Banca de DEFESA: LUANDA BARBARA FERREIRA CANARIO DE SOUZA

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : LUANDA BARBARA FERREIRA CANARIO DE SOUZA
DATE: 31/03/2020
TIME: 14:00
LOCAL: SALA 2 DO PPGCF
TITLE:

Influence of Eugenia uniflora Extract onAdhesionto Human BuccalEpithelialCells,BiofilmFormation, and CellSurfaceHydrophobicityof Candida spp. from the Oral Cavity of Kidney TransplantRecipients

KEY WORDS:

Candida spp., kidney transplantation, virulence factors, Eugenia uniflora, mechanism of action, synergism

PAGES: 214
BIG AREA: Ciências da Saúde
AREA: Farmácia
SUMMARY:

Oral candidiasis is an important clinical manifestation in kidney transplant recipients. Candida spp. it has virulence factors that contribute to the infectious process, including the ability to adhere to epithelial cells and form biofilm on biotic and abiotic surfaces. Considering the limited arsenal of antifungals available on the market, such as adverse and resistant species surgeries, it becomes necessary to find new antifungals that reach alternative targets, or synergistically to antifungals and that have less toxicity. In this context, natural products have gained importance. The extract activated from the leaves of Eugenia uniflora [acetone: water (7: 3, v/v)] has demonstrated antifungal activity against Candida spp.. Therefore, this study aimed to evaluate the effect of E. uniflora extract on virulence factors in vitro and to characterize the antifungal action in Candida spp. from the oral cavity of kidney transplant recipients. The E. uniflora extract was characterized by high performance liquid chromatography. Strains of Candida spp. belonging to a microorganism bank were reactivated and used in the analysis. The determination of the minimum inhibitory concentration of the extract was carried out by the broth microdilution method. Toxicity tests against erythrocytes and human epithelial bucal cells (HEBC) were carried out by determining the hemolysis index and counting 150 HEBC in an optical microscope, respectively. For in vitro virulence assays, yeasts were grown in the presence and absence of 1000 μg/mL of the extract. Adherence was quantified using the number of Candida cells adhered to 150 CEBH determined by an optical microscope. The biofilm formation was evaluated using two methodologies: XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) and the violet crystal assay, being analyzed later by scanning electron microscopy. The cell surface hydrophobicity (CSH) was quantified with the microbial adhesion test to hydrocarbons. In silico analysis was performed to predict antifungal activity of the major compounds determined in the extract. MIC was determined when in the presence of exogenous ergosterol, as well as in an osmoprotected environment by sorbitol (0.8 M). The action of the extract on the cell wall was evaluated from the growth of the cells treated with E. uniflora extract in ASD plus calcofluor, Congo red and sodium dodecyl sulfate (SDS). The loss of membrane integrity was assessed using propidium iodide (PI) staining. The combined action of E. uniflora extract with Fluconazole, Micafungin and Gallic acid was determined by the checkerboard method and finally, the time-kill curve of Candida spp. treated with E. uniflora extract and in combination with others antifungals drugs. E. uniflora extract was able to inhibit the growth of Candida spp cells by 50% and it has two marjoritary compounds: myricitrin and gallic acid. It was shown to be non-cytotoxic to erythrocytes and HEBC. Reduced adherence to HEBC and HSC for all Candida species was shown. There was also a statistically significant reduced ability to form biofilms using both methods of quantification. In silico analysis demonstrated that there are no molecular targets that correlate myricitrin to the antifungal action of E. uniflora extract, while gallic acid demonstrated a link to the CPY51 enzyme present in the fungal cell. The extract slightly increased MIC when the ergosterol binding assay was performed, discarding the direct binding of the extract with this molecule. In the assay in the presence of sorbitol, the MIC values for the extract were increased, suggesting that one of the possible mechanisms of action is to act on the fungal cell wall. The treatment of Candida cells with the extract conferred resistance to cell wall disturbers. There was a loss of fungal cell membrane integrity when cells treated with the extract were stained with PI. E. uniflora extract showed an additive synergistic action when combined with Fluconazole and Micafungin. The combination with gallic acid had an indeterminate effect (without interaction). The time-kill curve of the cells treated with the extract demonstrated a decrease in the number of CFU count in 48 hours and the combination of E. uniflora extract with fluconazole resulted in a significant drop in the CFU count. The E. uniflora extract may in some way be disturbing the cell membrane, having seen the loss of membrane integrity seen in the PI labeled assay. However, this process apparently does not occur through direct binding to ergosterol or only by inhibiting the enzyme CYP51 (14 α-demethylase), an action attributed to gallic acid in in silico analyzes. The extract interacts at the fungal cell wall level, having seen the increase in MIC in the sorbitol assay and the slight decrease in growth for some strains in the assay with cell wall disturbers. E. uniflora extract has shown promise as a great alternative therapy combined with fluconazole for the treatment of oral candidiasis. Further analysis should be carried out to confirm the evidence presented by the tests carried out regarding the mechanism of action of E. uniflora extract.

BANKING MEMBERS:
Externa à Instituição - EDELTRUDES OLIVEIRA LIMA - UFPB
Externa à Instituição - ELIZABETH CRISTINA GOMES DOS SANTOS - UNICEUNA
Presidente - 1715308 - GUILHERME MARANHAO CHAVES
Interna - 1055045 - MARCELA ABBOTT GALVAO URURAHY
Interna - 1490222 - SILVANA MARIA ZUCOLOTTO LANGASSNER