Resistance of artificial biofilms of Pseudomonas aeruginosa to imipenem and tobramycin

Viable cells of Pseudomonas aeruginosa were entrapped in alginate gel layers and incubated in a minimal glucose (15 g/L)-yeast extract (2 g/L)-salt medium to form artificial biofilm-like structures. After cultivation for 2 days, the biomass distribution inside the polymer was highly heterogeneous. The cell number reached approximately 10(11) cells/g gel in the outer regions of the gel structures whereas the inner areas were less colonized (c. 10(8) cells g/gel). Killing of immobilized organisms by imipenem and tobramycin were compared with free-cell experiments (inoculum c. 10(9) cells/ml). Sessile-like bacteria displayed a higher resistance to the two antibiotics used alone or in combination than did suspended cells. Exposure for 10 h to 20 x MIC imipenem and 15 x MIC tobramycin reduced the number of viable immobilized bacteria to 0.3% and 3%, respectively, of the initial cell population, whereas these antibiotic concentrations were much more efficient (bactericidal) against free-cell cultures (5 log kill in 6 h). A synergic effect of tobramycin and imipenem was detected on bacterial suspensions but not on biofilm-like structures. Effective diffusivity measurements showed that the diffusion of imipenem in the alginate layer was not hindered. A slight but significant enhancement of beta-lactamase induction in immobilized cells as compared with their suspended counterparts was insufficient to explain the high resistance of sessile-like bacteria.