Figure 3.

Studies with 3NH₂-PQS. (A) Dose-response study with preformed 10AF biofilm. Minimal dose-response. “RPMI control” was biofilm without test article or diluent, “RPMI + Ethanol control” contained same concentration of ethanol diluent as present in test article wells; no difference between these controls. Both controls were also included in sections (B) through (D) of Figure 3, but as the results of these two controls was always the same, for simplification, only one control is shown in sections (C) through (F). Twofold dilutions of 3NH₂-PQS were made from 50 to 0.78 μg/mL. Only at the very highest doses (12.5–50 μg/mL, shown) was there a suggestion of dose response, but there was no statistically significant difference among those bars. 3NH₂-PQS at 0.78 μg/mL was not different from controls shown, and bars showing results with 1.56–6.25 μg/mL were not different from that of 12.5 μg/mL, so are not shown in order to simplify the figure. All dilution results from 1.56–50 μg/mL were p < 0.001 compared to controls. (B). Dose-response study with 10AF biofilm formation. Same experimental design as in A, but test against Af biofilm formation. Dilutions 0.78–6.25 μg/mL resulted in slight inhibition, but p > 0.05 compared to controls, and no dose-response. 3NH₂-PQS 12.5 and 25 μg/mL p < 0.01 compared to controls, and 50 μg/mL p < 0.001 compared to controls. (C,D) Comparison with PQS, preformed biofilm, test reagents at 1.56 μg/mL. C, isolate 10AF. 3NH₂-PQS is less potent inhibitor than PQS (p < 0.01). Both are p < 0.01 compared to controls. D, isolate Af293. PQS more inhibitory (p < 0.001) compared to 3NH2-PQS. Both are p < 0.001 compared to controls. (E) 3NH₂-PQS (12.5 μg/mL) (left bar) inhibits preformed 10AF biofilm, p < 0.001 compared to control (right bar). Fe (0.1–1000 μM; numbers on x axis are these μM concentrations) reverses 3NH₂-PQS inhibition (3NH₂-PQS + Fe concentrations p < 0.001 compared to 3NH₂-PQS). Fe boosts Af metabolism, but 3NH₂-PQS + Fe does not increase metabolism above Fe alone. (F) Action of 3NH₂-PQS under hypoxic conditions. 3NH₂-PQS inhibitory to preformed Af293 biofilm (p < 0.05 compared to controls). PQS more inhibitory (p < 0.001) than controls, and more inhibitory compared to 3NH₂-PQS (p < 0.001). Both PQS and 3NH₂-PQS are at 1.56 μg/mL.