Avaliação das propriedades antimicrobianas do disseleneto de difenila e da associação de fármacos antifúngicos contra Pythium insidiosum e fungos patogênicos

Abstract

The increasing incidence of microbial infections associated with the development of antimicrobial resistance and treatment failure encourages the research for new alternatives for prophylaxis and treatment of infections caused by microorganisms. In this context, this thesis aimed to evaluate the antimicrobial properties of diphenyl diselenide ((PhSe)2), its structural analogues with functional groups containing chlorine, fluorine and methoxyl as well as the combination of antifungal drugs against pathogenic fungi and Pythium insidiosum in tests in vitro and in vivo. Additionally, the use of new culture media in the induction of zoosporogenesis and conservation of P. insidiosum has been assessed.Through the susceptibility tests based on the protocol M38-A2 of CLSI, with inoculum consisting of zoospores of P. insidiosum, can be observed that (PhSe)2 showed in vitro antifungal activity against P. insidiosum, with the minimum fungicidal concentration (MFC) ranging from 0.5 to 4 μg/mL and suggested a fungistatic activity in vivo treatment with 10 mg/Kg/day of (PhSe)2 in experimental pythiosis in rabbits. The biochemical and hematological laboratory evaluation of this experimental treatment showed no toxic characteristics of oral administration of (PhSe)2. The evaluation of in vitro susceptibility testing for yeast and filamentous fungi was based on protocols M27-A3 and M38-A2 of CLSI and indicated that (PhSe)2 showed fungistatic and fungicidal activity against several fungal species, including Candida albicans (MFC 8 to 32 μg/mL), Aspergillus fumigatus (MFC 16 to >64 μg/mL) and Fusarium solani (MFC 8 to 64 μg/mL). The introduction of functional groups in the molecule of (PhSe)2 produced a decrease or loss of antifungal activity, being (PhSe)2 > (p-CH3O-C6H4Se)2 > (m-CF3-C6H4Se)2 > (p-Cl-C6H4Se)2. Treatment of experimental pythiosis in rabbits was also conducted with the combined therapy with antifungal agents. In this context, the combination therapy of fluconazole (100 mg/Kg/day) + terbinafin (100 mg/Kg/day) + fluvastatin (1 mg/Kg/day) was the only one among the tested, which resulted in clinical cure of one rabbit and a possible fungistatic activity in other animals of this group. The laboratory evaluation of this treatment suggests the involvement of iron metabolism in view of different levels of transferrin saturation in the develop of pythiosis. Additionally, the evaluation of new protocols for the induction of zoosporogenesis in vitro showed that the sequential subculture of P. insidiosum in nutritionally rich (chocolate agar and egg yolk agar) and poor (0.2% yeast extract agar and cornmeal agar) media provides a recovery and stimulation of in vitro production of zoospores, which are essential for identification, susceptibility testing and induction of experimental infection in rabbits. The conservation of isolates of P. insidiosum in tubes containing sterile water and pieces of grass provide a favorable environment for the viability of this species for long periods.