Biofilm Formation of Legionella pneumophila in Complex Medium under Static and Dynamic Flow Conditions

Abstract

This chapter analyzes biofilm formation of Legionella pneumophila in rich medium which supports extracellular proliferation of the bacteria. To investigate biofilm formation of Legionella under static conditions, a 1:1 mixture of L. pneumophila labeled with the fluorescent proteins EGFP or DsRed-Express was inoculated in a glass-bottom 35-mm petri dish. Biofilm formation was analyzed with an inverted confocal microscope (Axiovert 200M, 100 X oil objective Plan Neofluar; Zeiss). Biofilm formation of L. pneumophila in rich medium was also quantified by crystal violet staining in upright polystyrene microtiter plates or on polystyrene pins of inverse lids under static (no medium exchange) or quasi-static conditions (medium replaced twice a day). The L. pneumophila fliA mutant strain reproducibly accumulated 30% less biomass within 5 days, demonstrating that bacterial factors contribute to biofilm formation. It is noteworthy that mutants lacking rpoS or letA, both of which are required for the expression of transmissive (virulence) traits of L. pneumophila, were not affected in biofilm formation. A continuous-flow chamber system was set up to further test the hypothesis that plank-tonic cells are crucial for biofilm formation on surfaces and to noninvasively monitor in real-time the adherence and biofilm formation of L. pneumophila under dynamic flow conditions.