Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions

Abstract

Legionella pneumophila persists for a long time in aquatic habitats, where the bacteria associate with biofilms and replicate within protozoan predators. While L. pneumophila serves 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 by L. pneumophila in a rich medium that supported axenic replication. Biofilm formation by the virulent L. pneumophila strain 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 an L. pneumophila fliA mutant lacking the alternative sigma factor sigma(28) was 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. pneumophila neither grew nor formed biofilms in the inverse system if the medium was exchanged twice a day. However, after addition of Acanthamoeba castellanii, the bacteria proliferated and adhered to surfaces. Sessile (surface-attached) and planktonic (free-swimming) L. pneumophila expressed beta-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-labeled L. pneumophila led 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 by L. pneumophila in a rich medium is due to growth of planktonic bacteria rather than to growth of sessile bacteria. In agreement with this conclusion, GFP-labeled L. pneumophila initially 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. pneumophila persisted in biofilms formed by Empedobacter breve or Microbacterium sp. but not in biofilms formed by Klebsiella pneumoniae or other environmental bacteria, suggesting that specific interactions between the bacteria modulate adherence.

FIG.1.

Biofilm formation by L. pneumophila under static conditions in rich medium. (A) Confocal laser scanning micrograph of a representative biofilm section formed in a glass-bottom dish by GFP-expressing L. pneumophila wild-type strain JR32. Unit cell, 13 μm. (B and C) Growth of planktonic L. pneumophila (OD₆₀₀) (○) and crystal violet staining (A₆₀₀) (•) of sessile bacteria in upright wells of polystyrene 96-well plates (B) or on polystyrene pins in an inverse biofilm assay (C). The assays were repeated at least three times with similar results.

FIG.1.

Biofilm formation by L. pneumophila under static conditions in rich medium. (A) Confocal laser scanning micrograph of a representative biofilm section formed in a glass-bottom dish by GFP-expressing L. pneumophila wild-type strain JR32. Unit cell, 13 μm. (B and C) Growth of planktonic L. pneumophila (OD₆₀₀) (○) and crystal violet staining (A₆₀₀) (•) of sessile bacteria in upright wells of polystyrene 96-well plates (B) or on polystyrene pins in an inverse biofilm assay (C). The assays were repeated at least three times with similar results.

FIG. 2.

Biofilm formation by clinical and environmental L. pneumophila isolates. (A) Crystal violet staining of 5-day-old biofilms of L. pneumophila wild-type strain JR32, clinical and environmental L. pneumophila isolates, and medium. The dashed line indicates the average biomass accumulation. (B) Crystal violet staining of biofilms formed by L. pneumophila JR32 (cross-hatched bars), clinical isolates (average, standard deviation) (light gray bars), and environmental isolates (average, standard deviation) (dark gray bars) at different temperatures. The experiments were done in triplicate.

FIG. 3.

L. pneumophila does not accumulate surface-attached biomass or grow under quasi-static conditions, but A. castellanii promotes biofilm formation. (A) Diagrams of static (no medium exchange) and quasi-static (medium exchanged twice a day) inverse biofilm assays. (B) Crystal violet staining of sessile L. pneumophila under static and quasi-static conditions and of noninoculated wells (medium). The results were reproduced in at least three independent experiments. (C) Growth of planktonic L. pneumophila (OD₆₀₀) in the absence (○) and in the presence (•) of 5 × 10⁴ A. castellanii cells per well and growth of A. castellanii alone (▴) in the inverse quasi-static biofilm assay. (D) Quantification of sessile, gfp-expressing L. pneumophila by flow cytometry in the absence (open bars) and in the presence (solid bars) of 5 × 10⁴ A. castellanii cells per well under static (no medium exchange) or quasi-static (medium exchanged once a day) conditions in the inverse assay (5 days of growth).

FIG. 4.

Expression of β-galactosidase activity by planktonic and sessile L. pneumophila. The β-galactosidase activities (in Miller units) of planktonic L. pneumophila, surface-attached, and resuspended biofilm bacteria were determined after induction with IPTG for 0.5 h (black bars) and 3.5 h (light gray bars) and after continuous induction with IPTG (cross-hatched bar and dark gray bar). P, planktonic bacteria; B-N, native biofilm; B-S, suspended biofilm. The data are representative of two independent experiments done in triplicate.

FIG. 5.

Distribution of GFP- and DsRed-labeled L. pneumophila in static and quasi-static cocultures. (A and B) Confocal laser scanning micrographs of GFP- and DsRed-expressing L. pneumophila JR32 on the surface of a glass-bottom dish at the time of inoculation (A) and after 3 days under quasi-static conditions (medium exchanged twice a day) (B). (C to E) Three-dimensional reconstruction (C) and sections at the surface (D) or through aggregates (E) of the biofilm formed after 3 days of growth under static conditions (no medium exchange). Bars, 10 μm; unit cell, 13 μm.

FIG. 6.

Adherence of L. pneumophila in a continuous-flow chamber system with a rich medium: fluorescence micrographs of GFP-expressing L. pneumophila in a continuous-flow chamber system operated at different flow rates with AYE medium (diluted 1:10). The bacteria initially adhered to surfaces and detached in a flow rate-dependent manner. The experiments were performed in triplicate and were independently reproduced at least twice with similar results. Bar, 50 μm.

FIG. 7.

Effect of defined cocultures on persistence and localization of L. pneumophila in a continuous-flow chamber system. (A) Microbacterium sp. increased and K. pneumoniae decreased the persistence of GFP-expressing L. pneumophila in a continuous-flow chamber system operated with AYE medium (diluted 1:10) at a flow of 50 μl/min. Bar, 50 μm. (B) Fluorescence micrograph of a Microbacterium-Legionella coculture biofilm stained with the dye SYTO62 14 days after inoculation. Single L. pneumophila bacteria (green) predominantly colocalized with the Microbacterium sp. biofilm (red). Bar, 30 μm. Similar results were obtained in at least two independent experiments done in triplicate.

FIG. 7.

Effect of defined cocultures on persistence and localization of L. pneumophila in a continuous-flow chamber system. (A) Microbacterium sp. increased and K. pneumoniae decreased the persistence of GFP-expressing L. pneumophila in a continuous-flow chamber system operated with AYE medium (diluted 1:10) at a flow of 50 μl/min. Bar, 50 μm. (B) Fluorescence micrograph of a Microbacterium-Legionella coculture biofilm stained with the dye SYTO62 14 days after inoculation. Single L. pneumophila bacteria (green) predominantly colocalized with the Microbacterium sp. biofilm (red). Bar, 30 μm. Similar results were obtained in at least two independent experiments done in triplicate.