Abstract
Cefodizime, ceftriaxone and cefonicid have similar pharmacokinetic profiles, allowing once daily dosing in humans [l]. The aim of this study was to evaluate the reliability of guinea pigs as an in vivo model of bacterial pulmonary clearance, by assessing the antibacterial activity of cefodizime, cefonicid and ceftriaxone in vitro and in vivo. Thus, after in vih:o testing, we investigated the efficacy of eradication of a clinical isolate of Klebsiella pnertmoniae causing pulmoinary infection in this experimental animal model and the time required for this process. I(. pnelrmotiiae was chosen because it may be responsible for bacterial pneumonia either in hospital or in the community, and there are many reports of its use in experimental animal models. Fifteen K. pneurnoniae strains, identified by API 20E (bioMerieux Italia, Rome, Italy), isolated from respiratory specimens, were tested for susceptibility to the three drugs and the most :sensitive strain (2603) was selected. Cefodzime (Hoechst: Marion Roussel, Lainate, Italy), cefonicid (SmithKlinl: Beecham Pharmaceuticals, Bollate, Italy) and ceftriaxone (Roche, Milan, Italy), as drug substances of stated potency, were used in this study. Killing kinetics were determined for the three antibiotics. An inoculum of 106 CFU/mL from a mid-logarithmic culture was exposed to different antibiotic concentrations (4, 2, 1, 0.5 and 0.25 MIC), and incubated at 3 7 T . At different time points (0, 3, 6 and 24 h), an aliquot from each tube was suitably diluted, spread on Mueller-Hinton agar plates in duplicate and incubated at 37’C for 24 h. Results were expressed as log CFU/mL of the mean. One hundred and fifty female Hartley guinea pigs (Charles River, Calco, Italy), weighing from 220 to 250 g, were used in the experiment. Guinea pigs were divided into five groups of 30 animals each. A positive control group was infected but received no antibiotic, while the negative control group was challenged with sterile phosphate-buffered saline (PBS). The remaining groups were infected and treated with cefodizime, cefonicid and ceftriaxone. The animals were anesthetized with diethyl ether, the trachea was exposed and an inoculum of 100 KL of bacterial suspension (10’ celldanimal) or PBS was administered via a sterile 76gauge needle, using an equal volume of air to push the bacterial solution directly into the lungs [2,3]. The skin incision was closed with surgical staples. The administered antibiotic dose was 15 mg/kg per day. Drug substance or sterile saline solution (for the control group) was administered intraperitoneally via a sterile needle, immediately and after 24 h. At 0, 1, 3, 6, 24 and 48 h after bacterial challenge, guinea pigs were anesthetized with inhaled ðyl ether and killed by cross-clamping. Lungs were harvested in aseptic conditions and homogenized in saline solution using a tissue homogenizer Uanke 81 Kunkel GmbH & Co, Staufen, Germany). Homogenates were placed in ice, and serial 10-fold dilutions was performed. One hundred microliters of each dilution were plated in duplicate on Mueller-Hinton agar plates and incubated for 18 h at 37OC, after which colonies were counted. The mean value obtained from each specimen was transformed in logarithms and expressed as CFU/g of homogenized lung. Sections of lung harvested at the same time points chosen for the bacterial count were perfused with 2 mL of 4% (vol/vol) paraformaldehyde (Sigma, St Louis, MO, USA) in PBS, embedded in paraffin, mounted on glass slides, and stained with hematoxylin and eosin. Histologic specimens were analyzed by light microscopy. Results for each group at each time point were compared by analysis of variance (ANOVA) and considered to be statistically significant when p < 0.05. The selected strain showed MIC values [4] of 0.125 mg/L, 0.062 mg/L and 0.25 mg/L for cefodi-
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