In-Vitro investigation of the efficacy of chitosan-supported metal nanoparticles in the treatment of multidrug-resistant Candida auris

Candida species are a natural element of the human microbiome and cause fatal infections when the immune system is weakened and the right opportunity arises. Candida auris is one of the five most important microorganisms on the list of the most serious microorganisms threatening humanity published by the Centers for Disease Control and Prevention (CDC) in 2019. Its resistance to many known antifungal drugs, antiseptic and disinfectant substances, its thermotolerance and osmotolerance properties, its ability to secrete enzymes such as phospholipase and hemolysin, its ability to form biofilms, and to colonize living and non-living surfaces make it a remarkable microorganism. Previous studies have shown the potential antifungal activities of metal nanoparticles (MNPs) against Candida species. In this study, the in vitro activity of gold (Au) and iron oxide (Fe3O4) nanoparticles and their chitosan-supported forms against C. auris planktonic and biofilm cells was investigated. In this study, a multidrug-resistant clinical C. auris isolate (K1575) and four reference isolates were used. The drug resistance profile of the isolates was revealed by broth microdilution testing. The genomic basis of the resistance profile of the clinical C. auris isolate was analyzed by whole genome sequencing. AuNP synthesis was carried out by citrate-reduction reaction, and chitosan-supported gold (Chi-AuNP) nanoparticles were synthesized by chitosan-supported reduction reaction. Fe3O4NP synthesis was carried out with oleyl amine and then colloidal stabilization was provided with citric acid and chitosan. The compatibility of all synthesized NPs with the literature was checked by X-Ray Diffraction (XRD), Fourier-Transform Infrared Spectroscopy (FT-IR), Scanning Electron Microscope (SEM), UV-Visible Spectroscopy (UV-Vis), Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS) tests. For in-vitro efficacy tests, cytotoxicity tests were tested on the NIH 3T3 fibroblast cell line by the MTT method. Within the scope of in vitro efficacy tests, five selected isolates were subjected to colony forming unit (CFU), spectrophotometric analysis, biofilm thickness determination by the XTT/Menadione method, SEM, reactive oxygen species (ROS) determination by iii H2DCFDA and demonstration of NPs on the biofilm formed by the isolates by confocal microscope experiments. The azole and echinocandin resistance detected with the broth microdilution test was confirmed with genomic analysis. Mutations were detected in the FKS1, ERG11, CDR1, TAC1b and FCY1 genes. In the CFU experiments initiated by determining the cytotoxic concentrations (CC) of the nanoparticles synthesized and quality controlled in accordance with the literature, it was determined that AuNPs and Chi-AuNPs had an inhibitory effect on the planktonic and biofilm forms of the clinical isolate K1575 at every dose applied, and that there was an inhibitory effect depending on the isolate at the doses applied on the planktonic and biofilm forms of the reference isolates. In the ROS experiments conducted, it was shown that this effect was provided by the ROS created by the interaction of the nanoparticles with the isolates. It was determined that the Fe3O4NP and Chi-Fe3O4NPs applied in the CFU experiments had no inhibitory effect on any of the isolates used. As a result, the genomic base of multidrug resistance in K1575 was shown by multiple mutations in the target antifungal genes. It was determined that K1575 and reference isolates showed inhibition in gold-based nanoparticles and that this inhibition was primarily related with ROS formation and fungal cell wall disruption. It was shown that gold-based nanoparticles should be considered as a potential for a new treatment method for multidrug resistant C. auris infections.