Journal of Antimicrobial Chemotherapy (2000) 46, 827-830
© 2000 The British Society for Antimicrobial Chemotherapy
Brief report |
In vivo activity of levofloxacin alone or in combination with imipenem or amikacin in a mouse model of Acinetobacter baumannii pneumonia
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
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We evaluated the in vivo activity of levofloxacin alone or in combination with imipenem or amikacin in a mouse model of Acinetobacter baumannii pneumonia using a susceptible strain and one with low-level resistance (MIC/MBC of levofloxacin: 0.06/0.06 and 4/4 mg/L, respectively). As demonstrated previously with other pathogens, the AUC/MIC ratio predicted the efficacy of fluoroquinolones against A. baumannii. This parameter correlated with bactericidal effect and survival. Combination therapy did not enhance the efficacy of levofloxacin.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In patients under mechanical ventilation, the proportion of nosocomial pneumonia cases caused by multiresistant strains of Acinetobacter baumannii is increasing,1 so an experimental model has been developed.2 The aims of the study were to evaluate the in vivo efficacy of levofloxacin alone or in combination against A. baumannii and to determine the major pharmacodynamic parameters correlated with the in vivo efficacy of levofloxacin. Two Acinetobacter isolates with different susceptibilities to this drug were used.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Challenge organisms
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 Mueller–Hinton broth (MHB) (Pasteur Mérieux, Marçy l'Etoile, France). The final inoculum was 106 cfu/mL. MBC endpoints were determined by subculture on to Mueller–Hinton 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 (18–20 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 concentration–time 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 Mueller–Hinton 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.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Pharmacokinetic parameters of levofloxacin, imipenem and amikacin are summarized in Table I
. For levofloxacin, a high peak serum concentration was observed with a good tissue distribution, as demonstrated by lung/serum ratios of 1.7, 1.9 and 2.0 for Cmax, AUC and t > MIC, respectively. These parameters were more favourable for the susceptible strain (MIC 0.06 mg/L) than for the intermediate strain (MIC 4 mg/L). The peak serum concentrations of imipenem and amikacin were very close to those observed in humans, but concentrations in lungs were relatively low.
|
Table II
shows the efficacy of various regimens against the two strains. Against the strain susceptible to levofloxacin, both levofloxacin and imipenem were weakly bactericidal, slightly reducing the lung bacterial counts compared with values at the start of the treatment. Combination of levofloxacin with imipenem or amikacin did not increase bactericidal activity. All the regimens tested significantly reduced mortality compared with controls (P < 0.01). Against Ab-40 (the intermediate strain), only imipenem (alone or combined) had a weak bactericidal effect and significantly reduced mortality compared with controls or with animals treated with levofloxacin (P < 0.01). The bactericidal effect of the levofloxacin–amikacin combination was not superior to that of amikacin alone. No mutants resistant to levofloxacin were isolated after treatment during the various experiments.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Because nearly half of the A. baumannii strains isolated in France are resistant to all available antibiotics except imipenem, new drugs have to be tested. In vitro and animal studies have shown that fluoroquinolones exhibit a concentration-dependent antibacterial activity.5 In our murine model, the pharmacodynamic properties of levofloxacin were studied using two A. baumannii strains of different susceptibility to this drug. In both serum and lungs, a low AUC/MIC ratio of levofloxacin was associated with lack of bactericidal effect and high mortality, as observed in animals challenged with the non-susceptible strain. The t > MIC in serum and lungs was longer in mice challenged with the susceptible strain. In a murine pneumococcal pneumonia model,6 the AUC/MIC ratio for ciprofloxacin or sparfloxacin was the best predictor of survival with a 100% clinical cure when it was
160, irrespective of the method of administration. In a neutropenic rat model of Pseudomonas aeruginosa sepsis,7 the impact of dose fractionation and altered lomefloxacin MICs on survival was examined. In this model, the significance of AUC/MIC varied with the Cmax/MIC ratio: a high Cmax/MIC ratio (10–20) was linked to survival, whereas at lower doses producing Cmax/MIC ratios of <10, the AUC/MIC was closely linked to outcome. Several clinical trials of patients treated with ciprofloxacin for Gram-negative pneumonia also identified the AUC/MIC ratio as the most important pharmacodynamic parameter.8 In our study, AUC/MIC ratios were 347 and 670 in serum and in lungs, respectively, in animals challenged with the susceptible strain but only 5.2 and 10, respectively, in those infected with the intermediate strain. Thus, we have confirmed the importance of the AUC/MIC when a fluoroquinolone is used to treat A. baumannii pneumonia. The poor efficacy of amikacin is disturbing given the good in vitro activity of this compound against A baumannii. Adaptive resistance and a reduced activity in tissue with low pH might explain these results and the absence of synergy between amikacin and either imipenem or levofloxacin.9 One potential benefit of the combination of levofloxacin with amikacin could be the reduction of selection of mutants resistant to levofloxacin during treatment. However, this point has not been addressed in our study.
|
Acknowledgments |
|---|
This work was supported by a grant from Hoechst Marion Roussel, Romainville, France.
|
Notes |
|---|
* Corresponding author. Tel: +33-1-4760-6012; Fax: +33-1-4760-6048.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 . Fagon, J. Y., Chastre, J., Domart, Y., Trouillet, J. L. & Gibert, C. (1996). Mortality due to ventilator-associated pneumonia or colonization with Pseudomonas or Acinetobacter species: assessment by quantitative culture of samples obtained by a protected specimen brush. Clinical Infectious Diseases 23, 538–42.[Web of Science][Medline]
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, 345–51.[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, 1190–7.
4 . Greenblatt, D. J. & Koch-Weser, J. (1975). Clinical pharmacokinetics. New England Journal of Medicine 293, 964–70.[Web of Science][Medline]
5 . Craig, W. A. (1998). Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial dosing of mice and men. Clinical Infectious Disease 26, 1–12.[Web of Science][Medline]
6
.
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, 29–35.
7
.
Drusano, G. L., Johnson, D., Rosen, M. & Standiford, M. C. (1993). Pharamacodynamics of a fluoroquinolone antimicrobial agent in a neutropenic rat model of Pseudomonas sepsis. Antimicrobial Agents and Chemotherapy 37, 483–90.
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, 1073–81.
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, 1155–64.
Received 7 January 2000; returned 18 April 2000; revised 22 May 2000; accepted 11 June 2000
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. A. Russo, J. M. Beanan, R. Olson, U. MacDonald, N. R. Luke, S. R. Gill, and A. A. Campagnari Rat Pneumonia and Soft-Tissue Infection Models for the Study of Acinetobacter baumannii Biology Infect. Immun., August 1, 2008; 76(8): 3577 - 3586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Peleg, H. Seifert, and D. L. Paterson Acinetobacter baumannii: Emergence of a Successful Pathogen Clin. Microbiol. Rev., July 1, 2008; 21(3): 538 - 582. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Perez, A. M. Hujer, K. M. Hujer, B. K. Decker, P. N. Rather, and R. A. Bonomo Global Challenge of Multidrug-Resistant Acinetobacter baumannii Antimicrob. Agents Chemother., October 1, 2007; 51(10): 3471 - 3484. [Full Text] [PDF]
|
|
|
|
 |
|
 R. Jain and L. H Danziger Multidrug-Resistant Acinetobacter Infections: An Emerging Challenge to Clinicians Ann. Pharmacother., September 1, 2004; 38(9): 1449 - 1459. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Montero, J. Ariza, X. Corbella, A. Domenech, C. Cabellos, J. Ayats, F. Tubau, C. Ardanuy, and F. Gudiol Efficacy of Colistin versus {beta}-Lactams, Aminoglycosides, and Rifampin as Monotherapy in a Mouse Model of Pneumonia Caused by Multiresistant Acinetobacter baumannii Antimicrob. Agents Chemother., June 1, 2002; 46(6): 1946 - 1952. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||