Saturability of Granulocyte Kill of Pseudomonas aeruginosa in a Murine Model of Pneumonia

Abstract

Outcomes for patients with dense bacterial burdens, such as ventilator-associated pneumonia (VAP) patients, are often critically influenced by the adequacy of antimicrobial chemotherapy and by the response of the immune system, particularly the granulocytes. Little information is available about the quantitation of kill of organisms over time by granulocytes. In this investigation, we examined the impact of the baseline bacterial burden on the ability of granulocytes alone (without chemotherapy) to keep the number of organisms in check or to kill them over a 24-h period. Pseudomonas aeruginosa ATCC 27853 was the study organism, and we employed a murine pneumonia model (granulocyte replete) for the study. We found that the ability of the immune system to kill P. aeruginosa was saturable. The burden at which the system was half saturated was 2.15 ؋ 10 6 ؎ 2.66 ؋ 10 6 CFU/g. Burdens greater than 10 7 CFU/g demonstrated net growth over 24 h. These findings suggest the need for aggressive chemotherapy early in the treatment of VAP to keep the burden from saturating the granulocytes. This should optimize the outcome for these seriously infected patients.We have recently demonstrated that the kill of both Pseudomonas aeruginosa and Staphylococcus aureus by granulocytes is saturable in a mouse thigh infection model in which the animals are granulocyte replete (1). As part of that evaluation, we speculated that the same would be true in the setting of pneumonia and that this phenomenon may explain why some antimicrobial agents that are predicted to be suboptimal based on the exposure achieved at the primary infection site still appear to work, particularly in trials where patients are drawn from the community and where the bacterial burden at the initiation of chemotherapy is modest.While we believed it was likely that the same finding would be demonstrable in the setting of pneumonia, the considerable pathological differences between a murine model reflecting mostly a skin or skin structure type of infection and a murine pneumonia model might be large enough that the findings in the pneumonia model might be substantially different.In this evaluation, we examined the impact of the initial bacterial burden on the ability of granulocytes to limit the expansion of the bacterial population over 24 h in a murine model of Pseudomonas pneumonia. MATERIALS AND METHODSMicroorganisms. Pseudomonas aeruginosa ATCC 27853 was employed. Stocks of the organism were stored in 10% glycerol at Ϫ80°C. The isolate was subcultured on blood agar plates twice before each experiment.Animals. Female 24-to 25-g outbred Swiss Webster mice (Taconic Farms, NY) were used in all in vivo studies. They received food and water ad libitum. All animal experimentation procedures were approved by our Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with its guidelines.Animal model. Animals were anesthetized with ketamine-xylazine (100 mg/kg ketamine and 6 mg/kg xylazine) via intraperitoneal (i.p.) injection. Pseudomonas aer...

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