Research Article

A Model of Intracellular Persistence of Pseudomonas aeruginosa in Airway Epithelial Cells

Figure 4

Intracellular infection kinetics and cytotoxicity of CF clinical isolates. (a) Intracellular bacterial burden, (b) relative bacterial survival, and (c) cytotoxicity over time following infection with PAO1 or clinical isolates CI180, CI455, CI565, or CI581. (d) Detection and 3D localization of intracellular P.a. (white arrowhead) by confocal microscopy, with evidence of clustered intracellular bacteria in AEC infected with CI180 at 24 h p.i. In AEC infected with CI180, (e) the percentage of AEC harboring intracellular P.a. signals among all AEC and (f) the percentage of AEC harboring a given number of bacteria per AEC at different time points (among all AEC with intracellular P.a.). For all experiments, BEAS-2B cells were infected with at MOI 1, except for uninfected (UI) controls. The intracellular bacterial burden was measured by viable CFU count (detection limit shown as green dashed line), and the relative bacterial survival was calculated as the intracellular bacterial burden at the indicate time normalized to the 4 h counts. The cytotoxicity was measured by LDH release assay. Confocal microscopy images were analyzed manually. Results in (a), (b), and (c) are from pooled data (≥ 6 biological replicates) from ≥3 independent experiments. Results in (b) are depicted as . For (d), images representative of 3 independent experiments are shown, with . For (e) and (f), results are from pooled data (≥ 8 fields of view per condition) from 3 independent experiments. ; ; .
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