Rhamnolipids r90

Zein, cholic acid, and rhamnolipids R90 (90%) were purchased from Sigma-Aldrich (Shanghai, China). Liraglutide was from GL Biochem (Shanghai). Mucin (from porcine stomach) was from meilun bio (Shanghai, China). Sulfo-cyanine 5 NHS ester (Cy5, analytical grade) was from Lumiprobe (Maryland, USA). Fluorescein isothiocyanate (FITC) was from Tokyo Chemical Industry (Tokyo, Japan). Anthrone was from Aladdin (Shanghai, China). Chlorpromazine, genistein, amiloride, and 1, 1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) were from Beyotime Biotechnology (Shanghai, China). All cell culture reagents and materials were from Thermo Fisher Scientific (Shanghai, China). All other reagents were from Sinopharm Chemical Reagent (Shanghai, China). Total bile acid (TBA) kits were from Nanjing Jiancheng Bioengineering Institute.

Both N-Acetyl-l-cysteine (Sigma-Aldrich, Prague, Czech Republic) and rhamnolipids (R90; Sigma-Aldrich, Prague, Czech Republic) were dissolved in sterile distilled water to a final concentration of 100,000 µg/mL (for NAC) and 2000 µg/mL (for RLs). (+)-Usnic acid (Sigma-Aldrich, Prague, Czech Republic) was dissolved in 12.5% DMSO (Fisher Scientific, Portsmouth, NH, USA) to a final concentration of 62.5 µg/mL. Dilution series of all tested antimicrobial substances were prepared by diluting the substances in an appropriate culture medium (BHI, or TSB + 1% Glc) in a 1:1 ratio to obtain 10 different concentrations. The maximal concentrations were given by the default purity and solubility of the substances. The concentration range was 100–100,000 µg/mL for NAC, 1.96–2000 µg/mL for RLs, and 0.06–62.5 µg/mL for UA. The highest available concentrations of all three tested substances were used only for the biofilm reduction testing, where they were directly applied to a preformed biofilm. The highest tested concentration of UA (62.5 µg/mL) was excluded from further analyses due to an inhibition effect of 12.5% DMSO in which UA was dissolved.