Figure 9.

Oxyclozanide induces permeabilization and depolarizes the Δψ of P. aeruginosa. Twenty-four hour biofilms were treated with oxyclozanide (100 μM) or tobramycin (500 μM), alone and in combination, for 2 h. (a) Cells were stained with TO-PRO-3 and analysed by flow cytometry to determine the number of cells that were permeabilized. (b) Cells were also stained with DiOC2(3) and analysed by flow cytometry to determine the number of cells maintaining a Δψ. Permeabilized cells were excluded from Δψ analysis. The experiment was performed two separate times in duplicate. The results are percentage averages plus the SEM. Percentage values indicate the average relative abundance of events within each gate normalized to the total number of events analysed, excluding artefacts, aggregates and debris. A one-way ANOVA followed by Dunnett’s multiple comparison post hoc test was used to determine statistical significance between each treatment and the untreated control (*P < 0.05) and oxyclozanide treatment alone was compared with tobramycin treatment alone in panel (a) (NS, not significant). A one-way ANOVA followed by Bonferroni’s multiple comparison post hoc test was used to compare tobramycin to the combination in panel (a) and (b) (*P < 0.05).