Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms

Abstract

Event Abstract Back to Event Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms Dalal Asker1, 2, Tarek Awad1, Perrin Baker3, Lynne Howell3, 4 and Benjamin Hatton1 1 University of Toronto, Materials Science and Engineering, Canada 2 Alexandria University, Food Science & Technology Department, Egypt 3 Hospital for Sick Children, Canada 4 University of Toronto, Dept. of Biochemistry, Canada Introduction: Implanted devices are at significant risk of developing bacterial biofilm-associated infections. Approximately 60% of all hospital-acquired infections (HAIs) are the result of biofilm formation on implanted medical devices and the bacterium Pseudomonas aeruginosa is one of the most prevalent isolated pathogens. Bacterial biofilms are communities of bacteria encased within a protective extracellular matrix. Exopolysaccharides (EPS) are the predominant component of biofilm matrix for many bacteria, contributing to initial adhesion, architecture, and resistance. Bacteria in biofilm are highly tolerant to antibiotics and can evade the host immune system. Recently we identified and produced several recombinant glycoside hydrolases that selectively target and hydrolyze biofilm EPS. Two of these enzymes - PslGh, and PelAh - hydrolyze the Psl and Pel polysaccharides from P. aeruginosa, respectively, and were found to be effective in solution at disrupting the biofilms from lab, clinical and environmental isolates. Current antibiotic therapy and antimicrobial surfaces are ineffective against biofilm infections. Therefore, we aim to use surface-immobilized enzymes as a means of preventing biofilm formation on implanted medical devices such as endotracheal tubes. Materials and Methods: We tested the immobilization of PslGh onto polymer biomaterial surfaces as a means of preventing biofilm growth for P. aeruginosa in long term static and flow culture. The enzyme was covalently bound with glutaraldehyde cross-linking to glass and PDMS surfaces and its attachment confirmed by ATR-FTIR and contact angle measurements. To evaluate anti-biofilm efficacy, our biomaterial surfaces were incubated in P. aeruginosa culture that overexpresses the Psl polysaccharide for varying duration. Fluorescence microscopy and fluorescent dyes (SYTOX Green) were used and for quantitative analysis, we used turbidity and crystal violet staining methods. Results and Discussion: The covalently-bound PslGh inhibited both cell attachment and biofilm formation of the P. aeruginosa (Psl-dependent) cells (Fig 1a), compared with the control (i.e., no enzyme) or a catalytically inactive enzyme (Fig. 1b) over a 24 hour period. This indicates that the anti-biofilm activity of PslGh is directly related its catalytic activity. The covalently-immobilized PslGh enzyme was also effective at preventing biofilm formation for an 8 day static culture. Quantitative analysis using crystal violet straining indicated significant anti-biofilm activity for the immobilized PslG, as compared with the control sample. Conclusion: These results indicate the high specificity of PslGh in targeting the Psl exopolysacchride in P. aeruginosa biofilms, which appears to greatly inhibit bacterial colonization and biofilm growth, even when the enzyme is covalently-immobilized on the polymer surface. Figure 1. Inhibition of P. aeruginosa (Psl-dependent) biofilm attachment and formation by covalently-attached PslGh on PDMS samples. (a) untreated PDMS; (b) catalytically-inactive mutant PslGh (mPslGh) covalently immobilized on PDMS; and (c) PslGh covalently immobilized on PDMS. Images were observed with 20X dry objectives. Scale bars, 40 µm. Keywords: Bacteria, Implant, biofunctionalization, bioactive interface, enzyme Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Interfacial phenomena Citation: Asker D, Awad T, Baker P, Howell L and Hatton B (2016). Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00131 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Google Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Google Scholar Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton PubMed Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.