ORIGINAL ARTICLE Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface Bruno Casciaro 1 | Debarun Dutta 2 | Maria Rosa Loffredo 1 | Stefania Marcheggiani 3 | Alison M McDermott 4 | Mark DP Willcox 2 | Maria Luisa Mangoni 1 1 Laboratory affiliated to Pasteur Italia- Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy 2 School of Optometry and Vision Science, University of New South Wales, Sydney, Australia 3 Department of Environment and Health, Istituto Superiore di Sanit a, Rome, Italy 4 Department of Applied Sciences, Northumbria University, Newcastle upon Tyne, United Kingdom Correspondence Maria Luisa Mangoni, Department of Biochemical Sciences, Sapienza University of Rome, via degli Apuli, 9-00185 Rome, Italy. Email: marialuisa.mangoni@uniroma1.it Funding Information Grant sponsor: Sapienza Università di Roma; National Health and Medical Research Council, Grant number: APP107620 Abstract Contact lens (CL) wear is a risk factor for development of microbial keratitis, a vision threatening infection of the eye. Adverse events associated with colonization of lenses, especially by the multi- drug resistant and biofilm forming bacterium Pseudomonas aeruginosa remain a major safety issue. Therefore, novel strategies and compounds to reduce the onset of CL-associated ocular infections are needed. Recently, the activity of the frog skin-derived antimicrobial peptide Esc(1-21) and its diastereomer Esc(1-21)-1c was evaluated against both planktonic and sessile forms of this patho- gen. Furthermore, Esc(1-21) was found to significantly reduce the severity of P. aeruginosa keratitis in a mouse model and preserve antipseudomonal activity in the presence of human basal tears. Here, we have analyzed the activity of the peptides on P. aeruginosa biofilm formed on soft CLs. Microbiological assays and scanning electron microscopy analysis indicated that the peptides were able to disrupt the bacterial biofilm, with the diastereomer having the greater efficacy (up to 85% killing vs no killing at 4 lM for some strains). Furthermore, upon covalent immobilization to the CL, the two peptides were found to cause more than four log reduction in the number of bacterial cells within 20 minutes and to reduce bacterial adhesion to the CL surface (77%–97% reduction) in 24 hours. Importantly, peptide immobilization was not toxic to mammalian cells and did not affect the lens characteristics. Overall, our data suggest that both peptides have great potential to be devel- oped as novel pharmaceuticals for prevention and treatment of CL-associated P. aeruginosa keratitis. KEYWORDS antimicrobial peptides, contact lens, keratitis, Pseudomonas aeruginosa 1 | INTRODUCTION Microbial keratitis is a severe sight-threatening event and can rapidly progress to corneal perforation within 2–3 days. [1] The use of soft con- tact lenses (CL), one of the most popular ways to correct vision disor- ders, accounts for approximately 12%–66% of all microbial keratitis cases. [2] This is mainly due to the colonization of lenses by microorgan- isms, such as Gram-positive and Gram-negative bacteria, including the ubiquitous Pseudomonas aeruginosa. [3–5] Indeed, P. aeruginosa is the most frequently isolated bacterium from CL-associated microbial kera- titis [6,7] accounting for 70% of all isolates. P. aeruginosa has the capacity to rapidly adhere to silicone hydrogel or hydrogel lenses form- ing sessile communities, that are called biofilms. [8] In addition, it can invade or directly kill host cells leading to damage and possible perfora- tion of the corneal tissue resulting in vision loss. [9,10] Invasion and cell death is a result of the production of bacterial virulence factors [10–13] and the recruitment/activation of host defense cells, primarily neutro- phils, which contribute to the clinical manifestations of the disease. [14] Abbreviations: AMP, antimicrobial peptide; CFU, colony-forming unit; CL, contact lens; DMEM, Dulbecco’s modified Eagle’s medium; EDC, 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride; LB, Luria–Bertani broth; MAA, methacrylic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; SAB, sodium acetate buffer. Biopolymers. 2017;e23074. https://doi.org/10.1002/bip.23074 wileyonlinelibrary.com/peptidesci V C 2017 Wiley Periodicals, Inc. | 1 of 9 Received: 3 August 2017 | Revised: 14 September 2017 | Accepted: 4 October 2017 DOI: 10.1002/bip.23074