Confirmation of AHL Degradation Activity by HPLC-MS

To confirm the QQ activity revealed in the well diffusion agar-plate assays by the AHL-degrading strains, we used high-performance liquid chromatography plus mass-spectrometry analysis (HPLC-MS; Romero et al., 2011). C₆-HSL and C₁₀-HSL were added to 500 μL samples of overnight cultures of AHL-degrading strains in MB medium to a final concentration of 10 μM and incubated for a further 24 h at 25°C and 150 rpm rotary shaking. The cultures were then centrifuged at 2,000 × g for 5 min, extracted twice with an equal volume of ethyl-acetate, evaporated under nitrogen flux at 50°C and suspended in 400 μL of acetonitrile for HPLC-MS analysis and quantification. As negative controls, AHLs (10 μM) were added to fresh MB medium and processed and extracted in the same way.

Analyses were carried out with a HPLC 1100 series (Agilent) equipped with a C8 pre-column (2.1 mm × 12.5 mm, 5 μm particle size) and a ZORBAX Eclipse XDB-C18 2.1 mm × 150 mm column (5 μm particle size) kept at 45°C. The mobile phase was composed of 0.1% v/v formic acid in water and 0.1% v/v formic acid in acetonitrile. MS experiments were conducted on an API 4000 triple-quadrupole mass spectrometer (Applied Biosystems, CA) equipped with a Turbolon source using positive-ion electrospray, multiple-reaction monitoring (MRM) mode. The MRM signals were used to generate relative quantification information by comparison with a calibration curve constructed for molecular-ion abundance, using each of the appropriate AHL synthetic standards.