Enhanced biofilm disruption in ESKAPE pathogens through synergistic activity of EPS degrading enzymes

Abstract Biofilm formation by Klebsiella pneumoniae, Acinetobacter baumannii, and methicillin-resistant Staphylococcus aureus (MRSA) significantly contributes to antimicrobial resistance (AMR), complicating infections associated with medical devices. This study investigates the potential of α-Amylase, DNase I, and Proteinase K in disrupting extracellular polymeric substances (EPS) within biofilms to enhance biofilm inhibition. Molecular docking studies revealed a strong interaction between α-Amylase and cellulose (−3.58 kcal/mol), suggesting effective targeting of biofilm polysaccharides. Biofilm inhibition was quantified using the crystal violet microtiter plate assay, and structural changes were visualized through confocal laser scanning microscopy (CLSM). The checkerboard synergy assay showed that enzyme combinations achieved up to 90 % biofilm inhibition, significantly outperforming individual enzymes (50–70 %). Notably, α-Amylase + DNase I and α-Amylase + Proteinase K exhibited synergy (FICI ≤ 0.5) in MRSA and A. baumannii , while DNase I + Proteinase K showed limited activity in A. baumannii (FICI = 2.0), suggesting biofilm composition differences influence enzymatic activity. Confocal microscopy analysis revealed that biofilm thickness was reduced by 50 % in K. pneumoniae and by 60 % in A. baumannii , further supporting enzyme-mediated biofilm disruption. These findings highlight the potential clinical applications of enzymatic therapy, particularly in preventing ventilator-associated pneumonia (VAP) by inhibiting biofilm formation and disrupting preformed biofilms in endotracheal tubes and improving antimicrobial efficacy. This study establishes α-Amylase as a potent biofilm-disrupting agent, with synergistic enzyme therapy offering a viable strategy to combat biofilm-associated infections. Currently, we are focusing on optimizing enzyme formulations for clinical application and evaluating their combination with antibiotics to enhance therapeutic outcomes.