Characterization Of EC16 Formulations For Their Antibiofilm and Antivirulence Effects on Pseudomonas aeruginosa

Biofilm-forming bacteria present significant public health challenges due to their resistance to antibiotics and environmental persistence. Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative opportunistic pathogen, is particularly known for its robust biofilm production and drug resistance mechanisms. This study evaluates the antibiofilm and anti-virulence properties of two formulations containing epigallocatechin-3-gallate-palmitate (EC16), a modified green tea polyphenol. Minimum inhibitory concentration (MIC) and IC₅₀ values were determined via microplate-based antibacterial assays. CF2 and F6 formulations exhibited MICs at 10% of their original concentrations. Cell viability was determined using fluorescent imaging analysis, analysis revealed 97% and 95% reductions in cell viability when treated with 10% CF2 and 10% F6 respectively. Congo red and crystal violet assays showed a 45% (CF2) and 84% (F6) decrease in biofilm attachment. Virulence factor attenuation was confirmed through decreased elastase activity and reduced pyoverdine and pyochelin fluorescence via spectrophotometry. Gene expression analysis using RT-PCR, RT-qPCR, and RNA sequencing revealed significant downregulation of key biofilm- and virulence-associated genes, including pqsA,rhlA, pslA, lasI, lasR, gacS, and vfr. These genes regulate quorum sensing, extracellular polymeric substance (EPS) synthesis, and global virulence pathways. STRING database analysis further confirmed their interaction within key regulatory networks. This study demonstrates that EC16 formulations effectively inhibit biofilm formation and suppress virulence in P. aeruginosa. These findings support the potential of EC16 as a promising antibiofilm agent and provide a foundation for future investigations targeting other clinically relevant biofilm-producing pathogens.