Crystal violet biofilm inhibition assay

A biofilm-inhibition assay was used to determine the capacity of the peptides (2Abz¹⁴S²⁹, 2Abz²³S²⁹, and HNP1ΔC18A), or antibiotics (amoxicillin and cefixime) to prevent or reduce biofilm formation against MDR E. coli isolate and P. aeruginosa PAO1, which have the high biofilm-forming ability. Bacterial cells from the overnight culture were inoculated into a fresh MHB media (1.100 dilutions). Antimicrobial agents were added at time zero (before adding the diluted, overnight cultures) into wells of 96-well microtiter plate at different concentrations. The plates were then incubated at 37°C for 24 h to allow biofilm formation. After the incubation, media and planktonic cells were removed, and each well was washed twice with sterile distilled water for removal of the free-floating cells. The plates were dried, and biofilm cells were stained with 0.1% CV for 15 min at room temperature. The excess of stain was removed, and all wells were rinsed three times. Then, the attached dye was solubilized by adding a mixture of the ethanol and acetone (80%/20%) to the wells. Finally, after 20 min incubation, optical density (OD₅₇₀ nm) values were measured using a microtiter plate reader. Percent inhibition of biofilm biomass of peptides and antibiotics alone and in combination was estimated using the following formula [15]. Experiments were repeated three times. % Inhibition of biofilm biomass = OD _control−OD _treatment /OD _control ×100.