Abstract
Natural products, such as the ethanolic propolis extract (PE), have been shown to be a safe and effective therapeutic alternative for the treatment of fungal skin and nail diseases. However, the presence of the resin and the physicochemical characteristics of the extract sometimes difficult the reading and determination of breakpoints of the in vitro tests, evidencing the need for alternatives that facilitate the reading. The present study aimed to standardize the use of resazurin in tests of susceptibility of PE with planktonic yeast cells and biofilm forms. The antifungal activity of PE was determined by minimum inhibitory concentration (MIC) and we observed that, for all Candida spp. tested, the most reproducible MIC results were obtained when resazurin was placed after 24 hours of incubation and remained more 24 hours with yeasts plus PE. For encapsulated yeasts, there was no dye reduction and color transition. Resazurin was also used for the evaluation of minimal biofilm inhibitory concentration and minimal biofilm eradication concentration and it was metabolized and reproduced the action of PE on Candida biofilms. In addition, microdilution checkerboard plates were made with the dye, which assisted reading the result of the interaction between PE and nystatin. We observed that the resin, the color and the turbidity of the PE slightly changed the color of the resazurin in high concentrations of the extract and did not impair the reading. Therefore, the resazurin standardization tests were proven to be efficient and grounds that it should be used as an auxiliary methodology for reading and interpretation of the susceptibility tests for non-encapsulated yeasts with natural products, which form turbidity or precipitation, such as propolis.
Highlights
Nowadays, skin diseases of various etiologies are the most common and prevalent disorders in the world
Determination of minimum inhibitory concentration (MIC) in planktonic cells using resazurin at different times The antifungal activity of propolis has been demonstrated in several studies carried out at different times and with different extracts and compositions (Negri et al, 2014)
Researches have shown the antifungal activity of propolis against different species and isolates of Candida, which corroborate our findings (Dalben-Dota et al, 2010; Falcão et al, 2013; Tobaldini-Valerio et al, 2016; Corrêa et al, 2020)
Summary
Skin diseases of various etiologies are the most common and prevalent disorders in the world For many years, these diseases were neglected and have recently been considered as being of relevance. The treatment of superficial fungal diseases is challenging for both patients and professionals, due to the difficulty in achieving a cure level and because of the high rates of recurrence. Another problem is the antifungal arsenal to be restricted to limited options and reports of clinical resistance to these drugs have already been related (Sanguinetti, Posteraro, & Lass-Flörl, 2015). Our group showed that PE is capable of breaking down the integrity of the cell membrane as well as the cell wall from the fungi, without causing damage to human cells (Corrêa et al, 2020)
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