Pharmacodynamic activity of fluoroquinolones against ciprofloxacin-resistant Streptococcus pneumoniae

George G. Zhanel,1,3,*, Danielle Roberts1, Andrew Waltky1, Nancy Laing1,3, Kim Nichol1,3, Heather Smith1, Ayman Noreddin1,2, Tracy Bellyou2,3 and Daryl J. Hoban1,3

Department of Medical Microbiology, Faculties of 1Medicine and 2Pharmacy, University of Manitoba; Departments of 3Clinical Microbiology and 4Medicine, Health Sciences Centre, Winnipeg, Manitoba, Canada

Received 15 March 2001; returned 3 July 2001; revised 4 October 2001; accepted 2 February 2002.


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The susceptibility and pharmacodynamic activity of ciprofloxacin and new fluoroquinolones were studied against low-level (MIC 4 mg/L) and high-level (MIC 16 mg/L) ciprofloxacin-resistant Streptococcus pneumoniae. An in vitro pharmacodynamic model simulating free fluoroquinolone (protein unbound) serum concentrations, using Cpmax and AUC0–24 achieved (in healthy volunteers) after standard oral doses that are used for community-acquired respiratory infections, was used to compare bacterial killing by five fluoroquinolones against six ciprofloxacin-resistant S. pneumoniae isolates (four different resistance mutant phenotypes: ParC, efflux, ParC with efflux, and ParC and GyrA) obtained from an ongoing Canadian respiratory organism surveillance study. The potency (MIC only) of fluoroquinolones was gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin > ciprofloxacin. Ciprofloxacin (free AUC0–24/MIC 0.9–3.5) produced no reduction of growth at 6, 24 or 48 h compared with the initial inoculum in all six strains. Levofloxacin (free AUC0–24/MIC 18–35) was bactericidal (>=3 log10 killing) at 6, 24 and 48 h for the ParC as well as the efflux mutants, but only bactericidal at 24 h for the ParC with efflux strain. Levofloxacin (free AUC0–24/MIC 4.4) demonstrated no reduction of growth relative to the initial inoculum against the ParC and GyrA mutants. Gatifloxacin and moxifloxacin (free AUC0–24/MIC 48 and 60, respectively) were bactericidal at 6, 24 and 48 h against the ParC, efflux, and ParC with efflux mutants, but demonstrated little to no growth reduction (free AUC0–24/MIC 6 and 7.5, respectively) in ParC and GyrA mutants. Gemifloxacin (free AUC0–24/MIC 67–133) was bactericidal (>=3 log10 killing) at 6, 24 and 48 h in all low-level ciprofloxacin-resistant S. pneumoniae mutants. Against two of the ParC and GyrA mutants, gemifloxacin (free AUC0–24/MIC 32) was bactericidal at 6, 24 and 48 h but against one ParC and GyrA mutant (free AUC0–24/MIC 16) gemifloxacin demonstrated reduced activity with initial killing at 24 h but with subsequent regrowth. These data indicate that ciprofloxacin produces no inhibition of growth of low- or high-level ciprofloxacin-resistant S. pneumoniae, whereas gatifloxacin, levofloxacin and moxifloxacin (moxifloxacin>gatifloxacin>levofloxacin) were bactericidal for low-level resistant strains but produced little or no inhibition of high-level resistant strains. Gemifloxacin at simulated free AUC0–24/MIC >=32, was bactericidal against low- and high-level resistant strains. When simulated free AUC0–24/ MIC was <16, gemifloxacin allowed regrowth of high-level ciprofloxacin-resistant strains.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ciprofloxacin was the first fluoroquinolone to be used extensively for the treatment of community-acquired respiratory infections.1 In addition to its excellent activity against typical respiratory pathogens, such as Haemophilus influenzae and Moraxella catarrhalis, this fluoroquinolone also demonstrates high penetration into pulmonary tissues and fluids, documented clinical efficacy, extensive safety experience and twice daily dosing (which enhances patient compliance).1 However, ciprofloxacin demonstrates poor potency against Streptococcus pneumoniae, an important pathogen in community-acquired respiratory infections.1,2 New fluoroquinolones with significantly greater activity than ciprofloxacin against S. pneumoniae have recently been developed.15 The enhanced pharmacodynamic potency (relative to ciprofloxacin) of these new fluoroquinolones results from their more potent intrinsic activity against S. pneumoniae (manifested by lower MICs), as well as greater areas under the curve (AUC0–24) due to higher bioavailability.69 These in turn result in greater area under the curve to MIC ratios (AUC/MIC) of the new fluoroquinolones for S. pneumoniae.69 We have recently demonstrated that these greater AUC/MIC ratios of fluoroquinolones for S. pneumoniae result in rapid and extensive bactericidal activity in an in vitro pharmacodynamic model with all new fluoroquinolones, with no re-growth over the 48 h study period.10 Ciprofloxacin, in comparison, demonstrated only a bacteriostatic effect against multidrug-resistant (ciprofloxacin-susceptible) S. pneumoniae, and regrowth occurred during therapy.10

Penicillin-resistant and macrolide-resistant S. pneumoniae have been reported worldwide.3,4,11 These strains are killed effectively with the new fluoroquinolones;1,10 however, ciprofloxacin-resistant (ciprofloxacin MIC >= 4 mg/L) S. pneumoniae have recently been reported.12 The purpose of the present study was to assess the activity of the new fluoroquinolones (gatifloxacin, levofloxacin, gemifloxacin and moxifloxacin) against ciprofloxacin-resistant S. pneumoniae using an in vitro pharmacodynamic model.


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

One ciprofloxacin-susceptible and six ciprofloxacin-resistant S. pneumoniae obtained from an ongoing cross-Canada respiratory organism susceptibility study were investigated for their susceptibility to new fluoroquinolones (Table 1).11 Both low-level (ciprofloxacin MIC 4 mg/L) and high-level (MIC 16 mg/L) ciprofloxacin-resistant S. pneumoniae were stu-died. Isolates represented four different resistant phenotypes (ParC mutant, efflux-only mutant, ParC and efflux mutant, and ParC and GyrA mutant). All strains were of different serotypes and from different regions of Canada.


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Table 1. Susceptibility of S. pneumoniae to fluoroquinolones and comparators
 
For the pharmacodynamic studies, logarithmic phase cul-tures were prepared at a density equivalent to a 0.5 McFarland (1 x 108 cfu/mL) standard by suspending several colonies in cation-supplemented Mueller–Hinton broth with 2.5% lysed horse blood (Oxoid, Nepean, Ontario, Canada). This suspension was diluted 1:100 and 20 µL of the diluted suspension was further diluted in 60 mL of cation-supplemented Mueller–Hinton broth with 2.5% lysed horse blood. Following overnight growth at 37°C, suspensions were further diluted 1:10 and c. 60 mL of the diluted suspension was added to the in vitro pharmacodynamic model. Viable bacterial counts consistently yielded a starting inoculum of c. 1 x 106 cfu/mL.10,13

Antibiotic preparations and susceptibility testing

Antibiotic agents were obtained as laboratory-grade powders from their respective manufacturers (ciprofloxacin and moxifloxacin: Bayer, Mississauga, Ontario, Canada; gatifloxacin: Bristol-Myers Squibb, Montreal, Quebec, Canada; gemifloxacin: GlaxoSmithKline, Toronto, Ontario, Canada; levofloxacin: Janssen Ortho, Ajax, Ontario, Canada); stock solu-tions were prepared and dilutions were made according to the NCCLS M7-A4 method.14 Following two subcultures from frozen stock, MICs of the antimicrobial agents for the isolates were determined by the NCCLS-approved broth microdilution method.14 All MIC assays were carried out in triplicate on separate days.

PCR amplification and DNA sequence analysis

Chromosomal DNA from each S. pneumoniae isolate was obtained by established methods and used as a template for PCR. For target gene amplification, the primers as described previously by Morrissey & George15 were used; PCR conditions consisted of initial incubation at 94°C for 5 min followed by 30 cycles at 94°C for 45 s, 55°C for 30 s and 72°C for 2.5 min, and a final extension at 72°C for 7 min. Amplified gyrA and parC fragments were analysed by agarose gel electrophoresis and purified with Microcon microconcentrators (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. DNA retrieval was verified by gel electrophoresis and the purified products were quantified using a spectrophotometer. DNA sequencing was carried out using an ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). Primers used for sequencing were as described by Morrissey & George.15 Sequencing conditions consisted of 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. Sequences were obtained using an ABI Prism 310 Genetic Analyzer (Applied Bio-systems) and analysed using Sequence Navigator (Applied Biosystems).

Pharmacokinetics of fluoroquinolones in the in vitro pharmacodynamic model

Experiments were performed simulating peak serum con-centrations (Cpmax) and AUCs of ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin and moxifloxacin achieved in human serum after standard oral doses [ciprofloxacin, 500 mg bd; gatifloxacin, 400 mg od; gemifloxacin, 400 mg (320 mg of active agent) od; levofloxacin, 500 mg od; and moxifloxacin, 400 mg od] (Table 2).1,10 Protein-free (unbound) serum concentrations were simulated using known protein binding fractions (ciprofloxacin, 30%; gatifloxacin, 20%; gemifloxa-cin, 60%; levofloxacin, 30%; and moxifloxacin, 50%).1,10 Clearance was simulated using reported serum half-lives (4 h for ciprofloxacin, 7 h for gatifloxacin, gemifloxacin and levofloxacin, and 12 h for moxifloxacin).1,10 The pharmacokinetics of fluoroquinolones were evaluated by dosing these agents using standard doses in the central compartment and sampling from this compartment at 0, 0.5, 1, 2, 4, 8, 12, 12.5, 13, 14, 16, 20 and 24 h. Drug concentrations in each sample were measured by disc diffusion bioassay using a susceptible strain of Bacillus subtilis.10,13 The linear range of the bioassay was 0.1–7 mg/L. The AUC0–24 (mg·h/L) for each fluoroquinolone was calculated using the trapezoidal rule.10,13 The AUC0–24/MIC ratio was calculated for each fluoroquinolone against the specific S. pneumoniae strain studied (Table 3).


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Table 2. Fluoroquinolone pharmacokinetic parameters simulated
 

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Table 3. Fluoroquinolone pharmacodynamic parameters simulated (free AUC0–24/MIC)a
 
In vitro pharmacodynamic model/pharmacodynamic experiments

The in vitro pharmacodynamic model used in this study has been described previously.10,16 The bacterial inoculum at approximately 1 x 106 cfu/mL was introduced into the central compartment (volume 610 mL) of the in vitro pharmacodynamic model and exposed to ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin and moxifloxacin simulating free (protein unbound) serum concentrations obtained after standard dosing (flow rates 0.59–1.77 mL/min). Pharmacodynamic experiments were performed in ambient air at 37°C. At 0, 1, 2, 4, 6, 12, 24, 26, 28, 30, 36 and 48 h, samples were removed from the central compartment and viable bacteria counted by plating 100 µL of serial 10-fold dilutions on to cation-supplemented Mueller–Hinton agar with 2.5% lysed horse blood. Plates were incubated overnight at 37°C in ambient air. The lowest limit of detection was 200 cfu/mL. Antibiotic carryover was prevented by adding 1% (w/v) MgCl2 to the Mueller–Hinton agar supplemented with 2.5% lysed horse blood to samples before plating.10


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The susceptibility patterns of the six ciprofloxacin-resistant S. pneumoniae are displayed in Table 1. Four different phenotypes were chosen, including ParC only, efflux only, ParC with efflux, and ParC and GyrA. These mutants represented both low-level (MIC 4 mg/L) and high-level (MIC 16 mg/L) ciprofloxacin-resistant S. pneumoniae and also demonstrated typical target site changes. The order of fluoroquinolone potency (MIC only) against ciprofloxacin-resistant S. pneumoniae was gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin > ciprofloxacin (Table 1).

The pharmacokinetic profiles of ciprofloxacin, gatifloxa-cin, gemifloxacin, levofloxacin and moxifloxacin in the central compartment of the pharmacodynamic model were within 15% of predicted pharmacokinetic values. For example, 0.5 h after a simulated 500 mg bd dose, the peak ciprofloxacin concentration was 2.2 ± 0.3 mg/L within the central chamber. The corresponding simulated free AUC0–24 was 14 mg·h/L (Table 2). The free AUC ranged from 4 mg·h/L with gemifloxacin to 35 mg·h/L with levofloxacin. The free AUC0–24/MIC ratio ranged from as low as 0.9 for ciprofloxacin to as high as 133 with gemifloxacin (Table 3). Based on free AUC0–24/MIC ratios, the order of potency was also gemifloxacin > moxifloxacin > gatifloxacin > levofloxacin > ciprofloxacin (Table 3).

The pharmacodynamic activity of fluoroquinolones against ciprofloxacin-resistant S. pneumoniae, simulating free serum concentrations, is displayed in Table 4. Ciprofloxacin (free AUC0–24/MIC 0.9–3.5) produced no reduction of growth at 6, 24 or 48 h compared with the initial inoculum against the six ciprofloxacin-resistant strains. Levofloxacin (free AUC0–24/MIC 18–35) was bactericidal (>=3 log10 killing) at 6, 24 and 48 h for the ParC as well as the efflux mutants, but only bac-tericidal at 24 h for the ParC with efflux strain. Levofloxacin (free AUC0–24/MIC 4.4) demonstrated no reduction of growth relative to the initial inoculum against the ParC and GyrA mutants. Gatifloxacin and moxifloxacin (free AUC0–24/MIC of 48 and 60, respectively) were bactericidal at 6, 24 and 48 h against the ParC, efflux, and ParC with efflux mutants, but demonstrated little to no growth reduction (free AUC0–24/MIC of 6 and 7.5, respectively) against ParC and GyrA mutants. Gemifloxacin (free AUC0–24/MIC 67–133) was bac-tericidal (>=3 log10 killing) at 6, 24 and 48 h. Against two of the ParC and GyrA mutants gemifloxacin (free AUC0–24/MIC of 32) was bactericidal at 6, 24 and 48 h but against one ParC and GyrA mutant (free AUC0–24/MIC of 16) gemifloxacin demonstrated reduced activity with initial killing at 24 h but with subsequent regrowth.


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Table 4. Fluoroquinolone killing of S. pneumoniae simulating free serum concentrations: log10 killing at 6, 24 and 48 h, respectivelya
 
The MICs of the various fluoroquinolones studied for S. pneumoniae in the in vitro model did not change during the 48 h period, even for strains where regrowth occurred.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As ciprofloxacin-resistant S. pneumoniae is increasing in Canada and other countries, it is important to assess the pharmacodynamic activity of new fluoroquinolones against this phenotype.12,17 Thus, we used an in vitro pharmacodynamic model to simulate the pharmacokinetic parameters Cpmax and AUC0–24 (in healthy volunteers) of new fluoroquinolones and ciprofloxacin given at standard oral doses for the treatment of community-acquired respiratory infections such as pneumonia (Table 2).1,10 The six ciprofloxacin-resistant S. pneumoniae selected for the present study were chosen because they displayed both low-level and high-level ciprofloxacin resistance and a variety of resistance phenotypes.

Against low-level ciprofloxacin-resistant S. pneumoniae, ciprofloxacin had little to no effect, whereas gatifloxacin, levofloxacin and moxifloxacin were bactericidal (moxifloxacin> gatifloxacin>levofloxacin). Against high-level ciprofloxacin-resistant S. pneumoniae, gatifloxacin, levofloxacin and moxifloxacin (AUC0–24/MIC 4.4–7.5) provided little to no inhibition. Gemifloxacin (when achieving AUC0–24/MIC >=32) was therefore the only fluoroquinolone to eradicate completely both low-level and high-level ciprofloxacin-resistant S. pneumoniae from the model and maintain this effect over 48 h. The lack of killing of high-level ciprofloxacin-resistant S. pneumoniae by ciprofloxacin, gatifloxacin, levofloxacin and moxifloxacin was not surprising as the free AUC0–24/MIC ratio for these agents ranged from 0.9 to 7.5 (Table 3). This is consistent with our previous work and that of other investigators that showed excellent eradication of S. pneumoniae with respiratory fluoroquinolones achieving an AUC0–24/MIC ratio of >=30. 8,10,13,1822


    Acknowledgements
 
This study was supported in part by GlaxoSmithKline.


    Footnotes
 
* Correspondence address. Department of Microbiology, Health Sciences Centre, MS673, 820 Sherbrook Street, Winnipeg, Manitoba R3A 1R9, Canada. Tel: +1-204-787-4902; Fax: +1-204-787-4699; E-mail: ggzhanel{at}pcs.mb.ca Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Zhanel, G. G., Ennis, K., Vercaigne, L., Walkty, A., Embil, J., Gin, A. et al. (2002). A critical review of the new fluoroquinolones: focus on respiratory infections. Drugs 62, 13–59.

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