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Chapter 96 : First Report of an Anti-Legionella Peptide Produced by Staphylococcus warneri
The chapter describes the partial characterization of a new bacteriocin, produced by Staphylococcus warneri RK, active against Legionella pneumophila. A bacterial strain, isolated from the environment, displayed an anti-Legionella activity by a spot on lawn assay. Various bacterial strains, gram-positive and gram-negative, were used as target organisms to test their sensitivity to S. warneri RK by a spot on lawn assay. The anti-Legionella molecule was then purified from the raw extract during a three-step purification procedure. First, the raw extract was diluted (50:50) with sodium acetate buffer (20 mM, pH 5) and applied to a weak cation exchange chromatography column (Hiprep 16/10 Carboxy-Methyl FF, Amersham Biosciences). Second, the latter fraction was applied onto a solid phase extraction C18 cartridge (Sep-pak plus, Waters), washed successively with 5 ml of 0, 10, 20, 30, 40, and 80% acetonitrile containing 0.1% trifluoroacetic acid. Third, the active fraction (80% acetonitrile) was lyophilized, solubilized with 1 ml of 40% acetonitrile, and injected on a Kromasil C8 reverse-phase HPLC analytical column (5μm, 100 Å, 4.6X250 mm, A.I.T.). The anti-Legionella activity has been eluted, during the last step, at 100% acetonitrile, indicating that the corresponding peptide is highly hydrophobic. This anti-Legionella-active fraction was subjected to mass spectrometry analysis, giving two molecular masses of 2613.8 Da and 2449.4 Da. This result shows that two peptides were present in the fraction, one of which likely corresponded to the anti-Legionella bacteriocin. The anti-Legionella molecule is therefore an original peptide.