Effect of GT-Peptide 10 and Triethyl Citrate on P. acnes Biofilm Formation, Viability, and Dispersion

June 2016 | Volume 15 | Issue 6 | Original Article | 778 | Copyright © June 2016

Hinnerk Eilers PhD and Oleg A. Alexeyev MD PhD

Department of Pathology, Umeå University, Umeå, Sweden

Abstract
BACKGROUND: P. acnes biofilms are emerging topics in acne vulgaris pathogenesis and may be responsible for antibiotic tolerance.
OBJECTIVE: To investigate the efficacy of GT peptide 10 either alone or in combination with triethyl citrate (TEC) in in vitro model of P. acnes biofilm.
METHODS: Six-day-old P. acnes biofilms were treated with various concentrations of these substances and biofilm dispersion and cell viability were monitored.
RESULTS: A 24-hour exposure of preformed biofilms to a combination of GT peptide 10/TEC led to killing of up to 92% of bacterial cells inside the biofilm. Neither the single substance nor the combination of both substances affected the biofilm integrity or resulted in biofilm dispersal.
CONCLUSIONS: A combination of GT peptide 10/TEC shows antibacterial effects in in vitro model of P. acnes biofilm.

J Drugs Dermatol. 2016;15(6):778-781.

INTRODUCTION

Acne vulgaris is a chronic inflammatory skin disease primarily targeting pilosebaceous units. The disease affects a majority of young adolescents and is associated with a profound psychological discomfort. The pathogenesis of the disease is complex and involves several factors.1 Colonization of hair follicles by P. acnes is thought to contribute to skin inflammation though there are opposing views on the pathogenic role of bacterium.2,3 P. acnes is believed to trigger inflammation and tissue injury originating in the sebaceous follicle through both innate and adaptive immune responses. Clinical effectiveness of topical and systemic antibiotics in acne vulgaris is a strong indicator for P. acnes involvement in acne pathogenesis. A relatively new concept in acne pathogenesis is a possible involvement of P. acnes biofilms. Biofilm is a microbial aggregate surrounded by extracellular matrix with phenotypic characteristics different from individual bacterial species that comprise it.4 An important feature of biofilms is their antibiotic resistance despite a high sensitivity of individual bacterial cells. For some, but not all, bacterial species the matrix can form a further hinder for antibiotic penetration.5 P. acnes biofilms have indeed been directly demonstrated in acne lesions.6-8 Antibiotic therapy directed against P. acnes has been a mainstay of acne treatment for many decades. Protracted courses of various antibiotics have led to accumulation of mutations in the genes encoding the 23S and 16S subunits of ribosomal RNA and emergence of resistant bacterial strains.9 Given a severe problem with antibiotic-resistant organisms whereby a therapeutic utility of antibiotics is gradually losing effectiveness, there is an urgent need for new strategies to treat acne vulgaris. The problem is further aggravated by the even greater resistance of biofilms. Another antibacterial therapy option is related to the usage of antibacterial peptides. Endogenous antimicrobial peptides such as defensins and cathelicidins are able to kill bacteria directly and modulate interactions between the innate and adaptive immune system.10 Exogenous and synthetic peptides are being developed with the goal of augmenting antibacterial effects.11 Few studies reported a possible usefulness of antimicrobial peptides against P. acnes.12,13 In this study, we evaluated anti-biofilm effects of a synthetic peptide GT peptide 10 either alone or in combination with and triethyl citrate (TEC) on formation, dispersal, and viability of P. acnes biofilm using an in vitro model.

METHODS

Reagents and Propionibacteria Species

GT peptide 10 (a 15 amino acid long peptide) and TEC are used in a product line Acnecare by General Topics (Italy) and were provided by the manufacturer. P. acnes IB (KPA171202) and clinical isolates (PRP60, PRP62, PRP78, 1663, 46361, and 50460), P. granulosum (DSM 20700), and P. avidum (DSM 4901) were grown anaerobically in BHI medium supplemented with 2g/l glucose at 37°C. Biofilm cultures were grown in 24 well plates (NUNC) for six days before susceptibility testing. A representative picture of the biofilm in shown (Figure 1).

Antibacterial Susceptibility Testing

The minimal inhibition concentrations (MIC) for GT peptide 10 and TEC on planktonic forms were determined by measuring the optical density at 590nm after a 48-hour growth.
The antibacterial susceptibility of preformed P. acnes biofilms was studied by a modified crystal violet assay as described.14