AHL quorum quenching activity assay

Cells recovered from motility plates of the indicated strains incubated under blue light or in the dark at 23 °C or 37 °C for 48 or 24 h respectively, were normalized to OD₆₆₀ = 1.5. The cells were recovered by centrifugation at 3000 g for 10 min and resuspended in 500 μl in swimming media (1% tryptone, 0.5% NaCl). The cells were sonicated and then centrifuged. Post-sonication supernatants were incubated with 2 µg of commercial standards, C8-AHL or C10-AHL, for 6 h in a shaker at 37 °C. Aliquots (500 μl) of the resulting supernatant were used to detect AHL degradation in a well diffusion assay in double agar plates, in which C. subtsugae CV026 or C. violaceum VIR07 were added to soft agar to detect inhibition of violacein³⁰. Quantification of violacein production in the different strains was determined by measuring the area and integrated density of each complete plate and subtracting the corresponding values measured in the negative control, using ImageJ software (NIH). The values were normalized to the positive control, which received the arbitrary value of 100. The data shown are the means of three independent experiments, and error bars represent the standard deviation of the mean.