a Department of Internal Medicine, Zieglerspital, Berne, b Department of Internal Medicine, Inselspital, Freiburgstrasse, 3010 Berne and c Institute for Medical Microbiology, University of Berne, Berne, Switzerland
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Abstract |
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Introduction |
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However, these antibiotics remain the first-line drugs for most pneumococcal infections, provided that their penetration into infected tissues is sufficient (e.g. in the lung).2 As a result of the limited penetration of penicillin into the subarachnoid space, the treatment of meningitis caused by resistant strains (MIC > 2 mg/L) is more difficult. A combination of high-dose cephalosporins (i.e. cefotaxime or ceftriaxone) and vancomycin is recommended.3
The problem of penicillin-resistant isolates is commonly complicated by the associated resistance to cephalosporins. Cases of treatment failure with cefotaxime and ceftriaxone have already been reported.4,5 Confronted with the limitations of the actual treatment recommendations, new therapeutic options are needed. Promising candidates among recently developed agents are the new quinolones. These substances are active against many Gram-positive microorganisms, including penicillin-resistant pneumococci.68
Grepafloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-5-methyl-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinoline carboxylic acid hydrochloride), a new broad-spectrum quinolone, has excellent activity against penicillin-sensitive and penicillin-resistant pneumococci in vitro9 and has been successfully evaluated in respiratory tract infections.1012 Little is known about the efficacy of grepafloxacin in meningitis caused by penicillin-resistant strains.
In the present study, we tested grepafloxacin alone and in combination with vancomycin against a pneumococcal strain resistant to penicillin (MIC > 2 mg/L) in the rabbit meningitis model and in vitro.
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Materials and methods |
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The meningitis model originally described by Dacey & Sande13 was used, with slight modifications. In brief, young New Zealand white rabbits weighing 22.5 kg were anaesthetized by intramuscular injections of ketamine 30 mg/kg and xylazine 15 mg/kg and were immobilized in stereotactic frames for induction of meningitis and cerebrospinal fluid (CSF) samplings. An inoculum containing 1 x 105 cfu of a penicillin-resistant pneumococcus serotype 6 originally isolated from a patient with pneumonia at the University Hospital of Berne, Switzerland, was injected directly into the cisterna magna. The MICs for this strain were as follows: penicillin G, 4 mg/L; vancomycin, 0.120.25 mg/L; ceftriaxone, 0.5 mg/L; and grepafloxacin, 0.06 mg/L. A long-acting anaesthetic (ethylcarbamate, 3.5 g/rabbit) was injected subcutaneously. The animals were then returned to their cages. Fourteen hours later, the cisterna magna was punctured again for periodic CSF sampling at 0, 0.75, 2.5, 4, 6 and 8 h. Antibiotics were administered through a peripheral ear vein as bolus injections at the following concentrations: grepafloxacin, 15, 30 and 50 mg/kg; vancomycin, 20 mg/kg; ceftriaxone, 125 mg/kg. Grepafloxacin (15 and 30 mg/kg) and ceftriaxone were injected at 0 h. Vancomycin and grepafloxacin (50 mg/kg) were administered at 0 and 4 h. Untreated controls received the same volume of saline.
Bacterial concentrations were measured by 10-fold serial dilutions of CSF samples, plated on blood agar plates containing 5% sheep blood and incubated overnight at 37°C. The antimicrobial activity of the regimens during the 8 h treatment was calculated by linear regression analysis and expressed as the decrease in log10 cfu per millilitre per hour (log10 cfu/mLh). In parallel, 20 µL of undiluted CSF were plated (limit of detectability: 50 cfu/mL). The different dilutions were compared in order to exclude significant carryover effects during therapy. We arbitrarily assigned a value of 1.7 (log10 of the limit of detectability) to the first sterile CSF sample and a value of 0 to any subsequent sterile sample. The results are expressed as mean ± standard deviation. Statistical significance was determined by the NewmanKeuls test.
Measurement of antibiotic concentrations in the CSF
Grepafloxacin concentrations in the CSF were determined by an agar diffusion method using antibiotic medium 11 (Difco Laboratories, Detroit, MI, USA). Standard curve experiments were performed in saline with 5% rabbit serum in order to mimic CSF protein concentrations during meningitis. Bacillus subtilis (ATCC 6633) was used as the test strain.14 The variability between days was <10%. The limit of detection was 0.2 mg/L.
In vitro assays
The pneumococcal strain was grown in C + Y medium15 to an optical density of 0.3 at 590 nm and then diluted 40-fold to 106 cfu/mL, corresponding to the CSF bacterial titre in rabbits before initiation of therapy. Grepafloxacin (MIC 0.06 mg/L) was added to a final concentration of 0.06 0.3 mg/L. The combination of grepafloxacin 0.06 mg/L and vancomycin 0.12 mg/L was also tested. Bacterial titres were determined at 0, 2, 4 and 6 h by serial dilution of samples, plated on agar plates containing 5% sheep blood and incubated at 37°C for 24 h. Experiments were performed in triplicate and results expressed in mean ± standard deviation. Synergy was defined as bactericidal effect of a drug combination significantly exceeding the sum of the bactericidal effects of each agent alone.
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Results |
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Discussion |
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The doses of ceftriaxone (1 x 125 mg/kg) and vancomycin (2 x 20 mg/kg) were standard doses that have been used in previous studies in the same model;22 they correspond to high-dose regimens in humans.3,23,24
Grepafloxacin 15 mg/kg had good bactericidal activity in vivo, comparable to that of vancomycin and ceftriaxone monotherapies.22,25 Higher doses (30 mg or 2 x 50 mg/kg) improved the killing rates only marginally. The reasons for the lack of efficacy of the higher doses are not clear but might be explained by the dose-dependent killing effects of quinolones in the CSF.17 The highest concentration used in this study (2 x 50 mg/kg) produced CSF:MIC ratios far above the dose-dependent killing range.
Addition of vancomycin tended to enhance the killing rate of grepafloxacin, although the differences were not statistically significant (P < 0.05).
To confirm the results obtained in vivo, combinations were also tested in vitro. For the timekill experiments over 8 h, we selected concentrations around the MIC that led to marginal killing rates with monotherapies. In this in vitro setting, addition of vancomycin produced a synergic effect (Figure 5).
The efficacy of this new quinolone in our animal model and its low side effect profile26,27 qualify grepafloxacin as a potential candidate for the treatment of meningitis with resistant strains. On the other hand, the addition of vancomycin did not produce a substantial benefit in vivo.
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Acknowledgments |
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Notes |
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References |
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2
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Received 12 July 1999; returned 2 November 1999; revised 22 December 1999; accepted 23 February 2000