Novel Low-cost ‘Green’ Antimicrobial Peptides for Topical Oral Biofilm Treatment

Objectives: Objectives: Protein drugs are minimally utilized in dental medicine due to high cost and invasive local delivery. There is limited clinical advancement in disrupting virulent oral biofilms, despite causing highly prevalent and costly diseases such as dental caries. Poor efficacy of antimicrobials following topical treatments or to penetrate and disrupt formed biofilms is a major challenge. Here, we developed an exciting low-cost approach using plant-made antimicrobial peptides (PMAMPs) for topical use to control biofilms.
Methods: Methods: PMAMP Protegrin 1 (PG1) was expressed in chloroplasts as GFP fusion proteins and purified by affinity chromatography. The antimicrobial functionality of PG1 against Streptococcus mutans and other oral microbes was analyzed by time-lapse killing assay. Morphology and dynamics of killing of S. mutans treated with PG1 were determined by scanning electron microscopy (SEM) and high-resolution confocal microscopy. Both cell viability and 3D architecture of the biofilms were determined via biochemical, microbiological and fluorescence-based methods.
Results: Results: PG1 rapidly killed S. mutans and other relevant bacteria (e.g. Actinomyces naeslundii) cells within 1h at concentrations ranging from 5-10µg/ml. Time-lapse confocal imaging showed that bacterial viability was affected as early as 10min. SEM imaging revealed disruption of S. mutans membrane surface, causing irregular cell morphology and extrusion of the intracellular content. More importantly, a single topical treatment of saliva-coated hydroxyapatite (teeth surrogate) with PG1 substantially impaired biofilm formation by S. mutans resulting in minimal accumulation of viable cells or EPS-matrix on the treated surface. Microbiological data revealed that most of the bacterial cells were killed by PG1 (vs. vehicle-control treated biofilms), demonstrating potent anti-biofilm effects despite brief topical exposure.
Conclusions: Conclusions: Our findings could lead to a novel and feasible platform for development of anti-biofilm therapeutics using low-cost, plant-produced AMPs for topical use.