Adherence of A. baumannii to A549 Cells

Adherence of A. baumannii strains to A549 cells (human type 2 pneumocytes) was determined as described previously [4 (link)], with minor modifications. In brief, ~ 10⁵ A549 cells were infected with ~ 10⁷ bacterial CFU and incubated for 60 min at 37 °C in 5 % CO2 (v/v) atmosphere. Non-adherent bacterial cells were removed by washing with PBS. Infected cells were lysed by the addition of 1 ml distilled water and serial 10-fold dilutions were plated on LB agar to determine the number of CFU of adherent bacteria. To determine adherent and invading bacteria, A549 cells were infected with A. baumannii strains as described above. The monolayers were then treated with 1 ml of fresh culture medium containing 5 mg/L of colistin sulfate (Sigma-Aldrich, Milan, Italy) for further 30 min, the shortest time point that resulted in the killing of all extracellular bacteria added to the monolayers. Afterwards, the cells were washed with PBS, harvested with trypsin, and lysed with sterile distilled water. Dilutions from harvested samples were inoculated on LB agar plates and bacterial colony counts were estimated after overnight incubation at 37 °C. Each experiment was performed in triplicate.