The tendency of bacteria to stick to surfaces and form surface-associated communities called biofilms has been well documented. A hallmark characteristic of biofilms is that they can be up to a thousand times more resistant to antimicrobial stress than free-swimming cells of the same species. Pseudomonas aeruginosa is an opportunistic human pathogen that has been implicated in nosocomial infections as well as chronic lung infections in people suffering from Cystic Fibrosis. P. aeruginosa uses a cell-to-cell signaling mechanism called quorum sensing to regulate virulence factor production, as well as biofilm formation. Therapeutic strategies directed at quorum sensing may be effective at combating P. aeruginosa biofilm infections. The proposed research will develop and utilize mathematical models for predicting acyl-HSL-regulated gene expression in a biofilm system. These models will be tested experimentally against actual biofilms grown under ecologically relevant conditions. The ultimate goal is to generate a versatile, predictive model that will allow clinicians to predict the onset of quorum sensing-regulated gene expression during the course of biofilm infections. A hierarchy of mathematical models will be developed for three different length scales: single unit cell, small clusters of cells, and full biofilm. The objective of these models will be to predict the level of acyl-HSL within an experimentally observed biofilm and its relationship to its environment. New hybrid numerical methods will be employed to simulate the mathematical models which will be based upon a coupling of the Level Set Method and the Extended Finite Element Method. The new method will be uniquely suited for simulating the growth of the biofilm and the synthesis of the signal acyl-HSL. Experimental data for parameter estimation in the models, as well as model testing will be done using a battery of biological reporter systems. These reporter strains utilize transcriptional fusions of quorum sensing-regulated promoters to the green fluorescent protein (GFP). The expression of GFP and the onset of fluorescence coincide with the accumulation of acyl-HSL signal to a critical threshold concentration. Use of these reporter systems in conjunction with scanning confocal laser microscopy will allow the examination of gene expression at the single cell level. Experimental results will be used to refine the mathematical models.
