Bacterial preparation

PA 762 strain was grown for 14 hours on a tryptic soy agar plate (BD Biosciences, Franklin Lakes, NJ, USA) and transferred to an Erlenmeyer flask containing 40 ml of tryptic soy broth (BD Biosciences). Bacteria were incubated for 60 min at 37°C and 125 rpm to allow bacteria to be in the logarithmic phase of growth. Bacteria were centrifuged at 2800 rpm for 10 min at 21°C. The bacteria were then resuspended in pre-warmed 40 ml of PBS (Thermo Fischer Scientific; Waltham, MA, USA). An O.D. of 0.04 was obtained using a spectrophotometer and the concentration of the bacteria was determined based on a standard absorption curve. Mice were infected with 10⁶ CFU/20 μl of bacteria. Prior to the inoculation, mice were anesthetized with 3% isoflurane in oxygen. Using a 31-gauge needle on a 1 ml syringe, each mouse was intranasally inoculated with 20 μl of PA.