Iron chelators selleck kinase inhibitor and/or tobramycin were subsequently added to the medium. After 16 hours of drug treatment, biofilm biomass was determined and compared with the biomass at T = 6 hours (immediately before addition of drugs). In the absence of any added drugs, P. aeruginosa caused extensive damage to CFBE cells and most airway cells detached from the glass coverslip (Figure 4A). Bacteria subsequently colonized those empty regions on the glass substratum and formed a flat biofilm on this abiotic substratum (Figure 4A). Neither DFO nor DSX alone prevented P. aeruginosa from damaging CFBE cells (Figures 4B and 4C). However, in the presence of DFO or DSX, P. aeruginosa did not form multicellular, biofilm-like aggregates, but rather they grew primarily as individual bacteria attached to the glass substratum (Figure 4B, DFO) or as a lawn of individual bacteria (Figure 4C, DSX).

The inability of P. aeruginosa to form biofilm-like aggregates in the presence of iron chelators is consistent with previous studies by Singh and colleagues (16). Figure 4. 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 … As opposed to the iron chelators alone, treatment of established biofilms with tobramycin protected the CFBE cells from being destroyed by P. aeruginosa (compare Figures 4A�C4C with Figure 4D).

This finding is consistent with our previous report showing that tobramycin reduces the virulence of biofilm bacteria both by reducing bacterial numbers and by altering the expression of virulence factors (38). However, despite the fact that the CFBE monolayer remained intact, P. aeruginosa biomass after a 16-hour exposure to tobramycin increased to approximately 140% of its pre-treatment size (Figure 5A). This finding is consistent with our previous work showing that P. aeruginosa biofilms grown on airway cells are highly resistant to tobramycin (20). Figure 5. 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), …

In contrast to tobramycin or iron chelators alone, the combination of tobramycin and either DFO or DSX significantly reduced the biomass of established biofilms by 90% of the pre-treatment value (Figures 4E, 4F, and and5A).5A). Thus, a combination of tobramycin and an iron chelator disrupted established, highly drug-resistant biofilms on human airway epithelial cells and, furthermore, prevented P. aeruginosa from Anacetrapib damaging the monolayer of CFBE cells, even after exposure to bacteria for 24 hours.