Figure 6. Limiting penetration of tobramycin protects biofilm cells.

A. Schematic of experiment. Biofilms of PAO1 were treated with 0, 10, 50, or 100 µg/ml tobramycin sulfate for 30 min followed by continuous treatment of 1 µg/ml tobramycin sulfate for 4 h. Biofilms were then stained with propidium iodide (PI) before imaging. Green shading, penetration of tobramycin due to pretreatment; red, area of cell death from 4 h tobramycin treatment. B. Representative images are shown. In the top row, the biomass is pseudo-colored green and PI, red. The bottom row is PI alone in grayscale. Underneath each cross-sectional image is a 4.25x magnified region (inset yellow box). Bar, 50 µm. C. Synergistic killing of tobramycin with MnSO₄ using the MBEC assay. Biofilms were grown on polystyrene pegs and challenged with various combinations of tobramycin and MnSO₄. Viable cell counts were determined by spot dilution plating. Mean log-kill was determined by subtracting the final from the initial log₁₀-transformed cell counts. For untreated control samples, the log₁₀-transformed mean viable cell count was 5.4 ± 0.2 CFU per peg at the end of the experiment. For clarity, only three concentrations of MnSO₄ are shown. Black circles, tobramycin with 0 mM MnSO₄; blue triangles, tobramycin with 0.16 mM MnSO₄; and red squares, tobramycin with 0.63 mM MnSO₄. Error bars represent standard deviation of results from three independent trials.