Domain driven mining of high activity anti-biofilm glycoside hydrolases in pathogenic bacteria

Biofilm formation represents a critical antimicrobial resistance mechanism contributing to persistent chronic infections. Glycoside hydrolases encoded within the exopolysaccharide synthesis gene clusters of pathogenic bacteria play pivotal roles in biofilm formation and dispersal processes, making the mining of anti-biofilm glycoside hydrolases from pathogen genomes a promising therapeutic strategy. In this study, we established a systematic pipeline for screening anti-biofilm glycoside hydrolases from Klebsiella pneumoniae and Acinetobacter baumannii genomes. Among 13 candidate enzymes screened, 7 were successfully expressed in soluble form, with PgaB_Ab1 and PgaB_Kp demonstrating potent anti-biofilm activity against Staphylococcus epidermidis biofilms. Among them, PgaB_Ab1 exhibited the highest anti-biofilm activity, with EC₅₀ values for the disruption and inhibition of S. epidermidis biofilms being 2.83 ± 0.65 nM and 4.53 ± 1.19 nM, respectively. Enzymatic characterization revealed optimal activity at pH 7.0 and remarkable stability at 37 °C for both enzymes. Mechanistic investigations confirmed that PgaB_Ab1 and PgaB_Kp mediate biofilm dispersal through enzymatic degradation of dPNAG. Notably, enzymatic pretreatment with these glycoside hydrolases significantly enhanced methicillin susceptibility in S. epidermidis. Our findings highlight the untapped potential of pathogenic bacteria as sources of therapeutic glycoside hydrolases and provide novel insights for addressing biofilm-associated chronic infections.