Intracellular targets of moxifloxacin: a comparison with other fluoroquinolones

Ekaterina Pestova, John J. Millichap, Gary A. Noskin and Lance R. Peterson*

Departments of Pathology and Medicine, Northwestern University Medical School, Divisions of Microbiology and Infectious Diseases, Northwestern Memorial Hospital, Chicago, IL 60611, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The in vitro activity of the novel 8-methoxyquinolone, moxifloxacin, against Streptococcus pneumoniae was evaluated, and the intracellular targets of this agent were studied. Analysis of mutant strains selected with moxifloxacin demonstrated that first-step mutants bore amino acid substitutions at position 81 in the GyrA subunit of DNA gyrase. This suggests that, unlike older fluoroquinolone agents such as ciprofloxacin and levofloxacin, but similar to other C-8 substituted quinolones like sparfloxacin and gatifloxacin, moxifloxacin targets the GyrA subunit of DNA gyrase as an initial lethal event. Such a mechanism results in high activity against increasingly common S. pneumoniae strains bearing substitutions in DNA topoisomerase IV. Moxifloxacin was active with an MIC of <= 0.25 mg/L against S. pneumoniae clinical isolates, and against mutants, selected in the laboratory with ciprofloxacin or levofloxacin, that bore a Ser-79->Phe/Tyr substitution in ParC. The moxifloxacin MIC for strains with mutations in the structural genes for both DNA gyrase subunit GyrA and DNA topoisomerase IV subunit ParC did not exceed 2 mg/L, a level within clinically achievable serum concentrations for this agent. We also found that moxifloxacin is a poor substrate for active efflux in S. pneumoniae. Therefore, the high activity of moxifloxacin against S. pneumoniae appears to be a result of both enhanced activity against DNA gyrase and topoisomerase IV, and reduced efflux from the bacterial cell.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluoroquinolone antimicrobial agents have been used in clinical practice since the 1980s, primarily for treating infections caused by Gram-negative bacteria. Recently, new derivatives have been developed with enhanced activity against Gram-positive species including Streptococcus pneumoniae, one of the most important pathogens responsible for respiratory tract infections, acute otitis media and meningitis. The application of fluoroquinolones to the treatment of pneumococcal infections has become important because of the dissemination of pneumococcal clones resistant to ß-lactam and macrolide antibiotics.1

Fluoroquinolones act by inhibiting homologous type II topoisomerases, DNA gyrase and DNA topoisomerase IV, enzymes that control DNA topology and are vital for chromosome function and replication. Each of these enzymes is a tetramer composed of two subunits: GyrA and GyrB forming an A2B2 complex in DNA gyrase; and ParC and ParE forming a C2E2 complex in DNA topoisomerase IV. Amino acid substitutions in any of the subunits of either gyrase or topoisomerase IV have the potential for associated fluoroquinolone resistance in S. pneumoniae. The ParC subunit of DNA topoisomerase IV is the initial, or primary, target of the older fluoroquinolones, such as ciprofloxacin.24 Changes in this enzyme are commonly associated with ciprofloxacin resistance in clinical isolates of S. pneumoniae.5

It was recently demonstrated, however, that fluoroquinolones with a different molecular structure could have other primary targets in S. pneumoniae. For example, the primary target of sparfloxacin is now believed to be GyrA, possibly as a result of a substituent change made at position C-8 (Figure 1Go).3 Furthermore, energy-dependent active efflux is increasingly seen as important in the development of bacterial resistance to many drugs, including fluoroquinolone antimicrobials.6,7 Our initial investigations on trovafloxacin suggested that the bulky substituent at position C-7 on this molecule may hinder an organism's ability to export that agent.8,9 Moxifloxacin has the bulkiest C-7 substituent of the currently available fluoroquinolone agents, leading to our hypothesis that it, too, will be poorly exported from the bacterial cell.



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Figure 1. Chemical structure of the quinolone/naphthyridone ring system, showing sites that are modified in various agents, and the structures of the fluoroquinolones investigated.

 
With the development of newer quinolones it is important to determine how their molecular structure affects the target preference and overall antimicrobial activity. It is also essential to find out if an alteration in a primary target of one fluoroquinolone affects cross-resistance to others in the same class. To assess this we analysed cellular targets of a novel 8-methoxyquinolone, moxifloxacin, which is a highly active antipneumococcal agent.10 In this work we measured the activity of moxifloxacin against selected clinical isolates of S. pneumoniae, determined its cellular targets by stepwise mutant selection and assessed the involvement of energy-dependent efflux in the response of S. pneumoniae to moxifloxacin compared with other fluoroquinolone agents.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and growth conditions

Clinical isolates of S. pneumoniae with varying fluoroquinolone sensitivity were obtained from the collection of strains at Northwestern Memorial Hospital in Chicago, IL, USA. A highly susceptible laboratory strain, CP1000, used in this study was described previously.11

This isolate was recovered before the introduction of fluoroquinolones, and we used it as a susceptible control and as the strain for selection of resistant mutants. Organisms were grown at 35°C in Todd–Hewitt broth (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% yeast extract (THBY), or on Tryptic Soy agar plates (Difco) supplemented with 5% sheep's blood. A casein hydrolysate–yeast extract–tryptone medium (CAT) was used for mutant selection.12 Archived strains were preserved at –80°C with 12% (v/v) glycerol.

Testing susceptibility to antimicrobial agents

The susceptibility of strains to antimicrobial agents was determined by a microdilution method, using Mueller– Hinton broth (Difco) supplemented with 5% lysed horse blood.13 The following agents were used: levofloxacin (Ortho-McNeil Pharmaceuticals, Raritan, NJ, USA), ciprofloxacin (Bayer Corporation, West Haven, CT, USA), sparfloxacin (Rhône-Poulenc Rorer R-D, Vitry-sur-Seine, France) and moxifloxacin (Bayer Corporation). Before testing, individual strains were incubated overnight at 35°C on Tryptic Soy agar plates (Difco) supplemented with 5% sheep's blood. These cultures were used to prepare an inoculum of 1–5 x 105 cfu/mL. Inoculated panels were incubated at 35°C for 24 h. All testing was in duplicate.

Selection of mutants

First-step mutants were obtained by exposing S. pneumoniae CP1000 to the MIC of each agent: ciprofloxacin, 0.5 mg/L; levofloxacin, 1.0 mg/L; sparfloxacin, 0.25 mg/L; moxifloxacin, 0.125 mg/L. Between 108 and 109 cells from an S. pneumoniae CP1000 culture grown in THBY were plated on to the top layer of CAT agar on a two-layer plate, with double the concentration of corresponding antimicrobial agent in the bottom layer of the agar. In each experiment, a total of 1 x 1010 cells (using multiple plates) were used for mutant selection. Individual clones were harvested after 48 h incubation at 35°C. Second-step mutants were obtained essentially by the same procedure, this time exposing first-step mutants to 2 x MIC of the respective selection agent for each strain. Fluoroquinolone-resistant mutants were tested for stability of the acquired resistance by two passages on drug-free blood agar plates (DiMed Inc., St Paul, MN, USA), incubated at 35°C for a total of 48 h. Organisms were then transferred on to blood agar plates containing a concentration of drug equivalent to half the new MIC in order to determine that resistance was stable after 48 h growth in a drug-free environment.

PCR amplification and DNA sequence analysis

To investigate whether the tested strains had amino acid substitutions in ParC, ParE, GyrA or GyrB, the nucleotide sequences of parC, parE, gyrA and gyrB gene fragments that include regions corresponding to quinolone resistance determining regions (QRDRs) of the respective proteins (amino acids 43–121 in GyrA, 361–511 in GyrB, 55–167 in ParC and 392–529 in ParE) were determined and compared with the corresponding sequences from the reference strain CP1000. The sequences of gyrA, gyrB, parC and parE were published previously and are available in the NCBI database. The QRDR regions that we assessed correspond to amino acids 47–125 in GyrA, 352–502 in GyrB, 59–171 in ParC and 383–520 in ParE of Escherichia coli.3 A 253 bp fragment of gyrA (bp 129–363), a 453 bp fragment of gyrB (bp 1080–1533), a 337 bp fragment of parC (bp 164–501) and a 413 bp fragment of parE (bp 1175–1587) were amplified using the following pairs of primers: GyrA1 (5'-CGTCGCATTCTCTACGGA-3') and GyrA2 (5'-CGTCGCATTCTCTACGGA-3'); GyrB1 (5'-CTCTTCAGTGAAGCCTTCTCC-3') and GyrB2 (5'-CTCCATCGACATCGGCATC-3'); ParC1 (5'- TGACAAGAGCTACCGTAAGTCG-3') and ParC2 (5-TCGAACCATTGACCAAGAGG-3'); and ParE1 (5'-ACGTAAGGCGCGTGATGAG-3') and ParE2 (5'-CTAGCGGACGCATGTAACG-3'). PCR amplifications were performed with AmpliTaq DNA polymerase (Perkin–Elmer Cetus, Foster City, CA, USA) on an MJ Research Peltier Thermal Cycler PTC-100. A 1 µL sample of the bacterial culture at a density of 1 x 108 cells/mL was used as a template in standard 50 µL PCR reactions. Sequencing was carried out on the amplified PCR products using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Cetus) and an ABI PRISM 310 Genetic Analyzer according to the protocol of the manufacturer.

Growth inhibition studies: evaluation of the effect of reserpine on susceptibility of S. pneumoniae CP1000 to fluoroquinolones

S. pneumoniae cultures were started at an optical density (OD550) of 0.001 (approximately 1 x 106 cells/mL) in THBY broth containing either ciprofloxacin 0.12 mg/L, levofloxacin 0.25 mg/L, sparfloxacin 0.06 mg/L or moxifloxacin 0.03 mg/L, or a combination of each of these fluoroquinolones with 10 mg/L of reserpine.8 After a 7 h incubation at 35°C, the OD550 reached by each culture was determined. A drug concentration of 0.25 x MIC was used as this inhibited growth of S. pneumoniae CP1000 cultures by two- to four-fold relative to a logarithmically growing culture without added antimicrobial agent, yet allowed enough growth over 7 h to observe the action of the drug, as well as the reserpine-mediated inhibition of growth.8 The extent of growth inhibition by each fluoroquinolone with or without reserpine was determined by comparing the optical density of cultures containing the corresponding fluoroquinolone, or the combination of this fluoroquinolone with reserpine, with the optical density of control cultures grown in the absence of antimicrobial agents, with reserpine added to one of the two controls.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activity of moxifloxacin against DNA gyrase and topoisomerase IV mutants

The activity of moxifloxacin against clinical isolates and laboratory mutants bearing changes in major fluoroquinolone target enzymes, DNA gyrase and topoisomerase IV, is shown in Table IGo. Strain SP30 is a recent clinical isolate susceptible to all fluoroquinolones tested. Clinical isolates 6406 and 6678 bear changes in the ParC subunit of DNA topoisomerase IV. Each of these strains has an additional substitution, Ile-460->Val, in ParE, which was found not to contribute to fluoroquinolone resistance using genetic transformation of the corresponding mutation into strain CP1000.9 Strains 1C1 and 1L1 are first-step mutants selected with ciprofloxacin (C) and levofloxacin (L) using laboratory strain CP1000.9,14 As is evident from Table IGo, moxifloxacin was the most active of the fluoroquinolones tested, against both the wild-type isolates and those with various mutations. It consistently had the highest activity against clinical isolates and first-step laboratory mutants bearing a Ser-79->Phe/Tyr substitution in ParC. These strains demonstrated only a two-fold increase in the MIC of moxifloxacin (to 0.25 mg/L), suggesting that an important target for moxifloxacin differs from that of ciprofloxacin and levofloxacin.


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Table I. Fluoroquinolone resistance in clinical isolates and resistant laboratory mutants of S. pneumoniae
 
Second-step laboratory mutants 2C2 and 2L1, with changes in the GyrA subunit of DNA gyrase and in the ParC subunit of DNA topoisomerase IV, had four-fold greater resistance to moxifloxacin than the parent strain CP1000. Similar levels of resistance were observed in clinical isolates RT1 and RT2, which were recovered from patients who failed therapy with levofloxacin (RT1) and ciprofloxacin (RT2); these strains had amino acid substitutions in GyrA/ParE and GyrB/ParC, respectively. The MIC of moxifloxacin for these strains, however, did not exceed 2 mg/L, while MICs for ciprofloxacin, levofloxacin and sparfloxacin increased to 8–16 mg/L. The maintenance of moxifloxacin's clinically useful level of activity against these strains suggests that multiple key cellular targets within the QRDR regions of DNA gyrase and topoisomerase IV may be available for new quinolones with the appropriate novel substituents.

Selection of resistance with moxifloxacin and sparfloxacin

To identify the molecular targets of moxifloxacin and to investigate whether the primary target of this antimicrobial agent differs from that of older fluoroquinolones, we performed stepwise selection of resistant mutants using moxifloxacin. For comparison, we also selected first-step mutants with sparfloxacin. The frequencies of direct mutant selection using moxifloxacin and sparfloxacin are shown in Table IIGo. Interestingly, moxifloxacin-resistant first-step mutants appeared with a frequency of 1.3 x 10–7 on plates containing 0.125 mg/L (the MIC) of this agent, while no mutant colonies were detected on those containing a higher concentration of moxifloxacin. In contrast, sparfloxacin-resistant mutants could be selected at concentrations up to twice the agent's MIC. However, using a first-step mutant (1M8) for selection of higher levels of moxifloxacin resistance, it was possible to obtain second-step mutants at 2 x MIC with a frequency of 1.8 x 10–7. Thus, once first-step resistance develops, the emergence of high-level, second-step resistance appears to be facilitated.


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Table II. Frequencies of resistance selection with moxifloxacin and sparfloxacin
 
Genetic analysis of the selected moxifloxacin-resistant mutants

Table IIIGo presents the results of sequence analysis of first-step (1M8, 1M15) and second-step (2M1, 2M2, 2M3, 2M4) mutants selected using moxifloxacin. We also analysed two first-step mutants (1S1, 1S2) selected by the same method with sparfloxacin. First-step mutants selected with either moxifloxacin or sparfloxacin bore changes in the gyrA gene that resulted in a deduced Ser->Phe substitution of the GyrA subunit. The acquisition of this amino acid substitution leads to a four-fold increase in the level of resistance to sparfloxacin and moxifloxacin for mutant strains, but only to a two-fold increase in the resistance to ciprofloxacin. No change in levofloxacin resistance was detected. The second-step mutants selected from strain 1M8 using 1 mg/L of moxifloxacin accumulated amino acid substitutions in both the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV (Table IIIGo). A combination of these two substitutions raised the MIC of sparfloxacin, ciprofloxacin and levofloxacin to >=8.0 mg/L, while the MIC of moxifloxacin rose only to 2.0 mg/L, again suggesting its enhanced activity against either these altered sites, or the remaining, non-mutated enzymes GyrB or ParE.


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Table III. Properties of the laboratory mutants selected with moxifloxacin and sparfloxacin
 
Evaluation of moxifloxacin efflux by growth-inhibition studies

To assess the role of active efflux as a cellular response to moxifloxacin, we studied the effect of the plant alkaloid reserpine combined with this fluoroquinolone on the growth of S. pneumoniae CP1000, and compared the results with those obtained with sparfloxacin, levofloxacin and ciprofloxacin. Our experiments were based on a known observation that several fluoroquinolones that are substrates of active efflux exhibit an increased antibacterial activity in the presence of reserpine. Being a specific inhibitor of an active multidrug efflux mechanism, reserpine potentiates the action of these agents by increasing quinolone intracellular accumulation.15,16 The extent of growth inhibition by each fluoroquinolone with and without reserpine was compared in 7 h S. pneumoniae cultures (Figure 2Go). As evident from this figure, growth with ciprofloxacin was inhibited nearly three-fold when combined with reserpine, and growth with levofloxacin and sparfloxacin in the presence of reserpine was reduced to approximately two-thirds of that without reserpine. Importantly, reserpine had little effect on the growth of S. pneumoniae CP1000 in the presence of moxifloxacin, suggesting that this agent is a poor substrate for active efflux by S. pneumoniae. Thus, in addition to the likelihood of multiple homologous type II topoisomerase targets for moxifloxacin, these data imply moxifloxacin's possibility of avoiding other bacterial resistance mechanisms, such as active efflux, in the cellular response after drug exposure.



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Figure 2. Growth inhibition curves with (a) ciprofloxacin, (b) levofloxacin, (c) sparfloxacin and (d) moxifloxacin with 0.25 x MIC of the agent alone ({circ}) or combined with reserpine 10 mg/L ({diamondsuit}).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In these studies we demonstrated that moxifloxacin is highly active against S. pneumoniae isolates, including strains bearing amino acid substitutions in the ParC subunit of DNA topoisomerase IV, considered be the primary or preferred target for the older fluoroquinolone agents, such as ciprofloxacin and levofloxacin.24,17 Although the presence of a Ser-79->Tyr substitution in ParC raised the MIC of ciprofloxacin and levofloxacin to 2 mg/L, the MIC of moxifloxacin was only 0.25 mg/L, showing an even higher potency than the other C-8-substituted quinolone, sparfloxacin. That moxifloxacin has a clinically useful level of activity even against strains with DNA gyrase and topoisomerase IV mutations indicates that multiple key targets within these two enzymes may be available for quinolones with novel substituents. The MIC of moxifloxacin for these strains did not exceed 2 mg/L, which is still below the maximum serum concentration of 3.6 mg/L achieved with an oral dosage regimen of 400 mg daily.18

The low level of resistance in ParC mutants to moxifloxacin suggests a different primary target of this fluoroquinolone in S. pneumoniae. Stepwise in vitro mutant selection demonstrated that, unlike older fluoroquinolone agents, moxifloxacin targets the GyrA subunit of DNA gyrase first. Similar target preference was demonstrated by an 8-fluoro-substituted quinolone, sparfloxacin, as shown here and reported in earlier studies.5 Recently this effect was noted for another fluoroquinolone, gatifloxacin.19 The C-8-substituted quinolones also exhibit enhanced bacteriostatic and lethal activities against GyrA mutants in Escherichia coli and Mycobacterium spp.20,21 Furthermore, in another Gram-positive bacterium, Staphylococcus aureus, the substitution was the most lethal for both wild-type cells and those with a pre-existing topoisomerase IV mutation.22 As shown in Figure 1Go, moxifloxacin and sparfloxacin carry a methoxyl group and fluorine, respectively, at position C-8, with levofloxacin (and ofloxacin) having a benzoxazine bridge between C-8 and N-1, while ciprofloxacin carries no C-8 substituent. Taken together, our results show that C-8-substituted quinolones appear to have an enhanced affinity within the Gram-positive bacterial cell for GyrA rather then ParC (compared with C-8-H agents), with increased lethality of the C-8-substituted agents persisting even against strains resistant to older quinolones. Of note is the observation that the fused C-8 substituent of ofloxacin and levofloxacin does not provide the same advantage, suggesting the need for a free substituent at C-8.

In this study, we also assessed the role of active efflux as a bacterial response to moxifloxacin. The involvement of active efflux as a key mechanism in the avoidance of S. pneumoniae resistance to moxifloxacin was suggested by the low frequency of resistance selection at drug concentrations exceeding the agent's MIC. With reduced active efflux, this drug appears to accumulate within the S. pneumoniae cell, leading to early death of the entire bacterial population and markedly reduced survival of first-step mutants. The involvement of active efflux as a mechanism of resistance to certain fluoroquinolone agents in S. pneumoniae was indicated in earlier studies.68,15 Reports now suggest that at least half of resistant clinical pneumococcal isolates express an active efflux phenotype.6,7 It was shown earlier that bulkiness at the C-7 substituent and the overall hydrophobicity of the fluoroquinolone molecule contribute to the reduction of the active efflux, therefore increasing their cellular accumulation in S. aureus.23 As further evidence, we recently demonstrated this trait for another quinolone with a bulky C-7 substituent, trovafloxacin, in S. pneumoniae8,9 Since moxifloxacin carries a bulky substituent, a bicyclic fused ring composed of a pyrrolidine and piperazine ring (Figure 2Go), we anticipated that the efflux of this compound would be impaired in S. pneumoniae. Our hypothesis was confirmed by the results of the growth inhibition experiments showing poor efflux of this fluoroquinolone agent. Of the fluoroquinolones tested in this study, ciprofloxacin has the least bulky C-7 substituent, a piperazine moiety, with levofloxacin and sparfloxacin each carrying slightly larger piperazine derivatives, a 4-methyl piperazine and 3,5-dimethyl piperazine, respectively, while moxifloxacin has the bulkiest C-7 substituent. In our study, moxifloxacin appeared to be least susceptible to efflux, while ciprofloxacin was the most sensitive of all fluoroquinolones tested.

In conclusion, the C-8-methoxyl substituent and a bulky bicyclic fused ring carried by moxifloxacin provide an advantageous increase in activity of this agent against the Gram-positive pathogen S. pneumoniae compared with older fluoroquinolones. This is probably the consequence of a combination of factors, including the increased lethality of moxifloxacin against parC mutants, as well as the decrease in the efficiency of its active efflux from the pneumococcal cell. The benefit of these structural innovations is most pronounced on wild-type pneumococcal cells with no pre-existing mutations in any QRDR. Here, the entire bacterial population appears to be killed at drug concentrations exceeding the MIC. This is in contrast to older compounds (ciprofloxacin and levofloxacin) where some resistant mutants can survive at even 8 x MIC.8 However, once a first-step resistant mutant is selected, whether by moxifloxacin or another fluoroquinolone, the development of more-resistant bacterial cells proceeds more readily. Thus fluoroquinolones with specific novel substitutions, like moxifloxacin, may be potent tools for maintaining long-term quinolone activity against Gram-positive organisms, but only if they are used preferentially over the older agents that appear more prone to selecting drug-resistant bacteria.


    Acknowledgments
 
We thank Richard B. Thomson, Jr, PhD, Evanston Northwestern Healthcare, Evanston, IL, USA for generously donating strains RT1 and RT2. This work was supported by grants from the Excellence in Academic Medicine (EAM) programme at Northwestern Memorial Hospital and the Pharmaceutical Division of Bayer Corporation; and supported by Northwestern University Medical School, Chicago, IL, USA. This material was presented, in part, in the abstracts of the Ninety-Ninth Annual Meeting of the American Society for Microbiology, Chicago, IL, May 30–June 3 1999 (Abstract #A-37).


    Notes
 
* Correspondence address. Microbiology Division, Department of Pathology, Wesley Pavilion, Room 565, Northwestern Memorial Hospital, 251 E. Huron, Chicago, IL 60611, USA. Tel: +1-312-926-2885; Fax: +1-312-926-4139; E-mail: lancer{at}nwu.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Quintiliani, R., Owens, R. C. & Grant, E. M. (1999). Clinical role of fluoroquinolones in patients with respiratory tract infections. Infectious Diseases in Clinical Practice 8, Suppl. 1, S28–41.[ISI]

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4 . Tankovic, J., Perichon, B., Duval, J. & Courvalin, P. (1996). Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro. Antimicrobial Agents and Chemotherapy 40, 2505–10.[Abstract]

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8 . Beyer, R., Pestova, E., Millichap, J. J., Noskin, G. A. & Peterson, L. R. (2000). A convenient assay to estimate the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: evidence for diminished moxifloxacin, sparfloxacin and trovafloxacin efflux. Antimicrobial Agents and Chemotherapy 44, 798–801.[Abstract/Free Full Text]

9 . Pestova, E., Beyer, R., Cianciotto, N. P., Noskin, G. A. & Peterson, L. R. (1999). Contribution of topoisomerase IV and DNA gyrase mutations in Streptococcus pneumoniae for resistance to novel fluoroquinolones. Antimicrobial Agents and Chemotherapy 43, 2000–4.[Abstract/Free Full Text]

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Received 21 May 1999; returned 23 September 1999; revised 29 October 1999; accepted 1 December 1999