Evaluation in vitro of the effect of denture desinfections solutions on capacity production of proteinase and phospholipase and formation of hyphae by Candida species

• Grant number: 16/05189-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2016
• End date: December 31, 2016
• Field of knowledge: Health Sciences - Dentistry - Dental Materials
• Principal Investigator: Cláudia Helena Lovato da Silva
• Grantee: Laís Ranieri Makrakis
• Host Institution: Faculdade de Odontologia de Ribeirão Preto (FORP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The object of this study is evaluate in vitro, the effect of hygiene solutions (Triclosan, Cloramina T, R. communis in 10% and 2% and Sodium Hypochlorite in 0,25%) at the proteinase and fosfolipase capacity production and hyphae formation by C. albicans, C. glabrata and C. tropicalis. Initially will be determinated the minimum inhibitory concentration from the Triclosan and Cloramina T solutions by the micro dilution method in cell culture plates with 96 wells, in duplicate. As an variation factor will be considerated an immersing (solution) in 5 levels: GHS= specimens contaminated and immersed in sodium hypochlorite 0,25%; GT= specimens contaminated and immersed in Trichlosan solution; GCt= specimens contaminated and immersed in Chloramine T solution; GRc10: specimens contaminated and immersed in R. communis 10% solution; GRc: specimens contaminated and immersed in R. communis 2% solution. The control groups will be formated to comprove the biofilm formation, where the contaminated specimens will be immersed in destilated water to comprove the body prove sterilization (n=6). The biofilms will be formated over thermoativated acrilic resin's circle specimens (13mm diameter x 4mm thickness) and then immersed in the solutions by 20 minutes, rinsed with PBS and immersed in a liquid culture medium (Letheen) for processing in accord with the protocol established for each variable. The proteinase quantification will be realized by the EnzChek® Protease Assay fluorimetric kit and the evaluation of the phospholipase production by the Candida species will be realized by the Amplex Red® phosphatidylcholine-specific phospholipase C assay fluorimetric kit. For the hyphae quantification, 100µL of Letheen celular suspension coming will be transferated for the medium "yeast extract peptona dextrose (YEPD) and cultivated for 2 days. Subculture will obtained in 5µL from the growth medium "yeast 23 ammonium dextrose" for 48 hours in 30ºC, under by agitation.Next, the culture will be centrifugated (5 minutes in 10ºC), washed twice with 5µL of PBS and transfered (3 x 106 cells/mL) for 5mL from the hyphae inductor medium (medium 199) plus with 10% of fetal bovine serum. The cells will be incubate in 37ºC for 3 hours. The cells counts (levedure + hyphae) will be realize in a Neubauer chamber. The analyses will be realized in triplicated. The data will be submeted by normality analyses (Shapiro-Silkis test) and homocedasticidate (Levene test) to define the appropriate statistical test. The analyses will be conduced in a 5% significancy level.