Evaluation of a novel CHX-EC16 antimicrobial formulation on biofilm-forming bacteria

The rise of antibiotic-resistant bacteria presents a growing threat to public health, particularly in clinical settings where overuse of antibiotics accelerates resistance. This study evaluated the antibacterial and antibiofilm efficacy of a novel formulation combining chlorhexidine (CHX) with EC16, a lipid-modified derivative of (-)-epigallocatechin-3-gallate, against Gram-negative Klebsiella aerogenes (K. aerogenes) and Gram-positive Staphylococcus epidermidis (S. epidermidis). The minimum inhibitory concentration (MIC) for K. aerogenes was 0.004% CHX/0.0002% EC16 and 0.0008% CHX/0.00004% EC16 for S. epidermidis, 0.02%. Checkerboard assays confirmed synergistic effects between CHX and EC16. When treating K. aerogenes with 0.002% CHX/0.0001% EC16, it was able to inhibit bacterial growth slightly better than 0.002% CHX by itself. Fluorescent microscopy was performed using DAPI to stain the A-T rich regions of the nuclear DNA. Results showed morphological changes of K. aerogenes shifting from rod-shaped to coccoid forms when treated with 0.004% CHX/0.0002% EC16. SYTO-9, a cell permeable green, fluorescent nucleic acid stain, and propidium iodide a cell impermeable red fluorescent nucleic acid stains were used to visualize the change in viable cells when treated with CHX-EC16. Flow cytometry confirmed a >97.8 % reduction in planktonic bacterial viability at 6 hours post-treatment and a 96.06% reduction in biofilm-associated cells at 24 hours for K. aerogenes. S. epidermidis saw biofilm reduction using lower concentrations of the formulation with 0.001% CHX/0.00005% indicating 89.84% inhibition and 0.008% CHX/0.00004% EC16 showing 85.25% inhibition 24 hours post-treatment. Congo red assays indicated minimal to no biofilm production in formulation-treated samples up to 48 hours. These findings support the potential of CHX-EC16 as an effective antimicrobial formulation. By enhancing efficacy while reducing the required CHX concentration, EC16 may mitigate side effects associated with prolonged CHX use, such as altered sense of taste, staining of teeth, and mucosal irritation, as well as offering a promising alternative for managing drug-resistant nosocomial infections.