Enzymatically Combating Biofilm Associated Microbial Infections: Towards Next-Gen Enzyme-Based Therapeutics

Abstract

Bacteria employ various defense mechanisms, one of the most significant being forming biofilms─structured communities of microorganisms encased in a self-produced extracellular polymeric substance (EPS) matrix. The widespread occurrence of biofilms across both natural and artificial environments poses a significant concern, as they adversely impact daily life. Biofilms protect bacterial communities, making them up to 1,000 times more antibiotic-tolerant than their free-floating (planktonic) counterparts. This increased resistance necessitates higher antibiotic dosages, which raises the risk of adverse side effects for patients and imposes a considerable economic burden. In most biofilms, 50–95% of the structure consists of a complex EPS matrix composed of polysaccharides, proteins, lipids, and extracellular DNA. Polysaccharides are often the predominant components, providing structural strength and inertness and contributing to the biofilm’s resistance to antibiotics and immune responses. Therefore, targeting polysaccharides of the EPS matrix presents a promising strategy to prevent and disrupt biofilms. Two key enzyme classes ─ glycoside hydrolases (GHs) and lytic polysaccharide monooxygenase (LPMOs), are known for their ability to degrade polysaccharides and are used in biomass deconstruction for biofuel production. In my research, we unveiled the untapped potential of the cow rumen microbial GH enzymes and microbial LPMOs to combat the biofilm-associated infections caused by clinically relevant pathogens. We have successfully expressed, purified, and biochemically characterized two cow rumen microbial GHs (GH-B2 and CRhAB) and an LPMO (Vc-LPMO). Among them, GH-B2 effectively degraded the biofilm matrix and exhibited potent activity against multiple clinical isolates of K. pneumoniae. It also significantly enhanced the efficacy of the broad-spectrum antibiotic meropenem and host immune cells, resulting in efficient biofilm clearance and wound healing in a murine wound infection model. Building on this approach, we evaluated the anti-biofilm activity of the next GH, CRhAB. This enzyme efficiently prevented the formation of A. baumannii biofilm on various medical devices. To access translational potential, we immobilized CRhAB onto wound dressing gauze, successfully preventing A. baumannii biofilm formation on wound sites in a murine model, highlighting its promise for wound care applications. Expanding this line of investigation, we explored the biofilm-disrupting potential of a recently discovered class of lytic polysaccharide monooxygenase (Vc-LPMO) and demonstrated its ability to disrupt biofilms formed by A. baumannii, K. pneumoniae, and their dual-species biofilms. Overall, our research opens new avenues for utilizing novel microbial enzymes as a robust tool for managing biofilm-associated infections, which are a global crisis.