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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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 manufacturers 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 0.17 mg/L. The AUC024 (mg·h/L) for each fluoroquinolone was calculated using the trapezoidal rule.10,13 The AUC024/MIC ratio was calculated for each fluoroquinolone against the specific S. pneumoniae strain studied (Table 3).
|
|
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 0.591.77 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 MuellerHinton 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 MuellerHinton agar supplemented with 2.5% lysed horse blood to samples before plating.10
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 AUC024 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 AUC024/MIC ratio ranged from as low as 0.9 for ciprofloxacin to as high as 133 with gemifloxacin (Table 3). Based on free AUC024/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 AUC024/MIC 0.93.5) produced no reduction of growth at 6, 24 or 48 h compared with the initial inoculum against the six ciprofloxacin-resistant strains. Levofloxacin (free AUC024/MIC 1835) 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 AUC024/MIC 4.4) demonstrated no reduction of growth relative to the initial inoculum against the ParC and GyrA mutants. Gatifloxacin and moxifloxacin (free AUC024/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 AUC024/MIC of 6 and 7.5, respectively) against ParC and GyrA mutants. Gemifloxacin (free AUC024/MIC 67133) was bac-tericidal (
3 log10 killing) at 6, 24 and 48 h. Against two of the ParC and GyrA mutants gemifloxacin (free AUC024/MIC of 32) was bactericidal at 6, 24 and 48 h but against one ParC and GyrA mutant (free AUC024/MIC of 16) gemifloxacin demonstrated reduced activity with initial killing at 24 h but with subsequent regrowth.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 (AUC024/MIC 4.47.5) provided little to no inhibition. Gemifloxacin (when achieving AUC024/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 AUC024/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 AUC024/MIC ratio of
30. 8,10,13,1822
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Bush, K. & Goldschmidt, R. (2000). Effectiveness of fluoroquinolones against gram-positive infections. Current Opinion in Investigational Drugs 1, 2230.[Medline]
3 . Doern, G. V., Pfaller, M. A., Kugler, K., Freeman, J. & Jones, R. N. (1998). Prevalence of antimicrobial resistance among respiratory tract isolates of Streptococcus pneumoniae in North America: 1997 results from the SENTRY antimicrobial surveillance program. Clinical Infectious Diseases 27, 76470.[ISI][Medline]
4 . Sahm, D. F., Jones, M. E., Hickey, M. L., Diakum, D. R., Mani, S. V. & Thornsberry, C. (2000). Resistance surveillance of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis isolated in Asia and Europe, 19971998. Journal of Antimicrobial Chemotherapy 45, 45766.
5 . Hooper, D. C. (2000). New uses for new and old quinolones and the challenges of resistance. Clinical Infectious Diseases 30, 24354.[ISI][Medline]
6 . Turnidge, J. (1999). Pharmacokinetics and pharmacodynamics of fluoroquinolones. Drugs 58, Suppl. 2, 2936.
7 . Pickerill, K. E., Paladino, J. A. & Schentag, J. J. (2000). Comparison of the fluoroquinolones based on pharmacokinetic and pharmacodynamic parameters. Pharmacotherapy 20, 41728.[ISI][Medline]
8
.
Wright, D. H., Brown, G. H., Peterson, M. L. & Rotschafer, J. C. (2000). Application of fluoroquinolone pharmacodynamics. Journal of Antimicrobial Chemotherapy 46, 66983.
9
.
McGowan, A., Rogers, C. & Bowker, K. E. (2000). The use of in vitro pharmacodynamic models of infection to optimize fluoroquinolone dosing regimens. Journal of Antimicrobial Chemotherapy 46, 16370.
10
.
Zhanel, G. G., Karlowsky, J. A., Walters, M. & Hoban, D. J. (2001). In vitro pharmacodynamic modelling simulating free serum concentrations of fluoroquinolones against multi-drug resistant Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 47, 43540.
11
.
Zhanel, G. G., Karlowsky, J. A., Palatnick, L., Low, D. E., the Canadian Respiratory Organism Susceptibility Study Group & Hoban, D. J. (1999). Prevalence of antibiotic resistance in respiratory tract isolates of Streptococcus pneumoniae: Results of a Canadian national study. Antimicrobial Agents and Chemotherapy 43, 25049.
12
.
Chen, D. K., McGeer, A., deAzavedo, J. C. & Low, D. E. (1999). Decreased susceptibility of Streptococcus pneumoniae to fluoroquinolones in Canada. Canadian Bacterial Surveillance Network. New England Journal of Medicine 341, 2339.
13
.
Lister, P. D. & Sanders, C. C. (1999). Pharmacodynamics of levofloxacin and ciprofloxacin against Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 43, 7986.
14 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.
15
.
Morrissey, I. & George, J. (1999). Activities of fluoroquinolones against Streptococccus pneumoniae type II topoisomerases purified as recombinant proteins. Antimicrobial Agents and Chemotherapy 43, 257985.
16
.
Zelenitsky, S. A., Booker, B., Laing, N., Karlowsky, J. A., Hoban, D. J. & Zhanel, G. G. (1999). Synergy of an investigational glycopeptide LY333328 with once daily gentamicin against vancomycin-resistant Enterococcus faecium in a multiple-dose in vitro pharmacodynamic model. Antimicrobial Agents and Chemotherapy 43, 5927.
17
.
Linares, J., de la Campa, A. G., Pallares, R., Peterson, D. E., Sahm, D. F., Chen, D. K. et al. (1999). Fluoroquinolone resistance in Streptococcus pneumoniae. New England Journal of Medicine 341, 15467.
18
.
McGowan, A., Bowker, K. E., Wootton, M. & Holt, H. A. (1999). Activity of moxifloxacin administered once a day against Streptococcus pneumoniae in an in vitro pharmacodynamic model of infection. Antimicrobial Agents and Chemotherapy 43, 15604.
19 . Garrison, M. W., Vance-Bryan, K., Larson, T. A., Toscano, J. P. & Rotschafer, J. C. (1990). Assessment of effects of protein on daptomycin and vancomycin killing of Staphylococcus aureus by using an in vitro pharmacodynamic model. Antimicrobial Agents and Chemotherapy 34, 192531.[ISI][Medline]
20
.
Lacy, M. K., Lu, W., Xu, X., Tessier, P. R., Nicolau, D. P., Quintiliani, R. et al. (1999). Pharmacodynamic comparisons of levofloxacin, ciprofloxacin and ampicillin against Streptococcus pneumoniae in an in vitro model of infection. Antimicrobial Agents and Chemotherapy 43, 6727.
21
.
Shah, P. M. & Schwarzel, R. (1999). Bactericidal activity of levofloxacin against Streptococcus pneumoniae in an in vitro model simulating serum pharmacokinetic parameters. Journal of Antimicrobial Chemotherapy 43, Suppl. C, 715.
22 . Bauernfeind, A. (1999). Pharmacodynamics of levofloxacin and ofloxacin against Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 43, Suppl. C, 7782.[Medline]