(a) Agar Well Diffusion Assay

Agar well diffusion assay using C. violaceum CV026 as biosensor strain was performed to screen anti-QS activity of antibiotics (n = 3) (Supplementary Figure 9). 50 μL of C6-HSL stock solution (10 mg/mL) was mixed with 10 mL of an overnight grown culture of C. violaceum CV026 (OD_{600 nm} = 1.0) and designated as AHL-CV026 solution. To prepare media plates, we slowly mixed 19 mL of tryptose soya agar (1%) with 1 mL of AHL-CV026 solution (avoid air bubble formation). Plate the mixture in a Petri plate and let the media cool down until agar is solidified. Use sterile pipette tip (200 μL) to puncher holes in solid agar. Press the pipette and twist to take the punched agar and invert the plate to remove the agar block. Discard the pipette tip and use fresh tips every time to make clean wells. Load different concentrations of antibiotics in each well and incubate the plates for 15 h at 37°C in a bacteriological incubator. We diluted the stock solution (1024 μg/mL) of antibiotic 10 fold and loaded 50 μL of each dilution in each well. The amount of antibiotics in well-1 to well-8 was as follows, well-1 = 51.2 μg; well-2 = 25.6 μg; well-3 = 12.8 μg; well-4 = 6.4 μg; well-5 = 3.2 μg; well-6 = 1.6 μg; well-7 = 0.8 μg and well-8 = 0.4 μg. After incubation, observe the plate in light back growth. Clear zone without bacterial growth and no pigment indicated antimicrobial zone and hazy zone with bacterial growth but without pigment production indicate anti-QS activity of antibiotics. The picture was taken by a Bio-Rad imager and compiled in one image with a scale bar (20 mm). Anti-QS zones were quantified following equation:

AQS Zone = (D_{Growth+No Pigment} - D_{No Growth/No Pigment}) [AQS Zone = Anti-QS zone (cm), D_{Growth+No Pigment} = Diameter of growth zone with no pigment production, D_{No Growth/No Pigment} = Diameter of no growth zone and no pigment].