In vitro approach to study the synergistic effects of tobramycin and clarithromycin against Pseudomonas aeruginosa biofilms using prokaryotic or eukaryotic culture media

Abstract

Recurrent Pseudomonas aeruginosa infections involving biofilm formation are frequent in cystic fibrosis, aggravating the respiratory distress. Co-administration of clarithromycin and classical tobramycin could improve the health status of patients. Antibiotic toxicity was assessed on epithelial (CFBE41o−) and macrophagic (THP-1) cell lines. Non-toxic concentrations of antibiotics alone or in combination were applied twice daily for 12 days on mature (12-day-old) biofilms of three P. aeruginosa strains, developed either in prokaryotic culture broth [tryptic soy broth (TSB)] or in a eukaryotic cell culture medium (RPMI-FCS) more similar to an in vivo environment. The antibiofilm and bactericidal effects of antibiotics were assessed. No toxicity of tobramycin was observed on eukaryotic cell lines at concentrations up to 500μg/mL, whilst 100μg/mL was selected as the clarithromycin upper safe limit. The amount of biofilm was strongly reduced by 100μg/mL and 500μg/mL tobramycin for each strain in both media, whilst clarithromycin was only effective in RPMI-FBS, with synergistic (PAO1 strain) and additive (PYO2 strain) effects detected when combining tobramycin 4μg/mL and clarithromycin 100μg/mL. Finally, tobramycin at ≥100μg/mL exerted strong bactericidal effects on each strain in both media. Clarithromycin also exerted bactericidal effects on each strain in both media; its effect was weaker than tobramycin in TSB but was similar in RPMI-FBS. Synergistic effects were observed on PAO1 and MUCO biofilms, e.g. when combining tobramycin 4μg/mL and clarithromycin 100μg/mL. These in vitro data show that co-administration of clarithromycin and tobramycin acts synergistically against in vitro P. aeruginosa biofilms.