Marine Fungal Extracts Obtained by OSMAC-Based Cultivation Reverse the Fluconazole-Resistance Phenotype in Yeasts

ABC transporters constitute a superfamily of transmembrane proteins that act mediating the translocation of several substrates across the membrane, using the energy of ATP hydrolysis. This mechanism of unrelated substrates efflux (multidrug resistance) has been associated with several diseases and it is a problem in chemotherapy efficacy, especially fluconazole chemotherapy. Recent studies demonstrated that marine organisms can be a good source of new natural products that can act as multidrug resistance inhibitors. Marine fungi are known mainly for the variety of secondary metebolite production with numerous pharmacological activities. However, under laboratory conditions, fungi produce only a fraction of their metabolic potential. Thus, many cultivation techniques are used to increase the production of secondary metabolites, such as the OSMAC (One Strain Many Compounds) technique. The aim of this study is to evaluate extracts from marine sponge-derived fungi, under different growing conditions, able to reverse the fluconazole-resistance phenotype mediated by efflux pumps. Initially, two marine sponges species were collected in June 2016 on the Ilha do Abraão – Rio de Janeiro (latitue: 23º6,899’S; longitude: 44°9,995’W). This material was used to isolate the marine fungi. Then, the strain M2-22DA isolated from the Malt extract agar were grown in Tubaki/Malt extract/Czapek/Cazapek-Glutamine/Potato Dextrose liquid medium with/without salt at 22°C/28°C for 12 days, under agitation or statically. Subsequently, the fermentation was terminated by the addition of EtOAc. The solvent was evaporated and the extract was dissolved in DMSO for the bioassays. Dish plates containing YPD medium, with or without fluconazole, were inoculated with fluconazole-resistant strains mediated by efflux pumps (2,5 x 106 cells/ml). After, paper disks containing 500 µg of each crude extracts were deposited on surface of YPD medium. The plates were incubated at 30°C (Saccharomyces cerevisiae mutant strain) or 37°C (clinical isolates of Candida spp.) during 48 hours. The extract from the culture in Tubaki medium with salt at 28°C under agitation proved to be a promising reversal of the fluconazole resistance phenotype. After the incubation period, a growth inhibition zone formed around the disc impregnated with the extract. As a perspective, a large-scale fermentation will be done under these ideal conditions for the identification and obtaining of the purified active compound