Fig 1.
Triclosan in combination with tobramycin results in increased permeabilization of cells within biofilms.
24-hr old biofilms were treated with triclosan (100 μM), or tobramycin (500 μM), alone and in combination for 2-hrs. Cells were stained with the live/dead cell indicator TO-PRO™-3 iodide to determine the number of cells that were permeabilized. The results are percent averages plus the standard deviation (SD, n = 4). Percent values indicate the average relative abundance of events within each gate normalized to the total number of events analyzed, excluding artifacts, aggregates and debris. A one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post-hoc test was used to determine statistical significance between each group and the untreated control and Bonferroni’s post-hoc test was performed to compare tobramycin vs tobramycin and triclosan. *, p<0.05. NS, not significant.
Table 1.
Bacterial strains used in this study.
Fig 2.
Mutants from experimental evolution are resistant to tobramycin and the combination.
24-hr old biofilms were treated with triclosan (100 μM) and tobramycin (500 μM) alone and in combination. The results represent the means plus SD (n = 6). A two-way ANOVA followed by Tukey’s posttest was used to determine statistical significance within each group compared to the untreated control for each strain. p<0.05.
Fig 3.
Triclosan reduces the activity of RND-type efflux pumps.
24-hr biofilms were stained with ethidium bromide to measure accumulation. Biofilms were treated with CCCP (100 μM), triclosan (100 μM), tobramycin (500 μM) alone and in combination. Fluorescence was read at 0,2,4, and 6-hrs. Results represent the average arbitrary fluorescence units ±SD (n = 6).
Fig 4.
Triclosan results in increased cellular accumulation and extrusion of Texas Red conjugated tobramycin (TbTR).
24-hr old biofilms grown in glass test tubes were treated for 30-mins with triclosan (100 μM), CCCP (100 μM) and TbTR (250 μg/mL). (A) Then cells were disrupted from the biofilms and lysed with 0.2% Triton-X 100 to measure intracellular accumulation of TbTR. (B) To measure extrusion, biofilms were first treated for 30-mins and washed three-times in DPBS. Biofilms then recovered in treatment free media for 30-mins and fluorescence of the media was measured. TbTR was measured by relative fluorescence units using excitation 595nm and emission 615nm. Results represent the average arbitrary fluorescence units plus the SD (n = 6). For panel A, a One-Way-ANOVA was performed followed by Tukey’s post-hoc test was used to determine statistical significance between each group compared to TbTR alone. For panel B, an unpaired t-test was performed comparing TbTR vs triclosan with TbTR. *, p<0.05. NS, not significant.
Fig 5.
Triclosan reduces the membrane potential surge induced by tobramycin at 2-hours.
24-hr old biofilms were treated with triclosan (100 μM), or tobramycin (500 μM), alone and in combination for 2-hrs. Cells were stained with DiOC2(3) to measure the membrane potential. Dead or permeabilized cells were excluded from membrane potential analysis by parallel staining with the dead cell indicator TO-PRO™-3 iodide. The results are percent averages plus the SD (n = 4). Percent values indicate the average relative abundance of events within each gate normalized to the total number of events analyzed, excluding dead cells, artifacts, 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, and Bonferroni’s post-hoc test was performed to compare tobramycin vs tobramycin and triclosan. *, p<0.05. NS, not significant.
Fig 6.
Triclosan and tobramycin hydrogels are more effective than tobramycin hydrogels in a murine wound model.
24-hr old bioluminescent biofilms formed within wounds were treated with triclosan (100 μM), or tobramycin (400 μM), alone and in combination for 4-hrs in a hydrogel. Reduction in the number of cells within biofilms was quantified using IVIS. The results are fold reduction of three separate experiments ±SD, no treatment n = 6, triclosan n = 6, tobramycin n = 10, triclosan and tobramycin n = 9. A one-way ANOVA followed by Dunnett’s multiple comparison test was used to determine statistical significance between each treatment and the untreated control and, and Bonferroni’s post-hoc test was performed to compare tobramycin vs tobramycin and triclosan. *, p<0.05.
Fig 7.
Triclosan sensitizes P. aeruginosa to tobramycin by acting as a protonophore, inhibiting efflux pump activity, and abolishing adaptive resistance.
(1) Within 2-hrs of exposure to tobramycin, adaptive resistance occurs, (2) which is due to the induction of RND-type efflux pumps and surge in membrane potential (Δѱ), resulting in reduced accumulation of tobramycin within the cytosol. (3) Triclosan shuttles protons across the inner membrane, collapsing the proton motive force (Δp) and depolarizing the Δѱ. Consequently, efflux pump activity is reduced and there is enhanced accumulation of tobramycin within the cytosol. Finally, tobramycin binds to the A-site of the ribosome, corrupting protein synthesis and causing membrane permeabilization. Overall, triclosan accelerates and increase the effectiveness of tobramycin by reducing the Δѱ and efflux pump activity. Proton gradient, ΔpH. Outer membrane protein (OMP), membrane fusion protein (MFP), inner membrane protein (IMP).