Dispersion

Dispersion assays were performed to compare the dispersion activity of each analog against 7-day biofilms. 2CP (stabilized version of C2DA) was compared with C2DA (active, positive control) and T2DA (inactive/active, negative control). Each well of a 96-well plate was seeded with 150 μl of bacterial culture (S. aureus, UAMS-1, or P. aeruginosa, PA-ATCC 27317) and incubated for 7 days at 37°C to result in biofilms. Media [Tryptic Soy Broth (TSB)] was changed after careful aspiration every 24 h. On day 7, each well was again carefully aspirated and then received 195 μl of TSB and 5 μl of fatty-acid stock ranging in concentration from 0 to 500 μg/ml. Fatty-acid concentrations were made in absolute ethanol; thus, the final ethanol concentration in wells was 2.5%. The plates were incubated for 24 h at 37°C, then turbidity was measured at 540 nm. Turbidity readings were performed using a Biotek Synergy^TM H1 microplate reader, with increased turbidity readings indicating a higher number of viable bacterial cells. Afterward, the media was aspirated and the plate was washed three times using phosphate-buffered saline (PBS). The relative percentage of attached cells as compared to non-treated controls were measured using BacTiter-Glo^TM (Promega) Luciferase assay for ATP production, with higher percentages indicating a higher quantity of attached biofilm cells.