FIG 5.
The total population of ofloxacin-tolerant wild-type P. aeruginosa localizes to vacuoles. (A) Images of human corneal epithelial cells containing PAO1F expressing pGFParabinose to show all intracellular bacteria in the presence of ofloxacin (0.25 to 4 μg/mL = 1 to 16× MIC) at 6 and 12 h postinfection versus control. After 3 h extracellular bacteria were killed with amikacin (200 μg/mL) (control), or amikacin (200 μg/mL) and ofloxacin were added to also target intracellular bacteria. Cytosolic PAO1F were cleared by ofloxacin at 1 μg/mL at 6 h and by 0.5 μg/mL at 12 h. Vacuolar bacteria persisted in the epithelial cells at 12 h postinfection at all ofloxacin concentrations. (B and C) Percentage of corneal epithelial cells containing at least one PAO1 with pGFParabinose bacterial cell at 6 h (B) and 12 h (C) post-infection under the above-described conditions. Despite ofloxacin clearance of cytosolic bacteria, the percentage of cells containing intracellular bacteria did not significantly differ between the control and ofloxacin-treated groups (one-way ANOVA with Dunnett’s multiple-comparison test). Data are shown as the mean ± SD of three biological replicates.