In vitro effect of an antimicrobial peptide against mono-species endodontic biofilms

Objective: To determine if the synthetic peptide BM2 has antimicrobial activity against mono-species biofilms of common endodontic pathogens. Methods: Strains of the pathogenic bacteria Enterococcus faecalis JH2-2, Streptococcus gordonii DL1, and Streptococcus mutans NG8 and the fungal pathogen Candida albicans ATCC 10261 were grown from glycerol stocks in closed tubes containing Todd Hewitt Broth (THB) or Tryptone Soy Broth (TSB) for 24 hours. Mono-species biofilms were prepared in 96-well plates for 72 hours before exposure to antimicrobial agents. Dilution series of the synthetic antimicrobial peptide BM2 (D-NH2-RRRFWWFRRR-CONH2) and the widely used endodontic antimicrobial agents sodium hypochlorite (NaOCl) and saturated calcium hydroxide (Ca(OH)2) were prepared as aqueous solutions. The efficacy of BM2 (10 µg/mL, 20 µg/mL and 40 µg/mL), NaOCl (2,500 µg/mL, 5,000 µg/mL and 10,000 µg/mL) and saturated Ca(OH)2 at limiting cell growth and possibly causing biofilm disruption was measured using a crystal violet assay at 24, 48 and 72 hours. A more clinically relevant biofilm formed on sterile root canal surfaces was exposed to BM2, NaOCl and Ca(OH)2, stained using a LIVE/DEAD assay™ kit and visualized using confocal laser scanning microscopy (CLSM). Results: Both the crystal violet biofilm assay and the CLSM assessment showed BM2 was an effective antimicrobial against all microorganisms tested. After 72h BM2 at 20 µg/mL caused reduction to the thickness of C. albicans biofilm and total viable cells by only 10%. BM2 at 40µg/ml was as effective as NaOCl (10,000 µg/mL) and saturated Ca(OH)2 against S. gordonii DL1 and S. mutans NG8. Mean biofilm thickness (A600nm) was reduced and the remaining cell showed 25% viability or less after 72 hour of exposure. BM2 at 40 µg/mL showed antimicrobial activity similar to saturated Ca(OH)2 or 2,500 and 5,000 µg/mL NaOCl in disrupting the E. faecalis JH2-2 biofilm. Although mean biofilm thickness was reduced slightly non-viable cells contributed 60% of to total biofilm volume. Conclusion: BM2 at 40 µg/mL showed robust antimicrobial activity against biofilms of major endodontic pathogens comparable to results achieved with current antimicrobial disinfectants. It is important to further investigate BM2 as an alternative antimicrobial agent in endodontic treatment