Eradication of Pseudomonas aeruginosa biofilms on cultured airway cells by a fosfomycin/tobramycin antibiotic combination

Abstract

Chronic biofilm formation by Pseudomonas aeruginosa in cystic fibrosis (CF) lungs is a major cause of morbidity and mortality for patients with CF. To gain insights into effectiveness of novel anti-infective therapies, the inhibitory effects of fosfomycin, tobramycin, and a 4:1 (wt/wt) fosfomycin/tobramycin combination (FTI) on Pseudomonas aeruginosa biofilms grown on cultured human CF-derived airway cells (CFBE41o-) were investigated. In preformed biofilms treated for 16 h with antibiotics, P. aeruginosa CFU per mL were reduced 4 log10 units by both FTI and tobramycin at 256 mg L(-1) , while fosfomycin alone had no effect. Importantly, the FTI treatment contained five times less tobramycin than the tobramycin-alone treatment. Inhibition of initial biofilm formation was achieved at 64 mg L(-1) FTI and 16 mg L(-1) tobramycin. Fosfomycin (1024 mg L(-1)) did not inhibit biofilm formation. Cytotoxicity was also determined by measuring lactate dehydrogenase (LDH). Intriguingly, sub-inhibitory concentrations of FTI (16 mg L(-1)) and tobramycin (4 mg L(-1)) and high concentrations of fosfomycin (1024 mg L(-1)) prevented bacterially mediated airway cell toxicity without a corresponding reduction in CFU. Overall, it was observed that FTI and tobramycin demonstrated comparable activity on biofilm formation and disruption. Decreased administration of tobramycin upon treatment with FTI might lead to a decrease in negative side effects of aminoglycosides.

Fig. 1

Destruction of preformed biofilms. Co-culture biofilms were formed for 7 h. These biofilms were then treated with the indicated antibiotic concentrations for an additional 16 h. Biofilm levels were determined by CFU remaining in the well after treatment. Controls reached approximately 3 × 10⁷ CFU per well before treatment. Tob, tobramycin; Fos, fosfomycin; Input = bacterial level at the start of antibiotic incubation (see text for details). *P < 0.05, compared to Input. Error bars represent standard deviation.

Fig. 2

Inhibition of biofilm formation. Antibiotics were added at the time of inoculation with Pseudomonas aeruginosa. Biofilm levels were determined at 16 h after inoculation. Tob, tobramycin; Fos, fosfomycin. *P < 0.05, compared to lowest antibiotic concentration. Error bars represent standard deviation.

Fig. 3

FTI, tobramycin, and fosfomycin exert little toxic effect on CFBE cells. CFBE cells were incubated with FTI, tobramycin (Tob), or fosfomycin (Fos) for 16 h, after which cytotoxicity was assessed by measuring LDH release. *P < 0.05, compared to lowest antibiotic concentration. Error bars represent standard deviation.

Fig. 4

Cytotoxicity after treatment of preformed biofilms with antibiotics. Conditions correspond to those in Fig. 1. Cytotoxicity was determined by measuring LDH release from the epithelial cells. 100% cytotoxicity was defined by complete lysis of all epithelial cells by 1% Triton-X 100. Tob, tobramycin, Fos, fosfomycin. *P < 0.05, compared to lowest antibiotic concentration. Error bars represent standard deviation.

Fig. 5

Cytotoxicity after biofilm inhibition studies. Conditions correspond to those in Fig. 2. Cytotoxicity was determined by measuring LDH release from the epithelial cells. Tob, tobramycin, Fos, fosfomycin. *P < 0.05, compared to lowest antibiotic concentration. Error bars represent standard deviation.