The animal experimental procedures were approved by the Institutional Animal Care and Use Committee at the Walter Reed Army Institute of Research (IB02-10). All research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to principles stated in reference 39 . Six-week-old female BALB/c mice (National Cancer Institute, Frederick, MD) were housed at 3 to 4 animals per cage and allowed access to food and water ad libitum throughout the experiment. The pulmonary infection model was adapted from reference 14 (link). Briefly, to promote infection, mice were rendered neutropenic via intraperitoneal administration of 150 mg/kg and 100 mg/kg cyclophosphamide in sterile saline on day −4 and day −1 prior to infection (day 0), respectively.
A. baumannii isolates AB4857, AB5075, AB5711, AB0057, and AB5256 were grown overnight in LB broth with aeration at 37°C, subcultured to mid-exponential phase, washed, and resuspended in PBS with optical density at 600 nm (OD600) values corresponding to 2 × 108 CFU/ml. For infection, mice were anesthetized with oxygenated isoflurane immediately prior to intranasal inoculation with 25 µl of bacterial cultures, corresponding to 5 × 106 CFU. For rifampin experiments, mice were injected IP daily, starting at 4 h postinfection. Animal morbidity was scored twice daily for 6 days using a system evaluating mobility, coat condition, and conjunctivitis as previously described (14 (link)). As mice became exceedingly moribund based on clinical score, they were humanely euthanized according to protocol.
To assess CFU burden in the lungs, mice were humanely euthanized according to protocol on days 2 and 3 postinfection via an injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). To quantify the pulmonary CFU burden, lungs were homogenized in 1 ml PBS, and serial dilutions were plated using the Autoplate spiral plating system (Advanced Instruments, Norwood, MA) onto LB agar supplemented with 50 µg/ml carbenicillin. Bacterial load was reported as CFU per gram of lung tissue.
A. baumannii isolates AB4857, AB5075, AB5711, AB0057, and AB5256 were grown overnight in LB broth with aeration at 37°C, subcultured to mid-exponential phase, washed, and resuspended in PBS with optical density at 600 nm (OD600) values corresponding to 2 × 108 CFU/ml. For infection, mice were anesthetized with oxygenated isoflurane immediately prior to intranasal inoculation with 25 µl of bacterial cultures, corresponding to 5 × 106 CFU. For rifampin experiments, mice were injected IP daily, starting at 4 h postinfection. Animal morbidity was scored twice daily for 6 days using a system evaluating mobility, coat condition, and conjunctivitis as previously described (14 (link)). As mice became exceedingly moribund based on clinical score, they were humanely euthanized according to protocol.
To assess CFU burden in the lungs, mice were humanely euthanized according to protocol on days 2 and 3 postinfection via an injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). To quantify the pulmonary CFU burden, lungs were homogenized in 1 ml PBS, and serial dilutions were plated using the Autoplate spiral plating system (Advanced Instruments, Norwood, MA) onto LB agar supplemented with 50 µg/ml carbenicillin. Bacterial load was reported as CFU per gram of lung tissue.
