Formation of Pseudomonas aeruginosa inhibition zone during Tobramycin disk diffusion is due to a transition from planktonic to biofilm mode of growth

Pseudomonas aeruginosa PAO1 (MIC 0.064µg/ml) was used to perform agar diffusion tests employing tobramycin containing tablets. The growth of the bacteria and the formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with Methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL which was studied by confocal laser scanning microscopy. The diffusion of tobramycin from the deposit was modelled by using a 3D geometric version of Fick's 2nd law of diffusion. The time-dependent gradual increase of Minimal Biofilm Eradication Concentration (MBEC) was studied by the Calgary Biofilm Devise. The early inhibition zone was visible after 5 h incubation. The corresponding calculated tobramycin concentration at the border was 1.9µg/ml and increased to 3.2µg/ml and 6.3µg/ml after 7 and 24 h incubation. The inhibition zone increased to the stable, final zone after 7 h incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained ≤≈64 cells and production of polysaccharide matrix including PSL had begun, thereafter the small bacterial aggregates were killed by tobramycin. The bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide containing matrix. The formation of the inhibition zone during the agar diffusion antibiotic susceptibility test is due to a switch from the planktonic to the biofilm mode of growth and gives clinical important information about the increased antibiotic tolerance of biofilms.