, gemifloxacin displays some attractive advantages over current quinolones, such as ciprofloxacin, in terms of both its antipneumococcal mechanism and its greatly enhanced potency. These features suggest that the drug could have a promising role in the treatment of S. pneumoniae infections.
ACKNOWLEDGMENTS
We thank Ken Coleman for helpful comments and Xiao-su Pan for purified enzymes.
This work was supported by a project grant from SmithKline Beecham.
FOOTNOTES
- Received 15 May 2000.
- Returned for modification 31 July 2000.
- Accepted 25 August 2000.
- Copyright © 2000 American Society for Microbiology
