Surfactin from Bacillus subtilis displays an unexpected anti-Legionella activity.

A contaminant bacterial strain was found to exhibit an antagonistic activity against Legionella pneumophila, the causative agent of Legionnaires' disease. The bacterial strain was identified as a Bacillus subtilis and named B. subtilis AM1. PCR analysis revealed the presence of the sfp gene, involved in the biosynthesis of surfactin, a lipopeptide with versatile bioactive properties. The bioactive substances were extracted from AM1 cell-free supernatant with ethyl acetate and purified using reversed phase HPLC (RP-HPLC). Subsequent ESI-MS analyses indicated the presence of two active substances with protonated molecular ions at m/z 1008 and 1036 Da, corresponding to surfactin isoforms. Structures of lipopeptides were further determined by tandem mass spectrometry and compared to the spectra of a commercially available surfactin mixture. Surfactin displays an antibacterial spectrum almost restricted to the Legionella genus (MICs range 1-4 µg/mL) and also exhibits a weak activity toward the amoeba Acanthamoeba castellanii, known to be the natural reservoir of L. pneumophila. Anti-biofilm assays demonstrated that 66 µg/mL of surfactin successfully eliminated 90 % of a 6-day-old biofilm. In conclusion, this study reveals for the first time the potent activity of surfactin against Legionella sp. and preformed biofilms thus providing new directions toward the use and the development of lipopeptides for the control of Legionella spread in the environment.

Intra-amoeba multiplication induces chemotaxis and biofilm colonization and formation for Legionella.

Legionella pneumophila, a facultative intracellular bacterium, is the causative agent of legionellosis. In the environment this pathogenic bacterium colonizes the biofilms as well as amoebae, which provide a rich environment for the replication of Legionella. When seeded on pre-formed biofilms, L. pneumophila was able to establish and survive and was only found at the surface of the biofilms. Different phenotypes were observed when the L. pneumophila, used to implement pre-formed biofilms or to form mono-species biofilms, were cultivated in a laboratory culture broth or had grown intracellulary within the amoeba. Indeed, the bacteria, which developed within the amoeba, formed clusters when deposited on a solid surface. Moreover, our results demonstrate that multiplication inside the amoeba increased the capacity of L. pneumophila to produce polysaccharides and therefore enhanced its capacity to establish biofilms. Finally, it was shown that the clusters formed by L. pneumophila were probably related to the secretion of a chemotaxis molecular agent.