İmplant Kaynaklı Enfeksiyon Etkeni Olan Staphylococcus Epidermidis’e Özgü Aptamer Geliştirilmesi ve Aptamerin Biyofilm Oluşumuna Etkisinin İn Vitro Koşullarda İncelenmesi

Abstract

One of the biggest problems encountered in the clinic regarding biomaterials that replace tissues or organs damaged or lost because of accidents along with various diseases is the occurrence of infection. Staphylococcus epidermidis is the leading cause of infection by forming a biofilm, and although this bacterium is an element of the normal skin flora, it enters the body during implantation and becomes a pathogen. While routine antibiotic treatments are not sufficient after biofilm formation, they also have many disadvantages. Therefore, alternative or supplemental methods to antibiotics are needed.

Within the scope of the thesis, aptamer selection was made against two different domains of the biofilm-associated Aap (accumulation associated protein) protein of S. epidermidis bacteria, which causes the formation of implant-associated osteomyelitis, with the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method and its effects on biofilm formation were investigated on both chitosan-coated and uncoated titanium surfaces. In the first stage, recombinant production was provided by making different combinations of Aap protein domains, A1.0, B1.0 and B1.5. Since the A1.0 and B1.0 domains were purified at a higher rate and amount, the immobilization process was carried out with these domains. In the next step, the aptamer library and primers to be used in the SELEX method were designed and synthesized. Polymerase chain reaction (PCR) optimization studies were carried out to amplify the primers and the aptamer library, and to determine their suitability. Sequence analysis was performed following cloning the aptamer pool obtained after 8 cycles of SELEX, and the dissociation constants of the selected candidate aptamers were determined using the ELONA (enzyme-linked oligonucleotide assay) method. In the last step, different molecular weights and concentrations were used in the coating of titanium surfaces with chitosan, and SEM, EDX and ATR-FTIR analyzes were used in the characterization of the surfaces. The effect of selected candidate aptamers on chitosan coated and uncoated titanium surfaces was determined by both microplate test using crystal violet and SEM analysis results. Among the 6 selected candidate aptamers, the aptamer with the highest binding affinity (10.2 nM) and the most effective on biofilm formation was determined to be the aptamer coded as AC31. In the presence of the AC31 aptamer, it was determined that the rate of preventing biofilm formation on uncoated titanium surfaces was at least 4-fold, and at least 2-fold on titanium surfaces coated with chitosan.

As a result, aptamer selection by SELEX method against two different domains of the biofilm-induced Aap protein of S. epidermidis bacteria was performed for the first time within the scope of this study. AC31 aptamer has been shown to prevent biofilm formation by showing its effect on the adhesion mechanism of S. epidermidis on titanium surfaces, and paved the way for its use as a therapeutic agent following in vivo studies.