FIG 7 .

Treatment of infected CF cells with hyperosmolar media reduces intracellular replication of P. aeruginosa. (a) Bacteria were allowed to internalize into CF cells before the media osmolarity was adjusted as described in the Fig. 5 legend. CFU counts taken at 6 h postinoculation showed a 75% loss in the total number of intracellular bacteria (***, P < 0.001 [ANOVA with Dunnett’s multiple-comparison post hoc analysis]; 3 to 6 independent experiments per condition). (b) Intracellular replication rates between 4 h and 7 h postinoculation in the same assay (gentamicin and hyperosmotic media were added simultaneously at 3 h post-inoculation). Wild-type bacteria replicated 4.6-fold ± 0.6-fold in iso-osmolar media, while replication diminished to 1.8-fold ± 0.3-fold in hyperosmolar media (P < 0.01, Student’s t test). Levels of T3SS mutant bacterial replication were similar in iso-osmolar media (0.99-fold ± 0.16-fold growth) and hyperosmolar media (1.08-fold ± 0.16-fold growth) (P = 0.6, Student’s t test). NS, not significant. Data are presented as means ± SEM. (c) Number of viable (propidium iodide-negative) epithelial cells following treatment with 600 mOsM NaCl or mannitol (P = NS, ANOVA with Dunnett’s multiple-comparison post hoc analysis). Data are expressed as means ± standard deviations of the results from three independent experiments. (d) Effect of hyperosmotic treatments on extracellular bacterial replication.