Pseudomonas aeruginosa biofilm structure, behavior and hydrodynamics

Abstract

Biofilm formation by bacterial pathogens is an important factor in the progression and treatment of many infectious diseases. Biofilm structural development is a dynamic process dependent on many cellular and environmental parameters including Quorum Sensing (QS) and hydrodynamics. Since QS is dependent on a threshold autoinducer concentration, it was hypothesized that the flow dynamics in the bulk fluid surrounding the biofilm would play an important role in expression of QS and the genes that are under its control. In order to investigate the relative contribution of hydrodynamics and QS on biofilm development, biofilms were grown from wild type Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows. When morphology of the biofilms were quantified using Image Structure Analyzer (ISA) software, a multivariate analysis demonstrated that both QS and hydrodynamics influenced biofilm structure, suggesting that QS was not required for biofilm development but affected structural heterogeneity in biofilms. GFP reporter based gene expression analysis of QS regulated lasB (coding for elastase) expression during biofilm development in laminar flow further supported these results. Detachment has been recognized as another factor that may define structural morphology of biofilms. Under flow conditions hollow biofilm clusters were formed as a result of active detachment process, termed as "seeding dispersal". A differentiation of a "seeding" microcolony into an interior motile, swarming, phenotype and a non-motile surrounding, "wall phenotype" formed as a prelude to the dispersal process in which the interior cells swarmed out of the microcolony from local break out points and spread over the wall of the flow cell. A critical microcolony diameter of approximately 100 æm was required for differentiation suggesting that regulation was related to cell density and mass transfer conditions. It was found that rhamnolipid (rhlA-) biosurfactant was not required and QS system (PAO1-JP2) was shown to be important in this process, possibly by sensing nutrient limitation within the biofilm microcolonies. These results strengthen a current view of multi-cellularity and coordinated behavior in prokaryotes as well as a dynamic network of overlapping pathways and cellular mechanisms that act on biofilm development in a complex interrelated manner.