Near-infrared light-enhanced protease-conjugated gold nanorods as a photothermal antimicrobial agent for elimination of exotoxin and biofilms

Abstract

Purpose: Treatment strategies to eliminate bacterial infections have long emphasized bacterial killing as a goal. However, bacteria secrete toxins that sustain chronic disease and dead cells release DNA that can promote the spread of antibiotic resistance even when viable cells are eradicated. Meanwhile, biofilms regulated by quorum-sensing system, protect bacteria and promote the development of antibiotic resistance. Thus, all of these factors underscore the need for novel antimicrobial therapeutic treatments as alternatives to traditional antibiotics. Here, a smart material was developed that incorporated gold nanorods and an adsorbed protease (protease-conjugated gold nanorods, PGs). When illuminated with near-infrared (NIR) light, PGs functioned to physically damage bacteria, prevent biofilm and exotoxin production, eliminate pre-existing biofilm and exotoxin, and inhibit bacterial quorum-sensing systems. Methods: PGs were incubated with suspensions of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria followed by exposure to 808-nm NIR laser irradiation. Bacterial viability was determined using a colony-forming unit assay followed by an exploration of cell-damage mechanisms using transmission electron microscopy, scanning electron microscopy, agarose gel electrophoresis, and SDS-PAGE. Quantification of biofilm mass was performed using crystal violet staining. A commercial enterotoxin ELISA kit was used to test inhibitory and degradative effects of PGs on secreted exotoxin. Results: Use of the remote-controlled antibacterial system reduced surviving bacterial populations to 3.2% and 2.1% of untreated control numbers for E. coli and S. aureus, respectively, and inhibited biofilm formation and exotoxin secretion even in the absence of NIR radiation. However, enhanced degradation of existing biofilm and exotoxin was observed when PGs were used with NIR laser irradiation. Conclusion: This promising new strategy achieved both the reduction of viable microorganisms and elimination of biofilm and exotoxin. Thus, this strategy addresses the long-ignored issue of persistence of bacterial residues that perpetuate chronic illness in patients even after viable bacteria have been eradicated.