a Service de Microbiologie, Hôpital Louis Mourier, 92701 Colombes; b Clinique de de Réanimation des Maladies Infectieuses; c Service de Pharmacie and e EPI 99 33, Hôpital Bichat-Claude Bernard, 75018 Paris; and d Hoechst Marion Roussel, 92000 Romainville, France
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Abstract |
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Introduction |
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Materials and methods |
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Two clinical isolates of A. baumannii with different susceptibilities to levofloxacin, Ab-40 and Ab-60, were studied; they were isolated from the blood of two patients.
In vitro tests
MICs were determined in MuellerHinton broth (MHB) (Pasteur Mérieux, Marçy l'Etoile, France). The final inoculum was 106 cfu/mL. MBC endpoints were determined by subculture on to MuellerHinton agar (Pasteur Mérieux). Escherichia coli ATCC 25922 was used as a control. For levofloxacin, breakpoints recently proposed by the French Committee on Antibiograms 1999 were used: susceptibility, <4 mg/L; resistance, 4 mg/L.
Mouse model
European guidelines on animal experimentation were followed throughout this study. Transiently neutropenic female C3H/HeN mice (1820 g) (Iffa-Credo Laboratories, L'Arbresle, France) were anaesthetized and infected by intratracheal instillation of 50 µL of a bacterial suspension containing 108 cfu/mL, as described previously.2 Surviving animals were killed on day 5 to avoid unnecessary pain.
Pharmacokinetic parameters in infected mice
A single dose of levofloxacin 100 mg/kg (Hoechst Marion Roussel, Romainville, France) and imipenem 100 mg/kg (Merck Sharp & Dohme, Paris, France) or amikacin 18 mg/kg (Bristol Myers Squibb, Paris, France) was given 3 h after infection. Serum and lung samples were collected 5, 10, 30, 60 and 120 min after injection (three mice per data point). Levofloxacin concentrations were determined by HPLC. The samples were extracted with dichloromethane after adding N-allylpefloxacin as an internal standard. Separation was performed on a Nova-pack C-18 column. The mobile phase was a mixture of methanol and 0.01 M potassium dihydrogen phosphate buffer with 0.025 M heptane sulphonate and 0.02 M triethylamine. Detection was performed by spectrofluorimetry (excitation at 309 nm and emission at 510 nm). Coefficients of variation for within- and between-day precision were 2% and 8%, respectively, and the lower limit of quantification was 0.1 µg/L and 0.1 µg/g for plasma and lung, respectively.
Imipenem and amikacin concentrations were determined using methods described previously.2,3 Pharmacokinetic parameters were evaluated by standard methods;4 maximum concentration observed (Cmax) and elimination half-life were calculated by linear least-squares regression. The inhibitory quotient (IQ) was calculated as Cmax/MIC. t > MIC is the time for which the antibiotic concentration exceeded the MIC in serum or lung. The area under the serum concentrationtime curve (AUC) was calculated by the trapezoidal rule.
Regimens
In vivo bactericidal effect of therapy.
Treatment was initiated 3 h after inoculation. Levofloxacin 100 mg/kg and amikacin 18 mg/kg were administered alone or combined as two ip doses 6 h apart; Imipenem 100 mg/kg was administered alone or combined with levofloxacin as four ip doses at 3 h intervals. The bacterial counts in lungs obtained 3, 6, 9, 12 and 24 h after the first dose were used to determine the maximum bactericidal effect of each regimen; 15 animals were used for each regimen (three animals per data point). Lungs were weighed and then homogenized in 10 mL of saline. Serial 10-fold dilutions of the homogenates were plated on to trypticase soy agar. The lower limit of detection was 102 cfu/g of lung.
Effect of therapy on survival rates.
Treatment was initiated 8 h after inoculation. Regimens were the same as those in the in vivo bactericidal experiments except for imipenem, which was administered as five ip doses (20 animals/ regimen).
Screening for emergence of resistant mutants during treatment
Etest strips of levofloxacin were not available, so an Etest strip of ofloxacin was used on MuellerHinton agar for screening for mutants resistant to levofloxacin (MIC > 4 mg/L); this was done when determining the bacterial count in lungs, 24 h after the first dose of levofloxacin.
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Results |
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Discussion |
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Acknowledgments |
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Notes |
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References |
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2 . Joly-Guillou, M. L., Wolff, M., Pocidalo, J. J., Walker, F. & Carbon, C. (1997). Use of a new mouse model of Acinetobacter baumannii pneumonia to evaluate the postantibiotic effect of imipenem. Antimicrobial Agents and Chemotherapy 41, 34551.[Abstract]
3 . Jolley, M. E., Stroupe, S. D., Wang, C. H., Panas, H. N., Keegan, C. L., Schmidt, R. L. et al. (1981). Fluorescence polarization immunoassay. I. Monitoring aminoglycoside antibiotics in serum and plasma. Clinical Chemotherapy 27, 11907.
4 . Greenblatt, D. J. & Koch-Weser, J. (1975). Clinical pharmacokinetics. New England Journal of Medicine 293, 96470.[ISI][Medline]
5 . Craig, W. A. (1998). Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clinical Infectious Disease 26, 112.[ISI][Medline]
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Bédos, J. P., Azoulay-Dupuis, E., Moine, P., Muffat-Joly, M., Veber, B., Pocidalo, J. J. et al. (1998). Pharmacodynamic activities of ciprofloxacin and sparfloxacin in a murine pneumococcal pneumonia model: relevance for drug efficacy. Journal of Pharmacology and Experimental Therapeutics 286, 2935.
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8 . Forrest, A., Nix, D. E., Ballow, C. H., Goss, T. F., Birmingham, M. C. & Schentag, J. J. (1993). Pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrobial Agents and Chemotherapy 37, 107381.[Abstract]
9 . Barclay, M. L., Begg, E. J., Chambers, S. T., Thornley, P. E., Pattemore, P. K. & Grinwood, K. (1996). Adaptive resistance to tobramycin in Pseudomonas aeruginosa lung infection in cystic fibrosis. Journal of Antimicrobial Chemotherapy 37, 115564.[Abstract]
Received 7 January 2000; returned 18 April 2000; revised 22 May 2000; accepted 11 June 2000