How to solve the problem of spontaneous bacterial clearance when testing new antibiotic treatment: results on experimental pneumonia due to a derepressed cephalosporinase-producing Enterobacter cloacae.

Because the magnitude of spontaneous bacterial clearance can be similar or even higher than treatment effect, depending upon experimental model and bacterial strain used, this work investigated the value of rendering rats immunosuppressed to facilitate bacterial implantation and reduce spontaneous bacterial clearance. In a first step, rats received a single intravenous cyclophosphamide dose 4 days before infection. Three different doses were tested: 10, 20, and 40 mg/kg. After modeling with NONMEM V, the cyclophosphamide dose required to maintain white blood cell count <1.0 × 10(3)/µL from day 4 to day 5 was 30 mg/kg. In a second step, influence of immunosuppression on lung bacterial titers was characterized. Rats were given one of the three intravenous cyclophosphamide doses (0, 10, 30 mg/kg), and after 4 days, they were infected by tracheal injection of 8.9 ± 0.1 log10 cfu Enterobacter cloacae before being sacrificed at different times. Bacteria in homogenized lungs were quantitatively cultured on Drigalski agar. Bacterial lung count was closely influenced by the grade of induced leukopenia. A single intravenous 30 mg/kg cyclophosphamide dose 4 days before infection suppressed the spontaneous clearance of E. cloacae for at least 30 h without significantly increasing animal mortality; this result seems to be linked to a white blood cell count maintained lower than 1.0 × 10(3)/µL for all the time. This modified animal model could be contributive in the evaluation of antibacterial agents, especially to simulate the behavior of intensive care unit immunocompromised patients.

Animal model methodology: immunocompetent or leucopenic rats, which is the best? Results from a model of experimental pneumonia due to derepressed cephalosporinase-producing Enterobacter cloacae.

BACKGROUND: The aim of this study was to compare the bactericidal activity of cefepime plus amikacin against experimental pneumonia induced by a stably derepressed cephalosporinase-producing Enterobacter cloacae strain in immunocompetent and leucopenic rats. METHODS: Sixty Wistar rats were used. Leucopenia was induced in half of them by a single intravenous administration of 30 mg/kg cyclophosphamide, while the remaining rats received the same volume of saline. All rats were infected 96 h later by tracheal instillation of 8 log(10) colony-forming units of E. cloacae. Twelve rats (6 immunocompetent and 6 leucopenic) were sacrificed 6 h later to assess the initial bacterial burden to the lungs. Then, the remaining 48 rats received a combination of 60 mg/kg cefepime twice a day and 25 mg/kg amikacin once a day given intraperitoneally or the same volume of saline. Six rats per group (leucopenic or not, treated or not) were sacrificed 12 and 30 h after therapy started. RESULTS: Spontaneous bacterial clearance with time was observed only in immunocompetent rats. Compared to untreated animals, antibiotic administration induced a decrease in lung bacterial titres in immunocompetent and leucopenic rats. The difference was statistically significant only in leucopenic rats. CONCLUSIONS: The use of leucopenic rats reduced spontaneous bacterial clearance in the lungs and increased the bactericidal effect of the antibiotic combination and ultimately the confidence in the reliability of the results.