a Department of Internal Medicine, Inselspital, Freiburgstrasse, 3010 Berne; b Department of Internal Medicine, Zieglerspital, Berne; c Institute for Medical Microbiology, University of Berne, Berne, Switzerland
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Abstract |
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Introduction |
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Among the recently developed drugs, oxazolidinone antibiotics are promising candidates. This new class of antibacterial agents acts by inhibiting protein synthesis.8,9 These substances are active against Gram-positive microorganisms, including penicillin-sensitive and -resistant pneumococci.10,11 The aim of the present work was to study the penetration of linezolid, a new fluorinated oxazolidinone derivative, into inflamed meninges and to test its efficacy against penicillin-sensitive and -resistant pneumococci in experimental meningitis and in vitro. Ceftriaxone monotherapy was used as standard regimen against a penicillin-sensitive isolate and ceftriaxone combined with vancomycin as a standard regimen against a penicillin-resistant strain.
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Materials and methods |
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A penicillin-sensitive and a penicillin-resistant pneumococcal strain were used in experiments in vitro and in the meningitis model. Both strains were originally isolated from patients with pneumonia at the University Hospital of Berne (Switzerland). The MICs against the penicillin-resistant strain serogroup 6 were the following: penicillin G 4 mg/L; vancomycin 0.120.25 mg/L; ceftriaxone 0.5 mg/L; and linezolid 0.5 mg/L. The MICs against the penicillin-sensitive strain were the following: penicillin G 0.06 mg/L; ceftriaxone 0.05 mg/L; and linezolid 0.5 mg/L. MICs were determined in liquid cultures.
Rabbit meningitis model
The meningitis model originally described by Dacey & Sande12 was used in a slightly modified way. The experimental protocol was approved by the Kantonales Amt für Veterinärmedizin, Berne, Switzerland. In brief, young New Zealand white rabbits weighing 22.5 kg (provided by the Zentraltierställe der Medizinischen Fakultät, Berne) were anaesthetized by im 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 1 x 105 cfu of either the penicillinsensitive strain or the penicillin-resistant pneumococcus was injected directly into the cisterna magna. A long-acting anaesthetic (ethylcarbamate or urethane, 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, 1, 2, 4, 5, 6 and 8 h, and a catheter was introduced in the femoral artery for serum sampling at 0, 0.25, 0.5, 1, 2, 4, 4.25, 4.5, 5, 6 and 8 h. Antibiotics were administered through a peripheral ear vein as bolus injections at the following concentrations: ceftriaxone 125 mg/kg; vancomycin 20 mg/kg; linezolid 20 mg/kg. Ceftriaxone was injected at 0 h. Vancomycin and linezolid were administered at 0 and 4 h. Antibiotics were purchased commercially (vancomycin, ceftriaxone) and linezolid was kindly provided by Pharmacia and Upjohn (Kalamazoo, MI, USA). Untreated controls received the same volume of saline. At the end of the experiment all animals received a lethal dose of nembutal.
Bacterial viability was measured by 10-fold serial dilutions of all CSF samples, plated on blood agar plates containing 5% sheep blood and incubated overnight at 37°C. The efficacy of the antimicrobial treatment was determined by decrease of viable cell count over 8 h. In addition, the antimicrobial activity of the regimens was calculated by linear regression analysis and expressed as decrease of log10 cfu/mL/h (log10 cfu/mL/h). In parallel, 20 µL of undiluted CSF was 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 following sterile sample. The results are expressed as mean ± S.D. Statistical significance was determined by the NewmanKeuls and the Tukey multiple comparison tests.
Measurement of antibiotic levels in serum and CSF
Linezolid concentrations in serum and CSF were determined by high-performance liquid chromatography (HPLC) (Drs M.-S. Kuo and R. Zielinsky, Pharmacia and Upjohn). The CSF penetration of linezolid was calculated by comparison of serum and CSF areas under the curve (AUC) for all animals (Systat software, SSPS Inc., Evanston, IL, USA).
In vitro assays
The pneumococcal strains (penicillin-sensitive and -resistant) were grown in C+Y medium13 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. Linezolid was added in concentrations ranging from 0.5 to 5 mg/L corresponding to 1 x, 5 x and 10 x MIC. 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 expressed as mean ± S.D.
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Results |
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Discussion |
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Little is known about the effectiveness of linezolid in pneumococcal meningitis.
In the rabbit meningitis model, we compared linezolid with the standard regimens: ceftriaxone against a penicillin-sensitive pneumococcal strain and ceftriaxone combined with vancomycin against a penicillin-resistant isolate.
Since the dosage of linezolid for the treatment of meningitis has not been clearly defined, we deliberately chose a dosage (2 x 20 mg/kg) higher than levels achieved in humans after one injection of 375 or 625 mg linezolid. Linezolid (20 mg/kg) produced peak levels ranging from 69 to 64 mg/L in rabbits, whereas 375 mg injected intravenously produces peak levels around 11 mg/L in humans. The trough levels (42.5 mg/L) in rabbits were reached after 4 h and corresponded to levels measured in humans after 8 h (2.5 mg/L).14 This provided the rationale for administering a second dose of linezolid after 4 h. The dosages of vancomycin (2 x 20 mg/kg) and ceftriaxone (1 x 125 mg/kg) used in our study were standard dosages used in previous studies with the same experimental model.15
With this dosing regimen, linezolid CSF levels remained above the MIC during the entire treatment period with CSF:MIC ratios ranging from 21 to 3.6.
In light of the good penetration (38 ± 4%) of linezolid into the CSF, the poor antibacterial activity of linezolid compared with the standard regimen (ceftriaxone) against penicillin-sensitive strains is unclear. The lack of efficacy of linezolid can be explained by the marginal bactericidal activity of linezolid documented in vitro, since a highly bactericidal activity is one of the major prerequisites for the treatment of pneumococcal meningitis.
It is interesting to note that linezolid seemed to show a slightly more pronounced activity against a penicillin-resistant strain, but based on our data in vitro (Figures 2 and 3), the reasons for this enhanced efficacy are unclear. On the other hand, the killing rates of linezolid were inferior to the standard regimen (ceftriaxone combined with vancomycin).
In summary, the place of linezolid in the treatment of pneumococcal meningitis is uncertain. On one hand, based on our experimental data, we cannot recommend the use of linezolid against penicillin-sensitive strains, but on the other hand, linezolid could be a conceivable alternative against penicillin-resistant pneumococci. The efficacy of linezolid in the treatment of pneumococcal diseases caused by penicillin-resistant strains deserves further investigation.
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Acknowledgments |
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Notes |
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References |
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2
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4
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Received 1 March 2000; returned 2 June 2000; revised 8 August 2000; accepted 2 September 2000