Membrane perturbation, altered morphology and killing of Staphylococcus epidermidis upon contact with a cytocompatible peptide-based antibacterial surface

Abstract

One possibility to prevent prosthetic infections is to produce biomaterials resistant to bacterial colonization by anchoring membrane active antimicrobial peptides (AMPs) onto the implant surface. In this perspective, a deeper understanding of the mode of action of the immobilized peptides should improve the development of AMP-inspired infection-resistant biomaterials. The aim of the present study was to characterize the bactericidal mechanism against Staphylococcus epidermidis of the AMP BMAP27(1-18), immobilized on titanium disks and on a model resin support, by applying viability counts, Field Emission Scanning Electron Microscopy (FE-SEM), and a fluorescence microplate assay with a membrane potential-sensitive dye. The cytocompatibility to osteoblast-like MG-63 cells was investigated in monoculture and in co-culture with bacteria. The impact of peptide orientation was explored by using N- and C- anchored analogues. On titanium, the ∼50 % drop in bacteria viability and dramatically affected morphology indicate a contact-killing action exerted by the N- and C-immobilized peptides to the same extent. As further shown by the fluorescence assay with the resin-anchored peptides, the bactericidal effect was mediated by rapid membrane perturbation, similar to free peptides. However, at peptide MBC resin equivalents the C-oriented analogue proved more effective with more than 99 % killing and maximum fluorescence increase, compared to half-maximum fluorescence with more than 90 % killing produced by the N-orientation. Confocal microscopy analyses revealed 4-5 times better MG-63 cell adhesion on peptide-functionalized titanium both in monoculture and in co-culture with bacteria, regardless of peptide orientation, thus stimulating further studies on the effects of the immobilized BMAP27(1-18) on osteoblast cells.

Keywords: Antimicrobial peptide-functionalized titanium; Bacterial morphology by FE-SEM; Bactericidal biomaterial; Fluorescence microplate assay; Osteoblast-bacteria co-culture.