Characterization and evaluation of the quorum quenching activity, using in vivo and in vitro models, of a chemically treated titanium surface functionalised with a lactonase enzyme.

Today, the increasing elderly population means a greater susceptibility to musculoskeletal disorders, typically managed, if they persist, through arthroplasty. These bone-contact titanium implants, despite their useful mechanical and physicochemical properties, remain susceptible to various complications, the most important of which are loosening and infection. Among these failures, prosthetic joint infection is the most significant, and the responsible bacterial species include Gram-positive and Gram-negative microorganisms. These infections often require prolonged treatment with broad-spectrum antibiotics, potentially exacerbating the critical problem of antibiotic resistance. This phenomenon not only increases treatment costs but also leads to higher levels of morbidity and mortality. There are several explanations for the increase in this problem, such as the growing global consumption of antibiotics, often without appropriate prescriptions, and the use of antibiotics in non-human settings. Looking closely at the processes that contribute to this resistance, multiple mechanisms can be identified, including biofilms, and quorum sensing. The last mechanism is bacterial signaling, which controls genes and functions useful for bacterial survival, such as the formation of the biofilm itself and the production of virulence factors. Interference with such communication, a process called quorum quenching, is one possible strategy to face bacterial infections. This technique involves acting on both signal molecules, called autoinducers, and other elements of the regulatory pathway, such as synthases or signal molecule receptors. Among the methods used for this purpose, enzymatic degradation of autoinducers is the most widely employed. These enzymes, including lactonases, degrade the signal molecules, thereby disrupting bacterial gene expression. This strategy is increasingly being studied because it decreases virulence and does not promote the emergence of resistant strains. The aim of this thesis is to functionalize a titanium surface with an active lactonase as a quorum quenching enzyme, and to conduct surface characterizations and tests to evaluate the action of this coating in preventing virulent biofilm formation. At the Institute of Molecular Genetics and Genetic Engineering (IMGGE, Belgrade), lactonase ST1-YtnP (extracted from Bacillus licheniformis) was produced, and Caenorhabditis elegans (worms) was used as an in vivo model. The enzyme in the solution exhibited no toxic effect on worms at different concentrations, and allowed the extension of the survival period of worms infected with Pseudomonas aeruginosa. Surface functionalization of a chemically treated titanium surface was investigated at two concentrations. Surface analysis of these sample by X-ray photoelectron spectroscopy showed no significant differences with the formation in both cases of a molecular layer of adsorbed enzyme. The functionalized samples had no toxic effect on worms, although they did not reduce the gene expression of different clinical pathogens present in the solution in which the samples were immersed, because the enzyme was not released from the surface in an efficient concentration for an action on planktonic bacteria. Further biological studies involved in vitro models with human cells and/or bacteria in contact with the surface of the functionalised samples for understanding the action of the grafted enzyme in preventing virulent biofilm formation.