Antifungal activity of crude extract and fractions of Streptococcus mutans on Candida albicans in models of in vivo study

Author(s)

Jéssica Diane dos Santos

• Document type: Master's Dissertation
• Press: São José dos Campos. 2018-02-16.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Ciência e Tecnologia. São José dos Campos
• Defense date: 2018-01-15
• Advisor: Juliana Campos Junqueira

Abstract

In vitro studies have shown that Streptococcus mutans can produce metabolites capable of inhibiting Candida albicans, becoming interesting the identification and development of new substances for the treatment of oral candidiasis. Thus, the objective of this study was to extract, fractionate and identify the substances produced by S. mutans and evaluate their effects on the pathogenicity of C. albicans and on the immune response in in vivo study models. Substances from the S. mutans culture supernatant were extracted with ethyl acetate and subsequently fractionated on a C-18 derivatized silica column (150 g, Φ = 3.5 cm) using different solutions of MeOH:H2O (36:64, 49:51, 60:40, 76:24, 100:0) as eluent, obtaining five different fractions (SM-F1, SM-F2, SM-F3, SM-F4 and SM-F5). The identification of the substances contained in the crude extract and fractions was performed by gas chromatography coupled to mass spectrometry (GC-MS). The crude extract products and the fractions of the supernatant of the S. mutans culture were assessed on experimental candidiasis induced in invertebrate model of Galleria mellonella and in immunosuppressed mice. For a choice of the concentration to be tested in the in vivo models, a determination of the Minimum Inhibitory Concentration (MIC) of the crude extract and fractions on C. albicans was performed. In the model of experimental infection with G. mellonella, the effects of extract and fractions were analyzed by the survival curve test, quantification of CFU/mL of C. albicans in hemolymph and determination of haemocyte density of G. mellonella larvae. The extract, as well as fractions with better results in invertebrate model were selected for the study of oral candidiasis in mice. In this experimental model, the development of candidiasis was evaluated by the tests of recovery and determination of CFU/mL of C. albicans from the mice’s oral cavity, macroscopic and microscopic examination of the tongue dorsum. The data obtained were statistically analyzed by Graph Pad Prism 5.0, with a significance level of 5%. In the determination of the minimal inhibitory concentration, only the crude extract and the SM-F2 fraction showed effect on C. albicans, with 10 mg/mL and 15 mg/mL, respectively. In the model of G. mellonella there was no increase in the larvae survival with the prophylactic use of the extract, occurring 100% of death in the first 24 h in both groups with infection. However, when we performed the post infection treatment with the crude extract there was an increase in survival from 18.75 to 25%. As there was no prophylactic effect for the crude extract, the fractions were administered only therapeutically, verifying increased survival of the larvae with the SM-F1 and SM-F2 fraction. In the CFU/mL count of C. albicans on larval hemolymph, a statistically significant difference was observed only with crude extract and SM-F2 fraction after 12 h of infection. In relation to hemocyte density, the groups treated with crude extract and fractions (SM-F1 and SM-F2) had a higher number of hemocytes circulating in the hemolymph compared to the group only infected with C. albicans. Therefore, the fractions SM-F1 and SMF2 were chosen for the tests in mice. In this study model, it was verified that the crude extract, SM-F1 and SM-F2 were able to significantly reduce the number of CFU/mL of Candida in oral cavity and lesions of candidiasis of the tongue dorsum, these effects were more prominent for SM-F2. Thus, it was concluded that the SM-F1 and SM-F2 fractions of the S. mutans extract contain antifungal substances with therapeutic action on experimental candidiasis, being able to be targets of new therapeutic strategies for oral candidiasis. (AU)

• FAPESP's process: 16/03395-4 - Antimicrobial activity of metabolites produced by Streptococcus mutans on Candida albicans in in vivo study models
• Grantee: Jéssica Diane dos Santos
• Support Opportunities: Scholarships in Brazil - Master