Covalent immobilization of furanone derivatives for the inhibition of biofilm formation on implant surfaces

A biofilm is essentially composed of microbial cells attached to a surface and completely covered with an extracellular polymeric matrix (EPS) produced by biofilm-forming bacteria. Uncontrollable and undesirable accumulation of cell aggregates constitute serious problems in industrial processes and biomedical applications. Studies concerning biofilm effects on human health are generally performed on water/systems (dentistry systems), prosthesis and implants. Biofilm formation and metabolic activities within may cause serious community health risks. Most of these health risks are nosocomial infections of gastrointestinal, eye and ear etc.Because of these reasons, there is a growing demand for developing strategies to remove and protect the surfaces against biofilm formation . The benefits of biofilm formation to microorganism include increased protection against antimicrobials and defence mechanisms. A biofilm cannot be considered as a homogeneous distribution of cells but it can be thought as a cluster of cells enclosed within a matrix with channels and pores. These channels facilitate the transport of oxygen and nutrients to the biofilm forming units. Removal of waste products and secondary metabolites also occur through these channels. Medical devices and implants are highly susceptible to microbial contamination and infection of biomedical devices is a considerable and increasing problem. The aim of our study is to prevent these infections from the application of a thin coating that prevents bacterial colonization on the surfaces of biomedical devices. The rationale of the study depends on the prevention of microorganism colonization on different surfaces by blocking the communication of these microorganisms through a non-biocidal method. The key molecule of this method is Acyl Homoserine Lactone (AHL), which is a signal molecule used for the communication of microorganisms. These AHL molecules are produced by the microorganisms and secreted out of their cells. These molecules are sensed by other microorganisms and when its density reaches the threshold level, it is taken back to the microorganisms and activates the expression of several genetic elements (virulence factors, antibiotic resistance genes etc.). This communication system is called as "Quorum Sensing". The blocking of this communication ("Quorum quenching") is vital to prevent the colonization of microorganisms and infection. In order to achieve this blocking, AHL molecule is the target in our study. Here, analogues of this AHL molecule has been used to block the biofilm formation. Inhibitory effect of AHL analogues 5- (dibromomethylene)-3-methylfuran-2(5H)-one (named as FU1), 3-(bromomethyl)-5- (dibromomethylene) furan-2(5H)-one (named as FU2), (Z-)-4-Bromo-5- (bromomethylene)-2(5H)-furanone (named as 4Z), 5-(Bromomethylene)-2(5H)- furanone (named as 2(5H)) have been examined. Synergistic antimicrobial agent and immobilized AHL analogues composition as inhibitors for adhesion of microorganism have also been investigated.xx Furanone molecules are a part of the chemical defense system produced by marine algae named as Delisea pulchra, which prevents the colonization on their surfaces [1]. Furanones have been found to interfere with quorum sensing mechanism of many human pathogens and in previous works, these compounds and their analogues have been shown to inhibit or prevent the growth of both Gram-negative and Grampositive bacteria [2-4]. These molecules act on intercellular communication system both as free molecules and also as surface immobilized molecules [5]. Natural and synthetic furanone compounds have been found to interfere with Quorum Sensing (QS) mechanism of many human pathogens and block this specific pathway. Thus, the strategy for the present study is covalently linking furanone derivatives on surfaces to assess the effect of the immobilized furanone molecules on the adhesion strength of Pseudomonas aeruginosa biofilms. Our goal is to inhibit formation of bacterial biofilm especially for medical applications. In the scope of the literature work, the strategy is to coat the surface with halogenated furanone derivatives. To obtain surface-immobilized furanones through the sol-gel processes, carbodiimide chemistry and azide-nitrene chemistry have been carried out. In the literature, the immobilization procedure of furanone molecules starts with amine plasma, following polyacrylic acid and 4-azidoaniline treatment to obtain nitrene groups on to the surface, which will be the linker of furanone under UV light. In this study, furanone molecules have successfully covalently bound to the substrate. Morphological differentiation at each step has been visualised by using AFM and SEM. FTIR results are also coherent with AFM and SEM. PAA attachment has radically increased surface roughness (from 2.059 ± 0.05nm SS-SC to 6.821 ± 0.75) and created niche areas for bacterial attachments. Change in surface morphology also effected CFU counts. CFU counts and CSLM images showed that FU1 and FU2 are highly effective against biofilm formation and FU2 has superior activity compared to FU1. FU2 has inhibited 78.9% of bacterial adhesion onto the surface while FU1 reduced 65.4% when compared the surface without furanone. 4-Z and 2-5(H) have no activity against biofilm formation. The total bacterial reduction is 4 times higher than AZA samples and 5 times higher when compared with uncoated SS-SC samples. Tobramycin asserts antibacterial activity when applied with furanone derivatives and it further reduced attachment of bacteria (96.3% reduction for FU2 and 79.4% reduction for FU1) when compared with tobramycin untreated samples (76% reduction for FU2 and 60% reduction for FU1). Furanone coated substrates were tested in vivo using foreign-body infection model. 68 % mice died before 24th hour of the experiment and furanone coated substrates had higher CFU counts compared to control samples. The relationship between eDNA concentration and CFU also assessed with this experiment and it has been found that eDNA concentration is proportional to CFU count. This project was performed as a collaboration between Istanbul Technical University in Turkey and the research group from Instituto di Chimica del Riconoscimento Molecolare at CNR (National Research Council) in Italy. Italian group worked on chemical grafting of protease and lysozyme on to the stainless steel coupons and carried out the ATR-FTIR imaging of both the surface coatings and the biofilm samples. Chemically grafted stainless steel surfaces were provided by CNR group.xxi Arcelik A.S. (partner company from industry) also partly contributed to the project by providing scholarships to researchers. CSLM experiments and animal studies were carried at the University of Copenhagen, Denmark.