Comparison of the in vitro activities of several new fluoroquinolones against respiratory pathogens and their abilities to select fluoroquinolone resistance

F. J. Boswell, J. M. Andrews, G. Jevons and R. Wise*

Department of Microbiology, City Hospital NHS Trust, Birmingham B18 7QH, UK

Received 18 September 2001; returned 1 March 2002; revised 12 April 2002; accepted 19 June 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study the in vitro activities and pharmacodynamic properties of moxifloxacin, levofloxacin, gatifloxacin and gemifloxacin were compared on recently isolated respiratory pathogens and strains of Streptococcus pneumoniae with known mechanisms of fluoroquinolone resistance. In addition, the resistance selection frequencies of moxifloxacin and levofloxacin on three recently isolated respiratory pathogens and four strains of S. pneumoniae with known mechanisms of fluoroquinolone resistance were investigated. The four fluoroquinolones had similar activities against both Moraxella catarrhalis (MIC90s 0.015–0.06 mg/L) and Haemophilus influenzae (MIC90s 0.008–0.03 mg/L). More marked differences in activity were noted with S. pneumoniae, with MIC90s of 0.25, 1, 0.5 and 0.03 mg/L for moxifloxacin, levofloxacin, gatifloxacin and gemifloxacin, respectively. With the S. pneumoniae strains, the four fluoroquinolones exhibited similar concentration-dependent time–kill kinetics. The resistance selection frequencies of levofloxacin were higher than those of moxifloxacin at concentrations equivalent to those at the end of the dosing interval. Therefore moxifloxacin may have less of an impact on the development of resistance than levofloxacin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Significant levels of antibiotic resistance, particularly to those antibiotics used to treat respiratory tract infections (RTIs) have emerged worldwide. Antimicrobials selected for the treatment of RTIs should ideally possess activity against common and atypical pathogens and have optimal pharmacokinetic and pharmacodynamic parameters that facilitate convenient dosage schedules and good tissue penetration. Consequently, in an attempt to increase quinolone efficacy and reduce adverse effects, hundreds of quinolone/naphthyridone ring analogues have been investigated. Newer agents have been introduced with broadened activity spectra and increased potencies compared with those of the older quinolones. Moxifloxacin, levofloxacin, gatifloxacin and gemifloxacin all have enhanced activity against Gram-positive bacteria whilst maintaining moderate activity against Gram-negative bacteria.14 The in vitro activities of these four newer fluoroquinolones were compared against recently isolated respiratory pathogens and isolates of Streptococcus pneumoniae with known mechanisms of fluoroquinolone resistance.

Both antimicrobial efflux mechanisms and mutations in topoisomerase IV and DNA gyrase lead to quinolone resistance in pneumococci.5,6 Efflux of antimicrobial prevents effective intracellular concentrations from being attained and, although not identified for all quinolones, resistance as a consequence of efflux has been reported for both Gram-positive and Gram-negative bacteria. Strains of S. pneumoniae expressing efflux mechanism resistance demonstrate reduced susceptibility to hydrophilic fluoroquinolones but not to hydrophobic fluoroquinolones.5

Routinely, in vitro antibacterial activities are most often described by determining MICs. However, although MICs are good predictors of antimicrobial potency, they do not reflect bactericidal activity or provide any data on the time-course of antimicrobial activity. The second part of the study compared the pharmacodynamic activities (time–kill kinetics) of the four fluoroquinolones.

The outcome of antimicrobial therapy relies on both the susceptibility of the infecting microorganism to the antimicrobial agent and the availability of the antibiotic at the site of infection. Therefore, adequate penetration of the antibiotic into potential sites of infection in the respiratory tract is important for therapeutic efficacy. For moxifloxacin, the concentrations in the epithelial lining fluid (ELF) and bronchial mucosa determined 2.2 h post-dose were 20.7 mg/L and 5.4 mg/kg,7 respectively, whereas those for levofloxacin 2 h post-dose were 9 mg/L and 6.5 mg/kg, respectively.8 In comparison, the concentrations in the ELF and bronchial mucosa determined at 24 h post-dose were 3.6 mg/L and 1.1 mg/kg for moxifloxacin,7 and less than the lower limit of detection of 0.06 mg/L for levofloxacin.8 In the third part of the current study the selection frequencies of fluoroquinolone-resistant mutants were compared for recently isolated respiratory pathogens and strains of S. pneumoniae with known mechanisms of quinolone resistance, at concentrations of moxifloxacin and levofloxacin equivalent to those in the ELF and bronchial mucosa towards the end of the dosing interval (using approximately 24 h post-dose levels). In addition, the fluoroquinolone resistance selection frequencies of a second-step high-dose exposure (using approximately 2 h post-dose levels) after a prior low-level exposure (to mimic in vivo dosing) were compared with single-step selection frequencies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains subjected to antibiotic susceptibility testing

A total of 279 recent, random, non-replicate, clinical isolates was studied (Table 1), consisting of 96 Moraxella catarrhalis and 95 Haemophilus influenzae, including ß-lactamase-producing strains, and 88 S. pneumoniae, including strains with different penicillin susceptibilities isolated routinely from various locations at City Hospital NHS Trust (Birmingham, UK). In addition, 24 S. pneumoniae clinical isolates shown previously to demonstrate efflux-mediated resistance were studied (efflux phenotype defined as a four-fold or greater reduction in norfloxacxin MIC in the presence of reserpine and an ethidium bromide MIC of >8 mg/L).5 Seven laboratory-generated mutants of S. pneumoniae were also evaluated. The laboratory mutants consisted of four parC mutants (2C2 with amino acid change Ser-79->Tyr in ParC,6 1N1, 1G11, 1G139), two parC gyrA mutants (3C6 with amino acid changes Ser-79->Tyr in ParC and Glu-87->Lys in GyrA, 3C7 with amino acid changes Ser-79->Tyr in ParC and Ser-83->Tyr in GyrA)6 and one parC gyrA parE mutant (amino acid changes Ser-79->Phe in ParC, Ser-83->Tyr in GyrA and Ile-460->Val in ParE). The type cultures used were H. influenzae ATCC 49247 and NCTC 11931 and S. pneumoniae ATCC 49619.


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Table 1.  In vitro activities (MIC, mg/L) of four fluoroquinolones against H. influenzae, M. catarrhalis and S. pneumoniae
 
Bacterial strains subjected to time–kill kinetic analysis and fluoroquinolone resistance selection analysis

One recent clinical isolate each of M. catarrhalis (ß-lactamase positive), H. influenzae and S. pneumoniae was evaluated, together with a laboratory-generated efflux mutant (1N27),10 and parC (1N1), gyrA (1B6 with amino acid change Ser-83->Tyr in GyrA) and parC gyrA (2B5 with amino acid changes Asp-83->Tyr in ParC and Ser-83->Tyr in GyrA) mutants selected from S. pneumoniae ATCC 49619.9 Strains used were a representative selection of resistant phenotypes. The clinical isolates had been evaluated in the susceptibility study.

Antimicrobial agents

The antimicrobial agents investigated were obtained from the following sources: moxifloxacin (Bayer, West Haven, CT, USA), levofloxacin (Aventis, Uxbridge, UK), gatifloxacin (Grünenthal, Aachen, Germany) and gemifloxacin (SmithKline Beecham, Worthing, UK). All antimicrobial agents were prepared and stored throughout these investigations following the manufacturers’ guidelines.

Fluoroquinolone susceptibility testing

The fluoroquinolone susceptibilities of clinical isolates of H. influenzae, M. catarrhalis and S. pneumoniae and laboratory-generated mutants of S. pneumoniae were determined by a standard agar dilution method.11 Briefly, IsoSensitest agar (Oxoid, Basingstoke, UK) was employed, supplemented with 5% defibrinated horse blood (Tissue Culture Services, Botolph Claydon, UK) and nicotinamide adenine dinucleotide (NAD) 20 mg/L (Sigma Chemicals, Poole, UK) for haemophili. All strains were tested at an inoculum of 104 cfu using a multipoint inoculator (Mast, Bootle, UK). Plates were incubated at 35–37°C in an atmosphere enriched with 4–6% CO2 (except M. catarrhalis, which were incubated in air) for 18–24 h. The MIC of the antimicrobial agent was defined as the lowest concentration inhibiting growth (two or three colonies were ignored).

Time–kill kinetics of fluoroquinolones for H. influenzae, M. catarrhalis and S. pneumoniae

Fluoroquinolone concentrations equivalent to 2 x, 4 x and 8 x MIC were added to logarithmic phase IsoSensitest broth cultures (20 mL; non-agitated; supplemented as necessary) of ~106 cfu/mL. Control cultures with no fluoroquinolone exposure were included for all strains investigated. Viable counts were determined (three 20 µL replicates) at 0, 2, 4, 6 and 24 h after the addition of the fluoroquinolone on Columbia agar (Oxoid; supplemented as necessary) following appropriate serial dilution of the culture in phosphate-buffered saline (PBS) pH 7.3 (Oxoid).12 The bacteria were enumerated after 48 h incubation at 35–37°C in an atmosphere enriched with 4–6% CO2 and the time–kill kinetics were plotted as log10 cfu/mL against time. The lower limit of detection was 2.70 log10 cfu/mL. Since bactericidal activity was defined as a 3.0 log10 decrease in cfu/mL (99.9% kill),13 we defined bacteriostatic activity as <99.9% kill.

Selection of fluoroquinolone-resistant mutants of H. influenzae, M. catarrhalis and S. pneumoniae

Single-step selection experiments to recover antibiotic (fluoroquinolone)-resistant mutants were performed in triplicate by inoculating 103–1013 cfu of the test strain on to Columbia agar (supplemented as necessary) containing moxifloxacin at 3.6 mg/L or 1.1 mg/L, or levofloxacin at 0.06 mg/L. The inoculum was prepared by harvesting the overnight growth on Columbia agar (supplemented as necessary) in a standard Petri dish into a minimal volume (2 mL) of Todd–Hewitt broth (Oxoid). Viable cell count was determined. Following incubation of selection plates for 72 h at 35–37°C in an atmosphere enriched with 4–6% CO2, the mutation frequencies were calculated as the proportion of the initial inoculum growing at the selective concentration of antibiotic.

Two-step selection experiments were performed (as above) by inoculating a mutant organism from each parent selected during the single-step selection experiments on to Columbia agar (supplemented as necessary) containing moxifloxacin at 20.7 mg/L or 5.4 mg/L7 or levofloxacin at 9.0 mg/L or 6.5 mg/L,8 equivalent to the 2.2 h and 2 h post-dose concentrations of moxifloxacin and levofloxacin, respectively, in ELF and bronchial mucosa.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibilities of H. influenzae, M. catarrhalis and S. pneumoniae to various fluoroquinolones

Against H. influenzae and M. catarrhalis, the activities of the four fluoroquinolones were similar and their MIC90s were within two dilution steps of each other (Table 1). For both bacterial species, the MIC90s of gemifloxacin and gatifloxacin were marginally less than those of levofloxacin and moxifloxacin. Differences in the activities of the fluoroquinolones were more noticeable against S. pneumoniae.

S. pneumoniae strains with parC mutations demonstrated MIC90s of gemifloxacin and levofloxacin four- and two-fold higher than those for the wild type strains (Table 1), respectively. The MIC90s of moxifloxacin and gatifloxacin were the same for S. pneumoniae strains with parC mutations and for wild-type strains. The two S. pneumoniae strains with parC gyrA mutations were found to have higher MICs of gemifloxacin, levofloxacin, moxifloxacin and gatifloxacin (16- and >64-fold, equal and 64-fold, 16- and 32-fold and 16- and 32-fold higher) than the MIC90s for wild-type strains. The S. pneumoniae strain with parC gyrA parE mutations demonstrated MICs of gemifloxacin, levofloxacin, moxifloxacin and gatifloxacin 32-, 64-, 32- and 32-fold higher than the MIC90s for the wild-type strains. S. pneumoniae strains with efflux mutations demonstrated MIC90s of gemifloxacin, levofloxacin and gatifloxacin that were four-, two- and two-fold higher than those for the wild-type strains. The MIC90 of moxifloxacin was the same for S. pneumoniae strains with mutations conferring an efflux phenotype and for wild-type strains.

The results for all the type cultures were equal to or within one dilution step of the expected MICs of the antimicrobial agents, where this information was known.11 The MICs for the organisms used for further studies are in Table 2.


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Table 2.  MICs (mg/L) of fluoroquinolones for microorganisms used in mutant selection and time–kill kinetics
 
Fluoroquinolone time–kill kinetics for H. influenzae, M. catarrhalis and S. pneumoniae

All four fluoroquinolones exhibited similar concentration-dependent time–kill kinetics at equipotent (x MIC) concentrations for each S. pneumoniae strain studied (Table 3). A bactericidal effect was seen with most S. pneumoniae strains after 6 h with fluoroquinolone concentrations equivalent to 4 x and 8 x MIC. Against H. influenzae, moxifloxacin was the most rapidly bactericidal fluoroquinolone and gemifloxacin the least. Indeed, at the lower concentrations tested (2 x and 4 x MIC) gemifloxacin was bacteriostatic. However, a bactericidal effect was seen after 6 h with the four fluoroquinolones at concentrations equivalent to 8 x MIC. Against M. catarrhalis, a bactericidal effect was observed with moxifloxacin, levofloxacin and gatifloxacin after 6 h at 8 x MIC; no bactericidal effect was observed with gemifloxacin.


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Table 3.  Bactericidal rates (change in log10 cfu/mL) of moxifloxacin, levofloxacin, gatifloxacin and gemifloxacin at concentrations equivalent to 2 x, 4 x or 8 x MIC for H. influenzae, M. catarrhalis and S. pneumoniae
 
Culture regrowth was observed occasionally (two S. pneumoniae strains with all four fluoroquinolones) at 24 h, and the colonies obtained were macroscopically identical to the controls. Further MIC testing to determine whether these cultures were fluoroquinolone resistant was not performed. The clinical importance of regrowth is unclear, particularly if it occurs after the usual dosing interval.13 Antibiotic carryover was not deemed to be a problem in our determinations. Problems may arise at concentrations >16 x MIC.13 In our experiments, serial dilution was employed, which minimizes residual antimicrobial effect. Furthermore, only a small volume (20 µL) was cultured to determine viable count.

Fluoroquinolone resistance selection frequencies

Fluoroquinolone resistance selection frequencies varied considerably with inoculum size and the selective concentration of the antibiotic (Table 4). Generally, the fluoroquinolone resistance selection frequencies recorded were lower for the higher moxifloxacin concentration (3.6 mg/L) than for the lower concentration (1.1 mg/L). In addition, the higher the multiple of the MIC used in the selection process, the lower the fluoroquinolone resistance selection frequency. High levofloxacin resistance selection frequencies were observed because the selective concentration was <0.1 x MIC for S. pneumoniae strains. The resistance selection frequencies were lower for H. influenzae and M. catarrhalis, consistent with the selective concentration of levofloxacin being 2 x to 4 x MIC.


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Table 4.  Single-step selection frequencies of moxifloxacin- and levofloxacin-resistant mutants of H. influenzae, M. catarrhalis and S. pneumoniae
 
Six strains (five S. pneumoniae and one M. catarrhalis) selected with levofloxacin and five strains (four S. pneumoniae and one M. catarrhalis) selected with moxifloxacin were used in a two-step fluoroquinolone resistance selection frequency determination (Table 5). The results from these experiments revealed that in the case of S. pneumoniae, the strains selected with moxifloxacin had higher MICs of both moxifloxacin and levofloxacin than those for the parent strains (Table 5). In the cases of those selected from the strain with just the gyrA mutation and that with parC gyrA mutations, the moxifloxacin resistance selection frequencies were higher than those for the parents. In some instances no organism growth was observed at the higher moxifloxacin concentrations.


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Table 5.  Two-step selection frequencies of moxifloxacin- and levofloxacin-resistant mutants of H. influenzae, M. catarrhalis and S. pneumoniae
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The four fluoroquinolones exhibited similar activities (MICs) to each other against the M. catarrhalis and H. influenzae strains studied; differences in activity were more pronounced against S. pneumoniae (Table 1). S. pneumoniae strains with parC, gyrA and mutations conferring an efflux phenotype demonstrated either a small increase (two to four-fold) or no increase in the MIC of the fluoroquinolones tested. These results correlate with current knowledge that parC and gyrA mutations and increased expression of the multi-drug efflux pump confer low-level quinolone resistance in pneumococci.5 However, the MIC of moxifloxacin was the same for S. pneumoniae wild-type strains and those with efflux pump mutations. These differences in the effect on MIC90, depending on the fluoroquinolone, may reflect the substrate specificity of the efflux pump or the rate of accumulation of different fluoroquinolones in the cell.14 Structural differences between the fluoroquinolones, notably overall molecular hydrophobicity and bulkiness of the C-7 substituent, are thought to influence the efficiency of efflux.15 In addition, it has been shown that the C-7 substituent determines the target preference of the fluoroquinolones.16 The primary targets of moxifloxacin and levofloxacin are GyrA and ParC, respectively.17 It is apparent from these data that the effectiveness of all fluoroquinolones is not equally affected by particular resistance mechanisms.

In general, moxifloxacin, levofloxacin, gatifloxacin and gemifloxacin exhibited the pharmacodynamic properties expected of fluoroquinolones, that is, concentration-dependent bactericidal activity.18 All four fluoroquinolones exhibited similar time–kill kinetics for each S. pneumoniae strain studied, in that all were bactericidal after 6 h at concentrations equivalent to 4 x and 8 x MIC. Against H. influenzae, moxifloxacin was the most rapidly bactericidal fluoroquinolone and gemifloxacin the least. Against M. catarrhalis, bactericidal activity was observed with moxifloxacin, levofloxacin and gatifloxacin only after 6 h at 8 x MIC; no bactericidal effect was observed with gemifloxacin. These observations concur with those of Wright and coworkers19 who concluded that fluoroquinolone pharmacodynamics are both quinolone and microorganism specific. The differences might be explained by the findings of Morrissey & George,20 who found that the bactericidal activity of fluoroquinolones did not correlate with their ability to inhibit topoisomerase IV, suggesting that inhibition of another target, such as DNA gyrase, may be responsible.

Because of low spontaneous mutation frequencies to fluoroquinolone of 10–9–10–10,21 the emergence of fluoroquinolone-resistant microorganisms during therapy is low. We observed that the resistance selection frequencies of moxifloxacin and levofloxacin ranged from 10–8 to 10–14 at concentrations >= 8 x MIC. The apparent high resistance selection frequencies observed with moxifloxacin and levofloxacin at concentrations below the MIC concur with those from previous in vitro studies.22 However, difficulties can arise when reporting apparently spontaneous mutational frequencies determined at sub-MIC selective concentrations, because some of the colonies that appear to be resistant may be the parent strain. Petersen23 has stated that resistance is more likely to emerge in a single mutational step when the organism that is exposed to a fluoroquinolone is only margin- ally susceptible. Our findings support this view, in that moxifloxacin-resistant mutants were more readily selected from parent strains with gyrA and parC gyrA mutations than from the wild-type parental strains.

There are three main aspects that relate to the ecological impact of a fluoroquinolone (in terms of its promoting or minimizing resistance): pharmacokinetics, potency and dissociated resistance.24 Fluoroquinolones that are highly potent are likely to prevent resistance emerging by killing both the parental organism and its less susceptible single-step mutants. Therefore, resistant respiratory pathogens are more likely to emerge as a result of exposure to levofloxacin than exposure to moxifloxacin, as levofloxacin is less potent and exhibits greater selective pressure at the end of the dose interval than moxifloxacin. Consistent with this, Firsov and coworkers25 have stated that to achieve equiefficient doses, levofloxacin requires greater dosing levels than moxifloxacin. Furthermore, Lubenko and coworkers26 observed that moxifloxacin was more efficient than levofloxacin at preventing the selection of resistant mutants with Staphylococcus aureus.


    Acknowledgements
 
We are grateful to N. Brenwald for his critical reading of this manuscript. We thank L. M. Fisher for supplying S. pneumoniae strains 2C2, 3C6 and 3C7. We would like to thank G. Tillotson and A. Dalhoff of Bayer for their financial support and advice.


    Footnotes
 
* Corresponding author. Tel: +44-121-507-4255; Fax: +44-121-551-7763; E-mail: r.wise{at}bham.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Woodcock, J. M., Andrews, J. M., Boswell, F. J., Brenwald, N. P. & Wise, R. (1997). In vitro activity of Bay 12-8039, a new fluoroquinolone. Antimicrobial Agents and Chemotherapy 41, 101–6.[Abstract]

2 . Child, J., Andrews, J., Boswell, F., Brenwald, N. & Wise, R. (1995). The in-vitro activity of CP 99,219, a new naphthyridone antimicrobial agent: a comparison with fluoroquinolone agents. Journal of Antimicrobial Chemotherapy 35, 869–76.[Abstract]

3 . Wise, R., Brenwald, N. P., Andrews, J. M. & Boswell, F. (1997). The activity of the methylpiperazinyl fluoroquinolone CG 5501: a comparison with other fluoroquinolones. Journal of Antimicrobial Chemotherapy 39, 447–52.[Abstract]

4 . Wise, R. & Andrews, J. M. (1999). The in-vitro activity and tentative breakpoint of gemifloxacin, a new fluoroquinolone. Journal of Antimicrobial Chemotherapy 44, 679–88.[Abstract/Free Full Text]

5 . Gill, M. J., Brenwald, N. P. & Wise, R. (1999). Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 43, 187–9.[Abstract/Free Full Text]

6 . Pan, X.-S., Ambler, J., Mehtar, S. & Fisher, L. M. (1996). Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 2321–6.[Abstract]

7 . Soman, A., Honeybourne, D., Andrews, J., Jevons, G. & Wise, R. (1999). Concentrations of moxifloxacin in serum and pulmonary compartments following a single 400 mg oral dose in patients undergoing fibre-optic bronchoscopy. Journal of Antimicrobial Chemotherapy 44, 835–8.[Abstract/Free Full Text]

8 . Andrews, J. M., Honeybourne, D., Jevons, G., Brenwald, N. P., Cunningham, B. & Wise, R. (1997). Concentrations of levofloxacin (HR 355) in the respiratory tract following a single oral dose in patients undergoing fibre-optic bronchoscopy. Journal of Antimicrobial Chemotherapy 40, 573–7.[Abstract]

9 . Brenwald, N. P., Gill, M. J. & Wise, R. (1999). Grepafloxacin vs. pneumococci resistant to fluoroquinolones by a putative efflux mechanism. Drugs 58, Suppl. 2, 117–8.[ISI]

10 . Brenwald, N. P., Gill, M. J. & Wise, R. (1998). Prevalence of a putative efflux mechanism among fluoroquinolone-resistant clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 2032–5.[Abstract/Free Full Text]

11 . British Society for Antimicrobial Chemotherapy Working Party Report. (2001). Antimicrobial susceptibility testing. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–28.[Abstract/Free Full Text]

12 . Miles, A. A., Misra, S. S. & Irwin, J. O. (1938). The estimation of the bactericidal power of the blood. Journal of Hygiene 38, 732–49.

13 . National Committee for Clinical Laboratory Standards. (1992). Methods for Determining Bactericidal Activity of Antimicrobial Agents: Tentative Guideline M26-T. NCCLS, Villanova, PA, USA.

14 . Gill, M. J. & Wise, R. (1998). Activity of grepafloxacin against pneumococci resistant to fluoroquinolones by a putative efflux mechanism. In Abstracts of the Thirty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, 1998. Abstract C-52, p. 83. American Society for Microbiology, Washington, DC, USA.

15 . Beyer, R., Pestova, E., Millichap, J. J., Stosor, V., Noskin, G. A. & Peterson, L. R. (2000). A convenient assay for estimating 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]

16 . Alovero, F. L., Pan, X.-S., Morris, J. E., Manzo, R. H. & Fisher, L. M. (2000). Engineering the specificity of antibacterial fluoroquinolones: Benzenesulfonamide modifications at C-7 of ciprofloxacin change its primary target in Streptococcus pneumoniae from topoisomerase IV to gyrase. Antimicrobial Agents and Chemotherapy 44, 320–5.[Abstract/Free Full Text]

17 . Pestova, E., Millichap, J. J., Noskin, G. A. & Peterson, L. R. (2000). Intracellular targets of moxifloxacin: a comparison with other fluoroquinolones. Journal of Antimicrobial Chemotherapy 45, 583–90.[Abstract/Free Full Text]

18 . Boswell, F. J., Andrews, J. M. & Wise, R. (1997). Pharmacodynamic properties of BAY 12-8039 on Gram-positive and Gram-negative organisms as demonstrated by studies of time–kill kinetics and postantibiotic effect. Antimicrobial Agents and Chemotherapy 41, 1377–9.[Abstract]

19 . Wright, D. H., Brown, G. H., Peterson, M. L. & Rotschafer, J. C. (2000). Application of fluoroquinolone pharmacodynamics. Journal of Antimicrobial Chemotherapy 46, 669–83.[Abstract/Free Full Text]

20 . Morrissey, I. & George, J. T. (2000). Purification of pneumococcal type II topoisomerases and inhibition by gemifloxacin and other quinolones. Journal of Antimicrobial Chemotherapy 45, Suppl. S1, 101–6.[Abstract/Free Full Text]

21 . Eliopoulos, G. M. & Eliopoulos, C. T. (1993). Activity in vitro of the quinolones. In Quinolone Antimicrobial Agents, 2nd edn (Hooper, D. C. & Wolfson, J. S., Eds), pp. 161–93. American Society for Microbiology, Washington, DC, USA.

22 . Fung-Tomc, J., Kolek, B. & Bonner, D. P. (1993). Ciprofloxacin-induced, low-level resistance to structurally unrelated antibiotics in Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 37, 1289–96.[Abstract]

23 . Petersen, L. R. (1993). Quinolone resistance in clinical practice: occurrence and importance. In Quinolone Anrimicrobial Agents, 2nd edn (Hooper, D. C. & Wolfson, J. S., Eds), pp. 119–37. American Society for Microbiology, Washington, DC, USA.

24 . Thomson, K. S. (2000). Minimizing quinolone resistance; are the new agents more or less likely to cause resistance? Journal of Antimicrobial Chemotherapy 45, 719–23.[Free Full Text]

25 . Firsov, A. A., Lubenko, I. Y., Vostrov, S. N., Kononenko, O. V., Zinner, S. H. & Portnoy, Y. A. (2000). Comparative pharmacodynamics of moxifloxacin and levofloxacin in an in vitro dynamic model: prediction of the equivalent AUC/MIC breakpoints and equiefficient doses. Journal of Antimicrobial Chemotherapy 46, 725–32.[Abstract/Free Full Text]

26 . Lubenko, I., Vostrov, S., Portnoy, Y., Zinner, S. & Firsov, A. (2001). Preventing the production of resistant mutants of Staphylococcus aureus with moxifloxacin and levofloxacin: dose ranging studies using an in vitro dynamic model. Clinical Microbiology and Infection 7, Suppl. 1, 166.