Quantitative analysis of biofilms and viable bacteria. P. aeruginosa biomass on CFBE cells was quantified with the COMSTAT program, as described in Materials and Methods. For each treatment (for which representative images are shown in Figure 2), six randomly chosen z-series image stacks were analyzed 6 hours after inoculation (i.e., 4 h after starting the drug treatment), and experiments were repeated at least three times. *P < 0.05 versus the untreated control; **P < 0.05 as indicated.

Combined treatment with tobramycin and iron chelators disrupt established P. aeruginosa biofilms. P. aeruginosa was grown on a confluent monolayer of airway cells for 6 hours. Established biofilms were then incubated for another 16 hours (A) in the absence of drug (Control) or were treated with (B) DFO (400 μg/ml), (C) DSX (1 μM), (D) tobramycin (1,000 μg/ml), or to both (E) tobramycin and DFO or (F) tobramycin and DSX at the concentrations indicated above. Images were acquired 22 hours after inoculation (i.e., 16 h after treatment). Differential interference contrast (DIC) images and the corresponding fluorescent images, pseudo-colored green, were merged. In A, B, and C, it is evident that the airway cells were damaged by exposure to the bacteria. However, in D, E, and F the airway monolayer was intact. Experiments were performed in triplicate and representative images are shown. Bar, 20 μm.

Prevention of P. aeruginosa biofilm formation by tobramycin and FDA-approved iron chelators. P. aeruginosa was grown on a confluent monolayer of airway cells for 2 hours in the absence of flow. The flow was then initiated and the indicated compounds were added to the input medium. Biofilms were allowed to develop for another 4 hours, after which images were captured. En face view of GFP-labeled P. aeruginosa PAO1 grown on human bronchial epithelial cells (CFBE41o⁻) homozygous for the ΔF508-CFTR mutation and stably overexpressing ΔF508-CFTR (CFBE cells) (A) in the absence of treatment (Control), after a 4-hour treatment with (B) deferoxamine (DFO) (400 μg/ml), (C) deferasirox (DSX) (1 μM), (D) tobramycin (1,000 μg/ml), or combined treatment with (E) tobramycin and DFO or (F) tobramycin and DSX at the concentrations indicated above. Experiments were performed in triplicate and representative images are shown. Bar, 20 μm.

DSX disrupts established biofilms by chelating iron. Experiments were conducted to determine if DSX reduced P. aeruginosa biomass by chelating iron. Increasing DSX concentration from 0 to 1 μM at constant [Fe, 0.1 μM] reduced biomass (compare first versus second bar). Increasing [Fe] from ∼ 0.1 to 8 μM at constant [DSX, 1 μM] increased biofilm formation (compare second versus third bar). Finally, increasing [DSX] from 1 to 20 μM at constant [Fe, 8 μM] once again decreased biofilm formation (compare third versus fourth bar). In all four experiments Tb was added to the medium at 1,000 μg/ml. Where indicated, ferric chloride (Fe) was added to the medium at a final concentration of 8 μM. Unsupplemented medium contains approximately 0.1 μM Fe (20). DSX was added to the medium at a final concentration of either 0, 1, or 20 μM. Biofilm biomass was quantified 22 hours after inoculation (i.e., 16 h after starting the treatment) using the COMSTAT program, and was compared with the biomass measured at 6 hours after inoculation. *P < 0.05 versus the tobramycin treatment, **P < 0.05; ns, not significantly different.

Tobramycin and conalbumin disrupt established biofilms. Green fluorescent protein (GFP)-labeled Pseudomonas aeruginosa biofilms were grown on cystic fibrosis (CF) human airway epithelial cells in a flow chamber for 6 h before drug treatment. Z-series image stacks were acquired with a 1-μm step, (A) immediately before injection of compounds (Control), 30 minutes after injection of either (B) tobramycin (Tb, 1,000 μg/ml) or (C) conalbumin (ConA, 20 μg/ml), or (D) the combination of tobramycin and conalbumin (Tb + ConA) at the concentrations indicated above. The biofilm biomass remaining after treatment is indicated under each panel as a percent of control (*P < 0.05 compared with control). The three-dimensional images are presented at a slight angle to facilitate viewing. Experiments were performed in triplicate and representative fields are shown. Grid unit, 14.5 μm.

Apical and basolateral application of DSX promotes tobramycin-mediated biofilm killing. (A) CFBE cells were grown on filters at an air–liquid interface and P. aeruginosa was added to the apical side of cells. Drugs were applied to 6-hour-old biofilms and biofilms were exposed to drugs for an additional 16 hours. Tobramycin was added to the apical compartment in all experiments, while DSX (1 μM) was added to the apical (DSX_AP) or to the basolateral (DSX_BL) compartment of airway cells. Bacterial CFU/well in the apical compartment were determined at the end of each treatment (22 h after inoculation). *P < 0.05 versus untreated control, **P < 0.05; ns, not significantly different. (B) The cytotoxic effects of P. aeruginosa on CFBE cells and the effect of tobramycin and DSX on P. aeruginosa–induced cytotoxicity were evaluated by measuring LDH release by airway cells, as described in Materials and Methods. Experiments were performed in triplicate. *P < 0.05 versus control (CTRL). (C) Effect of an extended basolateral application of DSX on P. aeruginosa CFU growing on CFBE cells. Seventy-two hours before inoculation with P. aeruginosa, polarized human airway cells were switched to serum-free medium or serum-free medium supplemented with DSX (800 μM) added to the basolateral side. P. aeruginosa was then added to the apical compartment and biofilms were allowed to develop for 6 hours. Subsequently, tobramycin was added to 6-hour-old biofilms. Biofilms were exposed to tobramycin for a total of 16 hours. Viable bacteria remaining in the apical side of airway cells were determined at the end of each treatment (CFU/well). Experiments were performed in triplicate. *P < 0.05 (tobramycin versus tobramycin plus DSX_BL).

Quantitative analysis of biofilm disruption by tobramycin and iron chelators. (A) Summary of P. aeruginosa biomass after treatments described in Figure 4. Image stacks were captured 22 hours after inoculation (i.e., 16 h after starting the treatment), and experiments were repeated at least three times. Data are expressed as percent of the biomass present at 6 hours after inoculation (i.e., before starting the treatment). Because P. aeruginosa damaged untreated airway cell monolayers and monolayers exposed to DFO or DSX only, biomass data for these conditions could not be determined and thus are not reported. The concentrations of DFO and DSX used in these experiments (400 μg/ml and 1 μM, respectively) chelate the approximately 0.1 μM Fe present in the growth medium. *P < 0.05 versus the tobramycin treatment. (B) Biofilms were grown on plastic microtiter dishes for 24 hours before being treated with tobramycin (1,000 μg/ml), DFO (400 μg/ml), DSX (1 μM), or to both tobramycin and DFO or tobramycin and DSX, at the concentrations indicated above. Treatment was maintained for 6 hours before remaining attached viable bacteria were dislodged from the surface via sonication, and the number of colony-forming units (CFU)/well was determined. *P < 0.05 versus the untreated control; ns, not significantly different.