a Department of Internal Medicine, Inselspital, 3010 Bern; b Department of Internal Medicine, Zieglerspital, 3007 Bern; c Institute for Infectious Diseases, University of Bern, 3010 Bern, Switzerland
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Abstract |
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Introduction |
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Cefepime is a broad-spectrum fourth-generation cephalosporin with good activity against a variety of human bacterial pathogens, including penicillin-resistant pneumococci, and has good penetration into the cerebrospinal fluid (CSF).4,5 The aim of this study was to test the bactericidal activity of cefepime against a pneumococcal strain resistant to penicillin and quinolones in the rabbit meningitis model. The standard regimen consisted of ceftriaxone combined with vancomycin.
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Materials and methods |
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The pneumococcal strain (WB4) was originally isolated from a patient with pneumonia at the University Hospital of Bern, Switzerland, and the quinolone-resistant mutant was obtained by sequential exposure of this strain to trovafloxacin in vitro. MICs were as followspenicillin-resistant strain: penicillin 4 mg/L, ceftriaxone 0.5 mg/L, cefepime 0.5 mg/L, vancomycin 0.120.25 mg/L, trovafloxacin 0.12 mg/L, ciprofloxacin 0.5 mg/L; penicillin- and quinolone-resistant mutant strain: penicillin 4 mg/L, ceftriaxone 0.5 mg/L, cefepime 0.5 mg/L, vancomycin 0.120.25 mg/L, trovafloxacin 4 mg/L, ciprofloxacin 32 mg/L.
Rabbit meningitis model
The meningitis model, originally described by Dacey & Sande,6 was slightly modified. The experimental protocol was accepted by the Veterinäramt des Kantons Bern. 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 CSF sampling. An inoculum containing c. 1 x 105 cfu of penicillin- and quinolone-resistant pneumococci serotype 6 was injected directly into the cisterna magna. A long-acting anaesthetic (ethylcarbamate = urethane, 3.5 g/rabbit) was injected subcutaneously and animals were returned to their cages. Fourteen hours later the cisterna magna was punctured again for periodic CSF sampling before and 1, 2, 4, 6 and 8 h after initiation of therapy. Anaesthesia was performed by repetitive intravenous injections of nembutal. Antibiotics were administered through a peripheral ear vein as bolus injections at the following dosages: ceftriaxone 125 mg/kg, vancomycin 20 mg/kg, cefepime 100 mg/kg. Ceftriaxone was injected once at 0 h and vancomycin was injected at 0 and 4 h according to Friedland et al.7 and Cottagnoud et al.8 Cefepime was administered twice (at 0 and 4 h) according to Gerber et al.9 Untreated controls received saline. All antibiotics and anaesthetic drugs were purchased commercially. Bacterial titres 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. In parallel, 20 µL of undiluted CSF samples were plated (limit of detectability, 50 cfu/mL). Comparison between different dilutions of CSF was used to exclude significant carry-over effects during therapy. The antimicrobial activity of the regimens during the 8 h treatment was calculated by linear regression analysis and expressed as decrease of log10 cfu per millilitre per hour (log10 cfu/mLh) and as killing rate over 8 h. A value of 1.7 (log10 of the limit of detection) was assigned to the first sterile CSF sample and a value of 0 to any following sterile sample. The results are expressed as mean ± s.d. Statistical significance was determined by the Newman Keuls test.
Measurement of antibiotic concentrations in the CSF
Antibiotic concentrations in the CSF were determined by the agar diffusion method. Standard curves were performed in saline with 5% rabbit serum in order to mimic CSF protein concentration.10 Bacillus subtilis (ATCC 6633) was used for the assay of cefepime.11 The intra- and inter-day variability of this method was <10%. The limit of detection was 1.5 mg/L for cefepime.
In vitro assays
The pneumococcal strains (a penicillin-resistant strain and a penicillin- and quinolone-resistant mutant) were grown in C+Y medium12 to optical density 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. Ceftriaxone and cefepime were added in concentrations corresponding to 5 x and 10 x MIC (2.5 and 5 mg/L, respectively). The MIC was the same for the two strains. Bacterial titres were determined at 0, 2, 4, 6 and 8 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 are expressed as mean ± s.d.
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Results |
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Discussion |
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Cefepime is a fourth-generation cephalosporin with good penetration into inflamed meninges5 and excellent activity against pneumococci.4 We have demonstrated recently that cefepime is very effective against penicillin-resistant pneumococcal strains in experimental meningitis, mostly due to its excellent penetration into inflamed meninges and its highly bactericidal activity against pneumococci.9 Because of the lack of guidelines in the literature about the treatment of pneumococcal infections due to penicillin- and quinolone-resistant strains, we arbitrarily chose the combination treatment based on ceftriaxone and vancomycin as standard regimen. The dosages of antibiotics (ceftriaxone, cefepime, vancomycin) used in this experimental model were standard ones that have been used in previous studies in the same model,79 corresponding to high dosages in humans.13,14 Cefepime (100 mg/kg) administered twice produced CSF concentrations between 5.3 and 10 mg/L, corresponding to levels described previously.9 The antibiotic concentrations used in vitro (5 mg/L, 10 x MIC) are comparable to the concentrations achieved in the CSF of rabbits during meningitis.
In this experimental meningitis model, cefepime showed a pronounced antibacterial activity in vivo against the penicillin- and quinolone-resistant mutant (-0.61 ± 0.24 log10 cfu/mLh), which is comparable to the standard regimen, despite this strain being killed more slowly by cefepime in vitro, measured by a decreased bactericidal efficacy of c. 3 log10 cfu/mL in time-killing experiments over 8 h (Figure 2
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In a previous study with the same animal model, cefepime produced similar antibacterial activity (-0.60 ± 0.14 log10 cfu/mLh) against the same penicillin-resistant parent strain used in the present study in time-killing experiments in vitro.9
Similarly, the penicillin- and quinolone-resistant mutant was killed more slowly by ceftriaxone in vitro (Figure 3), whereas the bactericidal activity of vancomycin remained unchanged (data not shown). In addition, this strain selected in the presence of trovafloxacin was also killed less rapidly by trovafloxacin and other quinolones tested in the same experimental setting (data not shown). The underlying mechanism of this common feature (diminution of bactericidal activity) between ß-lactam antibiotics and quinolones is not clear, but it seems to be different from the cross-tolerance observed between vancomycin and ß-lactam antibiotics,1517 since the bactericidal activity of vancomycin was maintained in vitro. Based on these preliminary data, it is too early to draw conclusions about the clinical significance of this cross-tolerance between quinolones and ß-lactam antibiotics.
In summary, the good penetration of cefepime into the CSF and its efficacy in our animal model mean that cefepime qualifies for further controlled trials, especially with respect to multiresistant strains, e.g. penicillin- and quinolone-resistant strains.
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Notes |
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References |
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Received 13 June 2001; returned 22 October 2001; accepted 8 November 2001