Klebsiella pneumoniae (Schroeter) Trevisan from ATCC catalog # 43816 (Manassas, VA), was stored in 1 ml aliquots at −80°C in trypticase soy broth obtained from Remel catalog # R112731 (Lenexa, KS) with 10% glycerol. For each experiment, the frozen culture was thawed and grown in 100 ml fresh trypticase soy broth at 37°C for 18 hours with a 150 rpm shaker, a process which allowed bacteria to reach log-phase growth. Bacteria were then centrifuged at 4500 rpm (3645 xg) to create pellet and washed twice with sterile phosphate-buffered saline (PBS). The bacteria were then re-suspended in a total of 10 ml of PBS, which made a concentration of 2 × 1010 colony-forming units (CFU) per ml. This value was determined by performing serial dilutions. This stock solution was used to make a final concentration of 105 CFU/ml for inoculation, and a total volume of 100 μl was inoculated in the airway of each rat using the method described below. The bacteria were plated on MacConkey agar (Remel catalog # R01550) to confirm their presence and concentration.
