University of Colorado Health Sciences Center, Antimicrobial Research Laboratory, Department of Pharmacy Practice, 4200 E. 9th Ave, Box C238, Denver, CO 80262, USA
Received 29 April 2002; returned 2 July 2002; revised 12 August 2002; accepted 20 August 2002
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Abstract |
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Keywords: Pseudomonas aeruginosa, synergy, fluoroquinolones, cephalosporins
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Introduction |
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We hypothesized that newer fluoroquinolones represent potentially effective agents for the treatment of P. aeruginosa infections when used in combination with potent antipseudomonal ß-lactam agents. Thus, the purpose of this study was to compare the synergic effects of cefepime or ceftazidime in combination with ciprofloxacin, levofloxacin, gatifloxacin or moxifloxacin against clinical isolates of P. aeruginosa using timekill methods. This technique was chosen because previous studies suggested that, compared with disc diffusion or chequerboard titration methods, timekill assays correlate best with cure in animal models.2,3
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Materials and methods |
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The MICs were determined in duplicate by the microbroth dilution method in cation-supplemented MuellerHinton broth (Difco, Sparks, MD, USA) according to NCCLS methods.4 Final antibiotic concentrations ranged from 0.0312 to 32.0 mg/L for cephalosporins and 0.0078 to 8.0 mg/L for fluoroquinolones. MIC microtitre plates were incubated aerobically at 35°C and read at 1824 h. The final bacterial inoculum in each well was 7.5 x 105 cfu/mL. The MIC was defined as the lowest concentration at which there was no visible growth in microtitre wells. Susceptibility testing was again performed on isolates recovered from microtitre plates after completion of the timeconcentration kill curve studies.
Bactericidal activity was determined using timekill experiments according to the NCCLS M26-A method.5 Antimicrobial concentrations tested were 0.5 x MIC as determined previously for each bacterial strain. The final inoculum was determined at time zero; viable counts were performed after 4, 8 and 24 h of incubation at 35°C. All tests were performed in duplicate. If discordant results were obtained, the experiment was repeated and quadruplicate data were used for analysis. The rate and extent of killing were determined by plotting viable colony counts (log10 cfu/mL) against time. The lower limit of detection for timekill assays was 1.3 log10 cfu/mL.
Synergy and antagonism were defined as a 100-fold increase or
100-fold decrease, respectively, in bacterial killing and assessed at 8 and 24 h with the combination of drugs compared with the most active single agent of the combination alone. Additivity (or indifference) was defined as a <10-fold change increase (or decrease) in killing at 8 and 24 h with the combination in comparison with the most active single antimicrobial alone. To examine any differences between antibiotic combinations in a more quantitative manner, areas under the log10 cfu/mLtime curves from time zero to 24 h (AUC024) were calculated by the linear trapezoidal summation method. Lower calculated AUC024 values represent more rapid and/or more complete bacterial killing over the 24 h assays compared with higher AUC024 values.
Statistical analysis
One-way analysis of variance (ANOVA) was used with the Tukey test for post-hoc analysis to compare AUC024 values between groups. A statistical P value of <0.05 was considered significant.
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Results |
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Activities of ceftazidime or cefepime in combination with each of the four fluoroquinolones are summarized in Table 1. Synergic and bactericidal activities were evaluated at both 8 and 24 h to account for any drug inactivation occurring by 24 h. Representative timekill curves of one isolate (P. aeruginosa number 00-14) are presented in Figure 1. All ceftazidime plus fluoroquinolone combinations yielded synergic activity against 80% of strains. Similarly, the combination of cefepime plus ciprofloxacin resulted in synergic activity against 60% of strains tested, while synergic activity was noted against 70% of strains with combinations of cefepime plus levofloxacin, gatifloxacin or moxifloxacin. There were no significant differences in synergic activity between combinations of ceftazidime plus fluoroquinolone or cefepime plus fluoroquinolone when regimens were compared on the basis of calculated AUC024 values (P = 0.83). Timekill results were not substantially different for strains of P. aeruginosa categorized as susceptible to all drugs, cephalosporin susceptible/fluoroquinolone resistant, cephalosporin resistant/fluoroquinolone susceptible or non-susceptible to all drugs tested. Antagonism was not observed with any antimicrobial combination.
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Discussion |
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Although synergy was not apparent when strains were susceptible to both agents in the combination, antibiotic combinations tested in the present study showed synergy in 34 of 37 cases (92%) when strains were resistant to one or both agents. In a previous report, ciprofloxacin plus ceftazidime was synergic in vitro by timekill studies against nine of 12 strains (75%) resistant to one or both agents; ceftazidime MICs for resistant strains in this study were 3264 mg/L and ciprofloxacin MICs were not given.9 This study also noted that synergy was infrequent (<10%) when isolates were susceptible to both agents. In a second report, timekill studies indicated synergy against all three strains resistant to ceftazidime (MIC 64128 mg/L) but susceptible to levofloxacin (MICs 0.52 mg/L) when the drugs were tested in combination.2 The potential for fluoroquinolones to act synergically with ceftazidime or cefepime against resistant isolates may prove advantageous when selecting empirical antibiotic therapy in institutions with high rates of drug resistance among P. aeruginosa.
The present study also demonstrated that combinations of ceftazidime or cefepime plus a fluoroquinolone effectively limited changes in antibiotic MICs and prevented the development of clinical resistance, even following exposure to drugs at sub-MIC levels. This may be attributable to the rapidly bactericidal activity that was observed with these drug combinations. These findings may have clinical relevance in that potentially synergic combinations of agents may help to slow or prevent the emergence of resistance among P. aeruginosa during antibiotic therapy, even among strains already possessing decreased susceptibility to one or both agents in the combination regimen.
Ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin all demonstrated equivalent synergic effects against P. aeruginosa in vitro when tested at concentrations of 0.5 x MIC. However, clinically achievable plasma concentrations at customary dosages may be important limitations to use of certain fluoroquinolones intended to provide synergic effects. Mean peak plasma concentrations of ciprofloxacin 400 mg intravenously (iv) every 8 h and levofloxacin 500750 mg iv every 24 h have been reported to be 5.0 and 8.012.0 mg/L, respectively.10 Thus ciprofloxacin and levofloxacin should be capable of achieving plasma concentrations that are at least 0.5 x MIC for strains with MICs of
8 and
16 mg/L, respectively, one doubling dilution above the NCCLS susceptibility breakpoints. Both ciprofloxacin and levofloxacin would theoretically have been suitable for use against the clinical strains used in this experiment, with some expectation of synergy as part of combination antibiotic regimens, although the weight of previous clinical experience would perhaps favour ciprofloxacin for this use. Gatifloxacin and moxifloxacin both achieve mean peak plasma concentrations of
4.06.0 mg/L during dosing regimens of 400 mg iv every 24 h.10 These drugs could be expected to achieve concentrations of 0.5 x MIC against strains for which the MICs is
8 mg/L (equal to the NCCLS breakpoint) but would not be expected to achieve adequate concentrations against more resistant strains. Thus gatifloxacin and moxifloxacin would have the greatest potential for synergy against intermediately susceptible strains of P. aeruginosa but would not be expected to achieve synergic effects against many more-resistant strains, as may perhaps be observed with ciprofloxacin and levofloxacin.
In conclusion, ciprofloxacin, levofloxacin, gatifloxacin and moxifloxacin were all shown to have synergic activity against clinical isolates of P. aeruginosa when tested in combination with ceftazidime or cefepime. No significant differences were found in rates of synergy between ceftazidime or cefepime, or between the various fluoroquinolones. Synergy was observed particularly when bacterial strains were resistant to one or both agents in the combination tested. Although synergy was demonstrated in vitro for all fluoroquinolones, ciprofloxacin or levofloxacin would be the most suitable for routine empirical clinical use, based on clinically achievable drug concentrations with commonly used dosage regimens.
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Acknowledgements |
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Footnotes |
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References |
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2
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