Temperature-regulated formation of mycelial mat-like biofilms by Legionella pneumophila

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25 degrees C, 37 degrees C, and 42 degrees C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37 degrees C or 42 degrees C than at 25 degrees C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25 degrees C than at 37 degrees C or 42 degrees C. On glass surfaces, the biofilms were formed faster but attached less stably at 37 degrees C or 42 degrees C than at 25 degrees C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37 degrees C or 42 degrees C were mycelial mat like and were composed of filamentous cells, while at 25 degrees C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37 degrees C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.