Fig 2. Synergistic anti-biofilm activity of PMZ in combination with tobramycin.

P. aeruginosa static biofilms grown in 96-well plates were treated with increasing concentrations of tobramycin (TOB) with or without the addition of 100 μM promethazine (PMZ). (a) Representative maximum intensity projections of PA14 biofilms (triplicates) treated with TOB alone (upper panel), or in combination with PMZ (bottom panel). The biofilms were stained with the BacLight Bacterial Viability Kit, visualizing dead cells in red (propidium iodide) and living cells in green (Syto9). Isopropanol treatment (IPA) was used as a killing control. Biofilms were assessed in triplicate wells, and at least three independent experiments were performed. (b) To assess the viability of the bacteria upon synergistic treatment, colony forming units (CFU) were determined from the tobramycin treated (dark grey) and tobramycin plus PMZ treated (light grey) biofilms of P. aeruginosa PA14 as well as the PAO1 reference strain. Mean CFU counts were performed on pools of three wells in at least three independent experiments. Error bars represent the standard error of the mean, while the dashed line indicates the lower limit of detection of the assay.