Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms.

Xi Chen,Yuping Li,Helmut Hirt,Conrado Aparicio,Sven-Ulrik Gorr,Jens Kreth

doi:10.1371/journal.pone.0111579

Abstract

Infection is one of the most prevalent causes for dental implant failure. We have developed a novel antimicrobial peptide coating on titanium by immobilizing the antimicrobial peptide GL13K. GL13K was developed from the human salivary protein BPIFA2. The peptide exhibited MIC of 8 µg/ml against planktonic Pseudonomas aeruginosa and their biofilms were reduced by three orders of magnitude with 100 µg/ml GL13K. This peptide concentration also killed 100% of Streptococcus gordonii. At 1 mg/ml, GL13K caused less than 10% lysis of human red blood cells, suggesting low toxicity to mammalian cells. Our GL13K coating has also previously showed bactericidal effect and inhibition of biofilm growth against peri-implantitis related pathogens, such as Porphyromonas gingivalis. The GL13K coating was cytocompatible with human fibroblasts and osteoblasts. However, the bioactivity of antimicrobial coatings has been commonly tested under (quasi)static culture conditions that are far from simulating conditions for biofilm formation and growth in the oral cavity. Oral salivary flow over a coating is persistent, applies continuous shear forces, and supplies sustained nutrition to bacteria. This accelerates bacteria metabolism and biofilm growth. In this work, the antimicrobial effect of the coating was tested against Streptococcus gordonii, a primary colonizer that provides attachment for the biofilm accretion by P. gingivalis, using a drip-flow biofilm bioreactor with media flow rates simulating salivary flow. The GL13K peptide coatings killed bacteria and prevented formation and growth of S. gordonii biofilms in the drip-flow bioreactor and under regular mild-agitation conditions. Surprisingly the interaction of the bacteria with the GL13K peptide coatings ruptured the cell wall at their septum or polar areas leaving empty shell-like structures or exposed protoplasts. The cell wall rupture was not detected under regular culture conditions, suggesting that cell wall rupture induced by GL13K peptides also requires media flow and possible attendant biological sequelae of the conditions in the bioreactor.

Highlights

Dental implants have rapidly become the treatment of choice for patients who are in need to replace missing teeth
We have focused on developing an antimicrobial peptide coating with activity against pathogens associated with dental periimplantitis
In this work we have shown for the first time that antimicrobial GL13K peptide coatings induced cell wall rupture under dynamic salivary-flow rate conditions on a drip flow bioreactor, acting differently from GL13K in solution or tested under regular culture conditions where exposure did not result in cell lysis [20,21]

Summary

Introduction

Dental implants have rapidly become the treatment of choice for patients who are in need to replace missing teeth. Different cationic antimicrobial peptides derived from human proteins have been either physically adsorbed [15,16] or covalently attached [17] on implant surfaces. These implants displayed antimicrobial activity against pathogens related with orthopedic peri-implantitis. We bonded the antimicrobial peptide GL13K to titanium surfaces using silane coupling agents to produce coatings that have covalent attachment to the metallic substrate and that have significant antimicrobial activity against the Gram negative bacterium Porphyromonas gingivalis [18], an oral pathogen that is closely associated with the development of biofilms and dental peri-implantitis [19]. The coating had resistance to hydrolytic and mechanical challenges with no significant release of peptides from the titanium surface and was cytocompatible with osteoblasts and human gingival fibroblasts [18]

Methods

Results

Conclusion