The development and application of an in vitro model to determine the susceptibility of P. aeruginosa in biofilms to various antibiotics

Purpose: Cystic fibrosis patients often develop chronic infections with Pseudomonas aeruginosa (PA). PA forms biofilms which will lead to reduced antibiotic (AB) susceptibility. Instead of using planktonic bacteria as a model to test susceptibility, we developed and tested a more representative in vitro model using PA biofilms. Methods: PA biofilms were grown in 96-well plates and quantified with crystal violet staining followed by VIS- absorption. Different parameters were evaluated to come to a reproducible method to form and quantify PA biofilms. With the optimised model susceptibility to Tobramycin sul- fate (TS) and various ABs and AB-combinations was determined. In a 3 days dose regime experiment TS (1.5; 3; 6 /ig/ml) was used 3 times daily against 4 PA strains (1 ATCC and 3 laboratory strains). Results: Most reproducible PA biofilm formation and quantification was obtained in Greiner Bio-One 96-Well Plates, with 1% Peptone in 0.9% saline solution as culture medium. All 4 strains showed a decrease in biofilm after the completed dose regime. PA01WT biofilm decreased to less than 10% with TS concentrations 3 and 6 /ig/ml. Conclusions: A reproducible and easy to use in vitro model to test the susceptibility of PA biofilms to various ABs or AB- combinations has been developed. No synergy was observed using AB-combinations with TS compared to TS alone. Dose regime experiments showed a significant decrease in the amount of biofilm. The pathogenic PA01 strain showed the largest decrease