Surfactin from Bacillus subtilis displays an unexpected anti-Legionella activity

Abstract

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.

Lipid composition in a strain of Bacillus subtilis, a producer of iturin A lipopeptides that are active against uropathogenic bacteria

“…Mireles et al (2001) observed that surfactin (at 100 µg) could reduce biofilm formation on urinary catheters by Salmonella enterica . A reduction in biofilm biomass formation in the presence of surfactin (at 66 µg/mL) was also observed for Legionella pneumophila (Loiseau et al 2015). …”

Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

“…[26,106,107] Recently, ethyl acetate has been successfully used to recover LP produced by Pseudomonas [12,108] and Bacillus. [89] This organic solvent can be used to substantially reduce recovery expenses and minimize environmental hazards. [109] Identification, characterization and quantification of LP by various chromatographic and spectroscopic techniques There are many chromatographic techniques used to characterize and quantify molecules, including thin layer …”

Bioaugmentation for the treatment of waterborne pathogen contamination water

“…There is limited number of reports available regarding the chemical nature of diffusible molecules responsible for anti-Legionella activity. Surfactin (lipopeptide), a well-characterized biosurfactant acquired from Bacillus subtilis, was found active toward various Legionella species including the Lens strain (CIP 108286) (Loiseau et al, 2015). According to previous reports, L. pneumophila was found to be sensitive to a variety of membrane-active biomolecules (Berjeaud et al, 2016).…”

“…A reference strain L. pneumophila serogroup 1 Lens CIP 108286 (Cazalet et al, 2008) was used for anti-Legionella strain selection. Spot-on-lawn assay was used for strain selection described previously by Loiseau et al (2015). Various inhibition zones of different diameters were observed, and stain showing maximum diameter was identified as most potent L. pneumophila competitor.…”