N butanoyl l homoserine lactone c4 hsl

Colony biofilms of H103 strain and its derivative mutants exposed or not to tobramycin were resuspended in 0.9% NaCl (three-colony biofilms per 10 ml of 0.9% NaCl). Biofilm suspensions were vortexed for 2 min and the AHL and HAQ molecules were extracted following the technique described in a previous study.^{70 (link)} AHLs and HAQs were quantified by liquid chromatography coupled to mass spectrometry (LC-MS/MS).^{29 (link),71 (link)} The obtained data were normalized to OD of biofilm suspensions. AHL standards were obtained from Sigma [N-butanoyl-l-homoserine lactone (C₄-HSL) and N-3-oxododecanoyl-l-homoserine lactone (3-oxo-C₁₂-HSL)].

To examine the QQ activity of ZP1 isolate an overnight bacterial culture in LB was washed three times with PBS pH 7.2–7.4 and then resuspended in PBS containing 10 μM N-(3-oxodecanoyl)-L-homoserine lactone (3oxo-C12-HSL; Sigma-Aldrich, St. Louis, MO, United States), 10 μM N-(hexanoyl)-L-homoserine lactone (C6-HSL; Sigma-Aldrich, St. Louis, MO, United States), or N-butanoyl-L-homoserine lactone (C4-HSL; Sigma-Aldrich, St. Louis, MO, United States). Cultures were incubated for 20 h at 30°C on a rotary shaker at 180 rpm. After centrifugation, supernatants containing AHLs were filtered and the filtrates were used for the evaluation of AHL levels either in C. violaceum disk assay or in assays with P. aeruginosa biosensors. Control samples containing 10 μM AHLs in PBS were either immediately placed at 4°C and used to quantify the starting amount of AHLs or incubated under the same conditions as samples with bacteria and used as control of spontaneous degradation.