Studies with 3Cl-PQS on preformed Af biofilm metabolism, assessed by XTT assay. (A) Dose response study, strain 10AF, control (same amount of DMSO as in test articles). Designations 1.56, etc. are μg/mL. Dose-response is shallow; 1.56 μg/mL, p < 0.01 compared to control, and other concentrations p < 0.001 compared to control. (B,C), comparison to PQS, all at 1.56 μg/mL, and to ethanol control (shown) identical to DMSO control. 3Cl-PQS is less inhibitory than PQS. In (B), using 10AF, 3Cl-PQS is p < 0.01, and PQS p < 0.001, compared to control; PQS is p < 0.001 compared to 3Cl-PQS. In (C), studying strain Af293, 3Cl-PQS is p > 0.05, and PQS p < 0.001, compared to control; PQS is p < 0.001 compared to 3Cl-PQS. (D) Effect of Fe on 3Cl-PQS (12.5 μg/mL) inhibition of strain 10AF. Control (same amount of DMSO as in test article), numbers 0.01–5000 are Fe μM concentration. 3Cl-PQS inhibits Af metabolism (left bar, p < 0.01). Fe alone boosts Af metabolism (white bars), starting at Fe 0.1 μM, all p < 0.01–0.001 compared to control. Fe reverses 3Cl-PQS inhibition in combinations (black bars), beginning at Fe 0.01 μg/mL, where Fe 0.01 μM + 3Cl-PQS is now p > 0.05 compared to control or to 3Cl-PQS alone, and all other 3Cl-PQS + Fe combinations are greater (p < 0.001) compared to control or to 3Cl-PQS alone. 3Cl-PQS + Fe combinations do not boost Af metabolism above same concentration of Fe alone, unlike previously reported with PQS [18] (and at two concentrations, Fe 0.1 or Fe 2500 μM + 3Cl-PQS, are significantly, p < 0.05, less than corresponding Fe alone). (E,F), effects in hypoxic environment. DMSO and ethanol control results shown identical. (E) is Af isolate 10AF, (F) is Af isolate Af293, both tested at 1.56 μg/mL of test articles. In (E), 3Cl-PQS is p < 0.05 compared to control or to PQS, and PQS p < 0.001 compared to controls. In (F), PQS and 3Cl-PQS both p < 0.001 compared to controls, and PQS and 3Cl-PQS differ by p < 0.001.