Evaluation of the effects of antimicrobial peptides on endodontic pathogens in vitro

Abstract

Aim - To assess the in vitro antimicrobial efficacy of antimicrobial peptides against common endodontic pathogens in the planktonic phase and biofilms.

Method - Strains of Enterococcus faecalis, Streptococcus gordonii, Streptococcus mutans, Streptococcus sanguinis, Candida albicans, Staphylococcus aureus, Escherichia coli, and Fusobacterium nucleatum were grown from glycerol stocks in closed tubes containing Todd Hewitt Broth (THB) for 24 h. Dilution series of the artificial peptides BM2, BM2L and BM2-2W, the natural peptides magainin 2 and buforin 2, as well as the widely used endodontic irrigant sodium hypochlorite (NaOCl) were prepared in aqueous solution. Values for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using a broth micro-dilution method. The D-decapeptide BM2 (D-NH2-RRRWFFWRRR-CONH2) showed the most potent antimicrobial action and was assessed further using two monoculture biofilm models prepared with E. faecalis strain JH2-2. The efficacy of BM2 and NaOCl at causing biofilm dettachment was measured using a crystal violet- based assay. A more sophisticated biofilm formed on a collagen coated 8-well chamber slide was exposed to BM2 or NaOCl, stained using a LIVE/DEAD assayTM kit and visualised using confocal laser scanning microscopy (CLSM).

Results - The five peptides tested showed antimicrobial activity that was different for each microbial species and strain. BM2 was most the potent antimicrobial against the microbial isolates tested. The strains most susceptible to BM2 were S. sanguinius ATCC 10557 (MIC 1 μg/mL, MBC 4 μg/mL) and S. mutans NG8 (MIC 2 μg/mL, MBC 2 μg/mL). The strain least susceptible to BM2 was S. aureus C55 (MIC 32 μg/mL, MBC 32 μg/mL). BM2L (the L-enantiomer of BM2) and BM2-2W (BM2 with its two D- tryptophans replaced with L-tryptophans) performed similarly, showing antimicrobial activity against most isolates but not the E. faecalis strains. The strain most susceptible to BM2L was S. mutans NG8 (MIC 2 μg/mL, MBC 4 μg/mL). The strains that were most susceptible to BM2-2W were S. mutans NG8 (MIC 2 μg/mL, MBC 4 μg/mL), S. mutans UA159 (MIC 2 μg/mL, MBC 4 μg/mL), and E. coli 9086 (MIC 2 μg/mL, MBC 4 μg/mL), S. mutans NG8 (MIC 2 μg/mL, MBC 4 μg/mL). Magainin 2 and buforin 2 at 128 μg/mL were bacteriostatic against some strains but were not bactericidal. NaOCl (1250- 5000 μg/mL) was bacteriostatic and had bactericidal activity between (2500- 5000 μg/mL). The crystal violet biofilm assay showed NaOCl (5000-50000 μg/mL) was much more effective at E. faecalis biofilm detachment than BM2 (4-128 μg/mL). CLSM assessment of the effects BM2 (160 μg/mL) on the collagen-attached biofilm model demonstrated almost complete loss of cell viability but only a slight reduction in biofilm height and limited cell detachment from substratum.

Conclusion - The artificial cationic BM2 peptide at concentrations at least several hundred-fold lower than the endodontic irrigant NaOCl had microbicidal activity against both planktonic cultures of oral pathogens and biofilms of the significant endodontic pathogen E. faecalis. This supports further investigation of this artificial cationic peptide as a potential endodontic antimicrobial.