Planktonic Replication Is Essential for Biofilm Formation byLegionella pneumophilain a Complex Medium under Static and Dynamic Flow Conditions

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Abstract
Legionella pneumophilapersists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. WhileL. pneumophilaserves as a paradigm for intracellular growth within protozoa, it is less clear whether the bacteria form or replicate within biofilms in the absence of protozoa. In this study, we analyzed surface adherence of and biofilm formation byL. pneumophilain a rich medium that supported axenic replication. Biofilm formation by the virulentL. pneumophilastrain JR32 and by clinical and environmental isolates was analyzed by confocal microscopy and crystal violet staining. Strain JR32 formed biofilms on glass surfaces and upright polystyrene wells, as well as on pins of “inverse” microtiter plates, indicating that biofilm formation was not simply due to sedimentation of the bacteria. Biofilm formation by anL. pneumophila fliAmutant lacking the alternative sigma factor σ28was reduced, which demonstrated that bacterial factors are required. Accumulation of biomass coincided with an increase in the optical density at 600 nm and ceased when the bacteria reached the stationary growth phase.L. pneumophilaneither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition ofAcanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming)L. pneumophilaexpressed β-galactosidase activity to similar extents, and therefore, the observed lack of proliferation of surface-attached bacteria was not due to impaired protein synthesis or metabolic activity. Cocultivation of green fluorescent protein (GFP)- and DsRed-labeledL. pneumophilaled to randomly interspersed cells on the substratum and in aggregates, and no sizeable patches of clonally growing bacteria were observed. Our findings indicate that biofilm formation byL. pneumophilain a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeledL. pneumophilainitially adhered in a continuous-flow chamber system but detached over time; the detachment correlated with the flow rate, and there was no accumulation of biomass. Under these conditions,L. pneumophilapersisted in biofilms formed byEmpedobacter breveorMicrobacteriumsp. but not in biofilms formed byKlebsiella pneumoniaeor other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.