Antibacterial assay. (A) Disc without treatment; (B) 20% DMSO; (C) Ultrapure water; (D) 100% DMSO; (E) 0.1 mg/mL caffeine; (F) 0.2 mg/mL caffeine; (G) 0.3 mg/mL caffeine; (H) 0.4 mg/mL caffeine; (I) 0.1 mg/mL; (J) 0.2 mg/mL (+)-catechin; (K) 0.3 mg/mL (+)-catechin; (L) 0.4 mg/mL (+)-catechin. None of the tested caffeine concentrations showed growth inhibition zones except for paper disc treated with 100% DMSO (positive control). This assay was conducted in three independent triplicates.

Anti-QS properties of caffeine. (A) untreated well; (B) 20% DMSO; (C) ultrapure water; (D) 0.1 mg/mL (+)-catechin; (E) 0. 2 mg/mL (+)-catechin; (F) 0.3 mg/mL (+)-catechin; (G) 0.1 mg/mL caffeine; (H) 0.2 mg/mL caffeine; (I) 0.3 mg/mL caffeine. Result showed that caffeine promoted QS inhibitory effect in concentration dependent manner. This assay was conducted in three independent triplicates.

Caffeine inhibits CV026 violacein production by anti-QS. The violacein production was measured spectrophotometrically as described in Materials and Methods and quantified by reading the OD values of the solution at (a) 585nm and (b) bacterial growth at 600 nm.The statistical significant of each test (n = 3) was evaluated by conducting one-way ANOVA test and a P value of P < 0.05 being significant. Inset: Structure of caffeine.

Caffeine did not degrade C6-HSL. Disc (A) C6-HSL treated with ultrapure water (negative control); (B) C6- HSL treated with 20% DMSO (negative control); (C) PBS; (D) C6-HSL treated with 0.1 mg/mL caffeine; (E) C6-HSL treated with 0.2 mg/mL caffeine; (F) C6-HSL treated with 0.3 mg/mL caffeine; (G) C6-HSL treated with 0.1 mg/mL (+)-catechin; (H) C6-HSL treated with 0.2 mg/mL (+)-catechin; (I) C6-HSL treated with 0.3 mg/mL (+)-catechin. The result showed that both caffeine and (+)-catechin have no effect on C6-HSL. This assay was conducted in three independent triplicates.

(a) Caffeine inhibited P. aeruginosa PA01 QS signal production. Filter paper A and B were added with 20 μL AHL extracted from P. aeruginosa PA01 culture (A); P. aeruginosa PA01 culture treated with ultrapure water (B). In (C) only PBS buffer (20 μL) was spotted on the CV026 lawn. Filter paper (D); (E); (F); (G); (H); (I); (J); (K); (L); and (M) were added with 20 μL AHLs extracted from P. aeruginosa PA01 culture treated with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 mg/mL caffeine, respectively. This assay was conducted in three independent triplicates and representative data are shown. This result shows that caffeine interfered AHLs production of P. aeruginosa PA01 in a concentration dependent manner as evident. (b) OD_600nm reading of P. aeruginosa PA01 overnight culture treated with various concentrations of caffeine. The statistical significant of each test (n = 3) was evaluated by conducting one-way ANOVA test and a P value of P < 0.05 being significant. Our result indicated no significant differences between the treatments and the control at P < 0.05 suggesting that caffeine at these concentrations did not exhibit any antibacterial activity in P. aeruginosa PA01.

Swarming inhibition assays. Swarming agars of P. aeruginosa PA01 (a); supplemented with ultrapure water (b); (v/v, negative control); and caffeine of 0.1 mg/mL (c); 0.2 mg/mL (d); and 0.3 mg/mL (e). Images shown are P. aeruginosa PA01 swarming patterns and inhibition effects after 16 hours of incubation at 37 °C. This assay was conducted in three independent triplicates.