a Department of Internal Medicine, Zieglerspital, 3007 Berne; b Institute of Medical Microbiology, University of Berne; c Department of Internal Medicine, Inselspital, 3010 Berne, Switzerland
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Abstract |
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Introduction |
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Materials and methods |
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The experimental protocol was approved by the Veterinäramt des Kantons Bern. Young New Zealand White rabbits weighing 22.5 kg 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 collection of samples of cerebrospinal fluid (CSF). An inoculum containing 1 x 105 cfu of penicillin-resistant pneumococci, serotype 6, was injected directly into the cisterna magna. The pneumococcal strain had originally been isolated from a patient with pneumonia at the University Hospital of Berne, Switzerland. The MICs/MBCs (mg/L) were as follows: penicillin, 4/4; ceftriaxone, 0.5/0.5; cefepime, 0.5/0.5; vancomycin, 0.120.25/0.25; and gatifloxacin, 0.12/0.25. A long-acting anaesthetic, ethylcarbamate (also called 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 collection of CSF samples before and 1, 2, 4, 6 and 8 h after initiation of therapy. A catheter was introduced into a femoral artery for collection of serum samples. Antibiotics were administered through a peripheral ear vein as bolus injections at the following doses: gatifloxacin, 15 mg/kg; ceftriaxone, 125 mg/kg; vancomycin, 20 mg/kg; cefepime, 100 mg/kg. Ceftriaxone and gatifloxacin were injected once at 0 h and vancomycin and cefepime at 0 and 4 h according to Gerber et al.5 Untreated controls received saline. Cefepime, ceftriaxone and vancomycin were purchased commercially and gatifloxacin was kindly provided by Grünenthal GmbH, Germany.
Bacterial titres were measured by 10-fold serial dilutions of CSF samples, plated on tryptic soy agar plates (Difco; Becton Dickinson, Sparks, MD, USA) containing 5% sheep blood and incubated overnight at 37°C. In parallel, 20 µL of undiluted CSF samples was plated (limit of detection: 50 cfu/mL). Comparison between different dilutions of CSF was used to exclude any significant effects of carry-over during therapy. The antimicrobial activity of the regimens during the 8 h treatment was determined by measuring the decrease in viable cell count. This was calculated by linear regression analysis and expressed as the change () in log10 cfu/mL/h. A value of 1.7 (log10 of the limit of detectability) was assigned to the first sterile CSF sample and a value of 0 to any subsequent sterile sample. The results are expressed as mean ± S.D. Statistical significance was determined by the NewmanKeuls test.
Measurement of antibiotic concentrations in CSF and serum
Antibiotic concentrations in the CSF were determined by an agar diffusion method. Standard curve experiments were performed in saline with 5% rabbit serum in order to mimic CSF protein concentration.6 Escherichia coli ATCC 25922 in Antibiotic Medium 1 (Difco) was used as test strain for cefepime. Bacillus subtilis ATCC 6633 was used in Antibiotic Medium 1 (Difco) for vancomycin and in Antibiotic Medium 11 (Difco) for gatifloxacin. The intra- and interday variability of this method was <10%. The limit of detection was 0.5 mg/L for vancomycin, 1 mg/L for ceftriaxone and 0.25 mg/L for gatifloxacin.
In vitro assays
The pneumococcal strain was grown in conditioned medium with yeast extract (Difco; C+Y medium)7 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. Gatifloxacin was added in concentrations corresponding to 1 x, 5 x and 10 x MIC (MIC = 0.12 mg/L). Combinations of cefepime (at 0.5 mg/L, which is equivalent to the MIC) with gatifloxacin (0.12 mg/L) were also tested. 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; results are expressed as mean ± S.D. A drug combination was said to show synergy when the bactericidal effect of the drug combination was significantly greater than the sum of the bactericidal effects of each agent alone.
Determination of fractional inhibitory concentration (FIC) index
The same isolate used in the timekill experiments and in the animal model was grown in C+Y medium until logarithmic growth phase and then back-diluted. Suspensions of bacteria (containing 0.51 x 106 cfu) were pipetted into microtitre trays containing the same medium and concentrations of each antibiotic that ranged from 1/32 x MIC to 2 x MIC. Microtitre trays were incubated at 37°C for 24 h. After 6, 12 and 24 h, inhibition of bacterial growth on the plates was assessed. Experiments were performed in triplicate. FIC indices were calculated according to the method of Eliopoulos & Moellering.8 Synergy was defined as an FIC index of 0.5, indifference as an FIC of >0.5 to
4 and antagonism as an FIC of >4.
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Results |
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Discussion |
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Gatifloxacin 15 mg/kg showed greater bactericidal activity than trovafloxacin 15 mg/kg and grepafloxacin 30 mg/kg, two quinolones previously tested in the same model.11,12
Based on the good penetration of gatifloxacin into the CSF (49%) and its efficacy in our animal model and in vitro, alone or in combination with cefepime, gatifloxacin 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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Kaplan, S. L. & Mason, E. O. (1998). Management of infections due to antibiotic-resistant Streptococcus pneumoniae. Clinical Microbiology Reviews 11, 62844.
3
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Hoellman, D. B., Lin, G., Jacobs, M. R. & Appelbaum, P. C. (1999). Anti-pneumococcal activity of gatifloxacin compared with other quinolone and non-quinolone agents. Journal of Antimicrobial Chemotherapy 43, 6459.
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Cottagnoud, P., Acosta, F., Cottagnoud, M., Neftel, K. & Täuber, M. G. (2000). Synergy between trovafloxacin and ceftriaxone against penicillin-resistant pneumococci in the rabbit meningitis model and in vitro. Antimicrobial Agents and Chemotherapy.44, 217981.
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Gerber, C. M., Cottagnoud, M., Neftel, K., Täuber M. G. & Cottagnoud, P. (2000). Evaluation of cefepime alone and in combination with vancomycin against penicillin-resistant pneumococci in the rabbit meningitis model and in vitro. Journal of Antimicrobial Chemotherapy 45, 638.
6 . Nau, R., Kaye, K., Sachdeva, M., Sande, E. R. & Täuber, M. G. (1994). Rifampin for therapy of experimental pneumococcal meningitis in rabbits. Antimicrobial Agents and Chemotherapy 38, 11869.[Abstract]
7 . Lacks, S. & Hotchkiss, R. D. (1960). A study of the genetic material determining an enzyme in pneumococcus. Biochimica et Biophysica Acta 39, 50818.[ISI]
8 . Eliopoulos, G. M. & Moellering, R. C. (1996). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, 4th edn, (Lorian, V., Ed.), pp. 33096. Williams & Wilkins, Baltimore, MD.
9 . LaCreta, F. P., Sanjeev, K., Kollia, G. D., Duncan, G. F., Randall, D. M. & Grasela, D. M. (1999). Bioequivalence of 400 mg intravenous and oral gatifloxacin in healthy adult subjects. In Program and Abstracts of the Thirty-Ninth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 1999. Abstract 192, p. 10. American Society for Microbiology, Washington, DC.
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Lutsar, I., Friedland, I. R., Wubbel, L., McCoig, C. C., Jafri, H. S., Ng, W. et al. (1998). Pharmacodynamics of gatifloxacin in cerebrospinal fluid in experimental cephalosporin-resistant pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 42, 26505.
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Gerber, C. M., Tovar, L., Cottagnoud, M., Neftel, K., Täuber, M. G. & Cottagnoud, P. (2000). Grepafloxacin against penicillinresistant pneumococci in the rabbit meningitis model. Journal of Antimicrobial Chemotherapy 46, 24953.
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Rodoni, D., Hänni, F., Gerber, C. M., Cottagnoud, M., Neftel, K., Täuber, M. G. et al. (1999). Trovafloxacin in combination with vancomycin against penicillin-resistant pneumococci in the rabbit meningitis model. Antimicrobial Agents and Chemotherapy 43, 9635.
Received 19 July 2000; returned 30 September 2000; revised 26 October 2000; accepted 20 November 2000