FIG 5.

Tobramycin and chlorate target distinct populations in aggregate biofilms. (A) Cartoon of the agar block biofilm assay (ABBA), where cells suspended in agar medium grow as aggregate biofilms. At early incubations, aggregates are uniform in size, but oxygen gradients develop over time, both within the aggregate population and within individual aggregates, leading to a metabolically heterogeneous population. After 12 h of growth, aggregates are incubated with drugs for 6 h before they are stained and imaged via confocal microscopy. (B) Representative images of untreated aggregates and those treated with 40 μg/ml tobramycin, 10 mM chlorate, or 40 μg/ml tobramycin plus 10 mM chlorate (combined) are shown at three depths, where cells are stained with SYTO 9 (cyan; live) and propidium iodide (red; dead). The scale bar is 50 μm for all images. (C) Confocal images were used to generate a sensitivity profile for each treatment condition, where the proportion of dead cells (propidium iodide intensity divided by the sum of the propidium iodide and SYTO 9 intensities) was determined at each depth. The dashed arrow highlights a shift in the depth, where 50% of cells are killed by chlorate in chlorate-only samples and compared to combined-treatment samples. Data show the means from 6 independent experiments, and error bars indicate standard errors.