Planktonic Replication Is Essential for Biofilm Formation byLegionella pneumophilain 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 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 ␤-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.Bacteria belonging to the genus Legionella are ubiquitous in natural and man-made aquatic habitats, where they colonize biofilms and replicate intracellularly in a wide range of protozoa (reviewed in references 7, 11, and 39). If inhaled via contaminated aerosols, Legionella spp. can cause the severe pneumonia Legionnaires' disease. The clinically most relevant species is Legionella pneumophila, which proliferates within alveolar macrophages of the human lung, causing disease (17,24). The mechanisms of phagocytosis...

Transcriptional profiling of Legionella pneumophila biofilm cells and the influence of iron on biofilm formation

“…pillar-and mushroom-like structures) formed at 25 u C remained more stably attached than filamentous, mycelial-mat-like biofilms formed at 37 and 42 u C. Also, Mampel et al (2006) reported that biofilm formation at 37 u C was 30 % lower than at 23 and 30 u C. Furthermore, we have shown that adhesion is highest for late stationary phase cells. This is expected, as L. pneumophila shows increased expression of the flagellum and of type IV pili, both of which have been described in other bacteria to be involved in adhesion (O'Toole & Kolter, 1998;Pratt & Kolter, 1998;Watnick & Kolter, 1999), in stationary phase during low-temperature growth (Heuner et al, 1999;Soderberg et al, 2004).…”

“…Although these models do not reflect the complexity found in natural environmental biofilms, monospecies biofilms were the first step towards understanding adaptation to the sessile lifestyle for many bacteria, including two recent studies on L. pneumophila (Mampel et al, 2006;Piao et al, 2006). These models pave the way for the study of complicated microbial interactions and have proven to be useful for assessing the gene expression profile of sessile cells even if the different cell types present within a biofilm cannot be analysed independently by this approach (Schembri et al, 2003;Beloin et al, 2004;Waite et al, 2005;Whiteley et al, 2001).…”

“…pillar-and mushroom-like structures) whereas at 37 u C, filamentous, mycelial-mat-like biofilms were the structures observed. Mampel et al (2006) reported that biofilm formation by L. pneumophila in complex nutrient-rich media most likely relies on adhesion of bacteria grown in the planktonic phase. Also, the Tat secretion pathway has been shown to be involved in the biofilm formation ability of L. pneumophila (De Buck et al, 2005).…”

Antibodies protect against intracellular bacteria by Fc receptor-mediated lysosomal targeting

“…Lpn strains used in this study were JR32 (wt Philadelphia-1) (38), GS3011 (icmT deletion mutant lacking a functional Icm/Dot T4SS; ΔT) (39), thymidine-auxotroph derivatives of Lpn ΔT, and Corby (wt or flaA deletion mutant). Where indicated, Lpn constitutively expressed GFP from plasmid pMMB207-Km14-GFPc (8) or DsRed from plasmid pSW001 (40). Before infections, Lpn was grown for 3 d at 37°C on charcoal yeast-extract agar (CYE) plates.…”

Detection of Legionella pneumophila in water and biofilm samples by culture and molecular methods from man-made systems in São Paulo - Brazil

“…In addition, at least 20 other species of Legionella have been associated with human infections (16). Legionella bacteria do exist as free-living planktonic forms in the environment (36), intracellular parasites of protozoans (3), and/ or inhabitants of mixed-community biofilms (29).Thus, the diversity of types and the ubiquitous occurrence of legionellae in water environments or moist soil make it difficult to identify epidemic strains, and outbreaks of legionellosis have been associated mainly with contamination of man-made aquatic environments (2,16).…”