Synergistic activity of silver nanoparticles with metal ions and antifungal agents on Candida spp. biofilms: susceptibility test and cellular differentiation analysis

Abstract

Candida spp. are fungi of the oral microflora of humans capable of causing a range of infections, including Candida-associated denture stomatitis. They develop biofilm communities which are antimicrobial resistant. So that, the aim of this study wil be identify metal ions (Ag+, Co2+, Zn2+, TeO32-) and the most current available effective antifungal agents (polyenes (amphotericin B), triazoles (fluconazole, itraconazole, voriconazole, posaconazole), and echinocandins (caspofungin, micafungin, anidulafungin)) that might synergistically enhance the efficacy of silver colloid nanoparticles (SCN) against, Candida albicans, Candida glabrata, Candida tropicalis, and Candida parapsilosis biofilms. Synergistic activity will be evaluated by using a high-throughput technique for biofilm susceptibility testing which permits examining antimicrobial arrays representing different combinations of these toxic metal ions and antifungal drugs with SCN. By using both light microscopy and confocal laser scanning microscopy (CLSM) in conjunction with three-dimensional (3D) visualization, it will be examined the multicellular architecture of Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis biofilms before and after exposure to metal ions, antifungal agents, silver nanoparticles and SCN. This approach may be identify if the tested compounds have antimicrobial potential to block or to trigger a switch from yeast to hyphal cell morphotypes. SCN will be synthesized in the Exact Science and Technology Center, Federal University of São Carlos, São Carlos, Brazil, and silver nanoparticles produced by Sigma-Aldrich will be used as a reference control of SCN synthesized compound. The choice of those metal toxic ions was based on their oxidative toxicity through disparate modes-of-action, especially thiol-disulfide chemistry. The Candida species were selected because they are associated with biomaterial-related infections at the oral level, i.e. denture base resin in touch with palatal mucosa, that are characterized by the formation of biofilms on this surface leading to clinically refractory disease as denture stomatitis. One-way analysis of variances (ANOVA) will be used to analyse log10-transformed raw data with MINITAB® Release 14 (Minitab Inc., State College, PA). Alternate hypotheses will be tested at the 95% level of confidence. Mean and standard deviation calculations of quantitative data will be performed using Microsoft® Excel XP (Microsoft Corporation, Redmond, WA), and these data will be imported into SigmaPlot 10.0 (Systat Software Inc., San Jose, CA) for threedimensional graphical representation. (AU)