Protein digestion was observed by a clear zone surrounding the holes.

To determine swimming motility, 0.3% agar with 1% tryptone and 0.25% NaCl were used (Sperandio et al., 2002). BM2 swarming medium (62 mM potassium phosphate buffer at pH 7, 2 mM MgSO4, 10 μM FeSO4, 0.4% glucose, AG-14699 0.1% casamino acids and 0.5% agar) was used for swarming motility (Overhage et al., 2007) and LB with 1.0% agar for twitching motility (Overhage et al., 2007). Briefly, the P. aeruginosa strain was grown from diluted overnight cultures to a turbidity of 1.0 at 600 nm. Each experiment was performed using at least two independent cultures. Overnight cultures of P. aeruginosa PAO1 were diluted 1 : 100 and grown to a turbidity PD-166866 in vitro of 1.0 at 600 nm with 1 mM indole, 1 mM 7-hydroxyindole, 1 mM 7FI or DMSO (0.1%, v/v) as a negative control. Antibiotics (0.06 mg mL−1 gentamicin, 10 mg mL−1 kanamycin and 0.8 mg mL−1 tetracycline) in the final concentration were mixed with cells and incubated

for 60 min without shaking. The cells that survived in the presence of antibiotics were enumerated on LB agar plates. Two independent cultures were used for each strain. Thirty-one commercially available indole derivatives (15 natural and 16 synthetic indole compounds) were screened for their ability to inhibit the biofilm formation and hemolysis of P. aeruginosa PAO1. The screening demonstrated various abilities to control the biofilm formation and hemolysis of P. aeruginosa, as some indole compounds, e.g. 3,3′-dimethyleneindole, increased and some indole compounds decreased biofilm formation (Table 1). Among the indole compounds tested, 7FI was the most effective at reducing both the biofilm formation and hemolytic activity of P. aeruginosa (Table 1). Specifically, the addition of 7FI (1 mM) decreased biofilm cAMP formation fourfold and hemolytic activity 14-fold. As the fluoride at carbon position 7 of

indole caused the most significant results, more fluoroindole compounds [4-fluoroindole (4FI), 5-fluoroindole (5FI), 6-fluoroindole (6FI), 5-fluorooxindole, 8-fluoroquinoline] and indole derivatives with different functional groups at carbon position 7 were investigated. 4FI, 5FI and 6FI reduced hemolytic activity 10-fold but their antibiofilm activity was less potent than 7FI. As the most potent antibiofilm and antihemolysis compound, 7FI was focused on. 7FI clearly and dose-dependently inhibited the biofilm formation and hemolytic activity of P. aeruginosa (Fig. 1a,b). Although three fluoroindoles at 1 mM slightly delayed the cell growth of P. aeruginosa, growth recommenced after 24 h (Fig. 1c). The overall data (Table 1, Fig. 1) indicated that the antibiofilm and antihemolysis activity of fluoroindoles at 1 mM was not due to its antimicrobial activity. As P.