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Nanostructured Coatings for Controlling Bacterial Biofilms and Antibiotic Resistance

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Nanostructured Coatings for Controlling Bacterial Biofilms and Antibiotic Resistance Nanostructured coatings for controlling bacterial biofilms and antibiotic resistance Kristina Dimitrova Ivanova A thesis submitted in fulfilment of the requirements for degree of Doctor of Philosophy at the Universitat Politècnica de Catalunya Supervised by Dr. Tzanko Tzanov Group of Molecular and Industrial Biotechnology (GBMI) Department of Chemical Engineering Universitat Politècnica de Catalunya Terrassa (Barcelona) 2017 The work carried out during the thesis was financially supported by: Novel approaches for prevention of biofilms formed on medical indwelling devices, e.g. catheters FP7-HEALTH-2011-278402 Group of Molecular and Industrial Biotechnology Terrassa “My strength is my family” To the people I love the most, Stanimir and my parents Abstract The accelerated emergence of drug resistant bacteria is one of the most serious problems in healthcare and the difficulties in finding new antibiotics make it even more challenging. To overcome the action of conventional antibiotics bacteria develop effective resistance mechanisms including the formation of biofilms. Biofilms are bacterial communities of cells embedded in a self-produced polymeric matrix commonly found on medical devices such as indwelling catheters. When pathogens adopt this mode of growth on the surface, they effectively circumvent host immune defences and antibiotic therapy, causing severe and life threatening device-related infections. To reduce the incidence of biofilm-associated infections different strategies, such as often replacement of the device, utilisation of novel materials for its construction and/or design of functional coatings have been suggested. Besides the variable efficiency, they commonly involve the use of large quantities of antibacterials leading to side effects, hypersensitivity, inflammatory responses or emergence of resistance. Thus, new paradigms are urgently needed for more efficient and specific approaches to prevent and treat biofilm infections. This thesis focuses on the development of advanced nanoscale materials and coatings for controlling clinically relevant bacterial biofilms and the emergence of drug resistance. To this end, acylase and amylase enzymes degrading essential for the biofilm growth components, were innovatively combined into hybrid nanocoatings to impart antibiofilm functionalities onto indwelling medical devices. Alternatively, ultrasound-assisted nanotransformation of antimicrobials was used as a tool for enhancing their antibacterial efficacy and overcoming the intrinsic drug resistant mechanisms in Gram-negative bacteria. These strategies offer new perspectives for prevention and treatment of biofilm infections, limiting the selection and spread of antibiotic resistance. The first part of the thesis describes the building of enzyme multilayer coatings able to interfere with bacterial quorum sensing (QS) and prevent biofilm establishment on silicone urinary catheters. This was achieved by alternate deposition of negatively charged acylase and oppositely charged polyethylenimine in a Layer-by-Layer (LbL) fashion. The acylase-coated catheters degraded the bacterial signalling molecules and inhibited the QS process of Gram-negative bacteria. These coatings also significantly reduced the biofilm growth on urinary catheters under conditions mimicking the real situation in catheterised patients, without affecting the human cells viability. i Acylase was further combined with the matrix degrading α-amylase enzyme into hybrid multilayer coatings able to interfere simultaneously with bacterial QS signals and biofilm integrity. The LbL assembly of both enzymes into hybrid nanocoatings resulted in stronger biofilm inhibition as a function of acylase or amylase location in the multi- layer coating. Hybrid nanocoatings with the QS inhibiting acylase as outermost layer reduced the occurrence of single and multi-species biofilms on silicone catheters in vitro and in an in vivo animal model. The thesis also reports on the efficacy of nanomaterials for prevention and eradication of antibiotic resistant biofilms. Multilayer assemblies that contain in their structure and release on demand antibacterial polycationic nanospheres (NSs) were engineered on silicone surfaces. A polycationic aminocellulose (AC) conjugate was first transformed into NSs with enhanced bactericidal activity and then combined with hyaluronic acid to build bacteria-responsive layers on silicone material. When challenged with bacteria these multilayers disassembled gradually inhibiting both planktonic and biofilm modes of bacterial growth. The same AC NSs were also covalently immobilised on silicone material using epoxy-amine conjugation chemistry. The intact NSs on the silicone material were able to inhibit bacterial colonisation and biofilm growth, suggesting the potential of epoxy- amine curing reaction for generation of stable non-leaching coatings on silicone-based medical devices. Finally, ultrasound-assisted nanotransformation of clinically relevant antibiotic penicillin G was used as a strategy to boost its activity towards Gram-negative bacteria and their biofilms. The efficient penetration of the spheres within a Langmuir monolayer sustained the theory that NSs are able to cross the membrane and reach the periplasmic space in Gram-negative bacteria where they may exert their bactericidal activity “unrecognised” as a threat by bacteria. In such conditions it is very likely that the selective pressure is reduced and bacteria do not feel the need to defend, expressing resistant phenotypes. Keywords: enzymes, Layer-by-Layer coatings, sonochemistry, nanospheres, silicone, urinary catheters, quorum quenching, antibacterial activity, biofilm prevention, antibiotic resistance ii Table of Contents Abstract ........................................................................................... i Table of Contents ........................................................................... iii Abbreviation list .............................................................................. vi 1 Introduction .............................................................................. 1 2 State-Of-The Art ...................................................................... 4 2.1 Bacterial Biofilms Formation ........................................................................... 5 2.2 Quorum Sensing in Bacterial Virulence and Biofilm Growth ............................ 7 2.2.1 Quorum Sensing in Gram-negative Bacteria ............................................ 7 2.2.2 Quorum Sensing in Gram-positive Bacteria ........................................... 10 2.3 Antibiotic Resistance of Biofilms ................................................................... 12 2.4 Biofilms in Device-Related Infections ............................................................ 12 2.5 Current Strategies for Controlling Bacterial Biofilms ...................................... 17 2.6 Layer by Layer assembly: A versatile tool for engineering bacteria resistant materials ................................................................................................................. 18 2.7 Quorum Sensing Inhibitors in Next Generation Antibiofilm Coatings ............. 25 2.8 Enzymes in Biofilms Prevention .................................................................... 28 2.8.1 Quorum Quenching Enzymes ................................................................ 28 2.8.2 Matrix-degrading Enzymes .................................................................... 29 2.8.3 Enzymes Targeting Bacterial Cells ........................................................ 31 2.8.4 Enzymes Producing Biocides ................................................................ 33 2.9 Nanomaterials for Controlling Bacterial Biofilms ........................................... 34 2.9.1 Sonochemistry for Synthesis of Nanoparticles ....................................... 35 2.9.2 Nanoparticles Interaction with Bacteria and Biofilms .............................. 36 2.9.3 Metal and Metal Oxide Nanoparticles .................................................... 38 2.9.4 Nanoantibiotics ...................................................................................... 40 3 Objectives of the Thesis ......................................................... 42 4 Materials and Methods ........................................................... 45 4.1 Materials and Reagents ................................................................................ 46 4.2 Bacterial and Human cells ............................................................................ 46 4.3 Experimental Methods .................................................................................. 47 4.3.1 Enzymatic Degradation of Biofilm Components ..................................... 47 4.3.2 Enzyme Multilayer Coatings for Prevention of Bacterial Biofilm Formation on Silicone Foley Catheters ................................................................................. 48 iii 4.3.3 Nanomaterials for Controlling Planktonic Bacterial Growth and Biofilm Occurrence: Polycationic Aminocellulose Nanospheres ....................................... 57 4.3.4 Covalent Grafting Of Aminocellulose Nanospheres onto Silicone Material for Escherichia Coli Biofilm Prevention ................................................................ 60 4.3.5 Nano-sized Penicillin G for Overcoming Gram-Negative Bacterial Resistance ..........................................................................................................
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