Esc(1-21) a novel antimicrobial peptide for microbial keratitis?

Purpose: To investigate the antimicrobial efficacy of a novel amphibian antimicrobial peptide, Esculentin 1-21 (Esc 1-21) in vitro. Methods: Microbroth dilution assays were used to determine the minimal inhibitory concentration (MIC) of Esc(1-21) against PA strain ATCC 27853 and the effects of salt and tears (basal and reflex) on peptide antimicrobial activity. Esc(1-21) activity on a pre-formed biofilm was also tested using the Calgary Biofilm device and evaluated by analysis of re-growth, viable cell (CFU counts and MTT assay) and biomass. Results: The MIC for Esc(1-21) was 4uM (n=3). Esc(1-21), 1uM, retained the ability to kill 100% of ATCC 27853, within 20 min, in 150mM NaCl (n=3). When tested in the presence of 50 and 70% v/v reflex human tears, killing of ATCC 27853 by 20uM Esc(1-21) was 100% and 94% respectively, after 90 min of peptide treatment. When tested in the presence of 50 and 70% v/v basal human tears, killing was 98% and 70% respectively. The minimum biofilm eradication concentration (concentration inhibiting re-growth of bacteria from peptide treated biofilm) was 6uM (n=4) and the minimum bactericidal concentration (concentration required to reduce the number of viable biofilm cells by ≥3 log10) was 12uM (n=3). Biofilm biomass, evaluated by crystal violet staining, was 15% to 32% for 48-12uM peptide. Conclusions: Esc(1-21) is effective against both the free-living and sessile forms of PA in vitro and importantly retains significant bactericidal activity in the presence of human tears. As antimicrobial peptides are recognized to induce minimal pathogen resistance Esc(1-21) is a very promising candidate for a novel therapeutic for the treatment of microbial keratitis.