Evaluation of the ionic liquid cholinium L-alaninate as a promising biofilm control in clinically relevant bacteria

• At 0.9 % w/v concentration, ionic liquid cholinium alaninate inhibited over 95 % of biofilm growth. • Eradication of over 85 % of biofilm for all strains was achieved at a concentration of 0.9 % w/v of ionic liquid. • Biofilm inhibition and eradication tests showed significant differences between treatments and control ( p < 0.05), with Gram-negative bacteria showing the most effective results. Pathogenic biofilms pose a significant clinical challenge owing to their extreme antibiotic resistance. Multiple strategies, including ionic liquids (ILs), have been explored to inhibit or eradicate biofilms. Among them, choline and amino acid based ionic liquids are of interest due to their biocompatibility and low cytotoxicity. This study assessed the antimicrobial efficacy of the ionic liquid cholinium alaninate against planktonic cultures and biofilms of Escherichia coli, Staphylococcus aureus , and Pseudomonas aeruginosa . We determined the minimum inhibitory concentration (MIC) for planktonic cells using growth curve analysis. The inhibition and eradication of biofilm were assessed in microplates through stains to quantify total and viable cells. Treatments with different concentrations of ionic liquid were compared to controls. Results were analyzed using ANOVA and multiple comparison tests. A minimum inhibitory concentration of 1.3 % w/v cholinium alaninate was determined, with inhibition in the order E. coli < P. aeruginosa < S. aureus . For biofilms, a 0.9 % w/v treatment inhibited >95 % of biofilm growth. The sub-inhibitory treatment of 1.3 % w/v resulted in the removal of ≈92 % of biomass for all Gram-negative strains, while ≈85 % eradication was achieved for S. aureus . Approximately 3 % of Gram-negative bacteria and 5 % of S. aureus were metabolically active. Significant differences were observed for all treatments on biofilm ( p < 0.05), with the greatest effects on Gram-negative bacteria. The IL cholinium ʟ-alaninate ([Cho][Ala]) effectively prevented biofilm formation and eradicated preformed biofilms. We recommend investigating multidrug resistant pathogens and surfaces that exhibit enhanced adherence.