Pulmonary Clearance Model in Mice

The pulmonary clearance model in mice was carried out as previously described [42 (link)]. Briefly, five groups (n = 6) of 6–8-week-old female BALB/C mice were infected intranasally by injecting 40 μl of a bacterial suspension (∼5 × 10⁶ CFU) of each of the WT O35E, ΔfghA, ΔfghA/R, ΔadhC-fghA, and ΔadhC-fghA/R into the nostrils under anesthesia using 250 µL of 25 µg/mL 2,2,2-tribromoethanol. Four-and-a-half-hour post-inoculation, mice were sacrificed by an overdose of the anesthesia (750 µL of 25 µg/mL 2,2,2-tribromoethanol), followed by cervical dislocation. The lungs were excised, homogenized, serially diluted, and plated on TSB agar. Plates were incubated for 48 h followed by colony counting.