Differentiation and distribution of colistin/SDS tolerant cells in Pseudomonas aeruginosa biofilms

Center for Biomedical Microbiology, BioCentrum-DTU, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark; Department of Medicine, University of Washington, Seattle

	Abstract

During Pseudomonas aeruginosa flow-cell biofilm development the cell population differentiates into a non-motile subpopulation which forms micro colonies, and a migrating subpopulation which eventually colonizes the top of the micro colonies resulting in the development of mushroom-shaped multicellular structures. The cap-forming subpopulation was found to develop tolerance to membrane targeting anti-microbial agents such as the cyclic cationic peptide colistin and the detergent sodium dodecyl sulphate (SDS). The stalk-forming subpopulation, on the other hand, was sensitive to the membrane targeting antibacterial agents. All biofilm associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface associated motility, prevented the formation of regular mushroom-shaped structures in the flow-cell biofilms, and the development of tolerance to the antimicrobial agents was found to be affected as well. Mutations in genes interfering with LPS modification (pmr) eliminated the biofilm associated colistin tolerance phenotype. Experiments with a PAO1 strain harbouring a pmr-gfp fusion showed that only the cap-forming subpopulation in biofilms treated with colistin expresses the pmr operon. These results suggest that increased antibiotic tolerance developing in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties.