FIG 3.

Characterization of tobramycin enhancement of S. constellatus in mixed biofilms with P. aeruginosa. (A) Viability of S. constellatus 7155 grown as biofilms on CF airway cells from assays of single or mixed biofilms of heat-killed P. aeruginosa PA14 or non-heat-killed P. aeruginosa PA14, as indicated, treated with no tobramycin or with 5 μg/ml tobramycin. ****, P < 0.0001, as determined by ANOVA, with Tukey's posttest for multiple comparisons. WT, wild type. (B) P. aeruginosa and S. constellatus colocalize in vitro. Shown is a representative image of a mixed-species biofilm of P. aeruginosa (green, expressing GFP) and S. constellatus 7155 (red, stained with hexidium iodide) on the surface of CF airway cells (gray) at ∼3 h postinoculation with P. aeruginosa and S. constellatus 7155. (C) Representative image of a single-species biofilm of P. aeruginosa (green, expressing GFP) on the surface of CF airway cells (gray) at ∼3 h postinoculation with P. aeruginosa alone. Red staining indicates the nonspecific staining levels of hexidium iodide for CF airway cells. (D) Representative image of S. constellatus 7155 (red, stained with hexidium iodide) on the surface of CF airway cells (gray) at ∼3 h postinoculation with S. constellatus 7155 alone. (E) Tobramycin treatment of P. aeruginosa and S. constellatus 7155 mixed biofilms enhances S. constellatus biofilm formation on plastic. The data represent the viability of bacteria grown as biofilms on plastic from biofilm assays on single or mixed biofilms of P. aeruginosa and S. constellatus 7155 treated with no tobramycin or 5 μg/ml tobramycin. *, P < 0.05; ***, P < 0.001, as determined by the Mann-Whitney test. For panels A and E, the bars and error bars indicate the means and SD of the results from three biological replicates.