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26Background: Traditionally known as a common contaminant, Rhodotorula mucilaginosa is 27 among the leading causes of invasive fungal infections by non-candida yeasts. They affect 28 mainly immunocompromised individuals, often mimicking the cryptococcosis infection, 29 despite invasive infections by Rhodotorula are still not well explained. Thus, here we aimed to 30 characterize microbiologically clinical isolates of R. mucilaginosa isolated from colonization 31 of a patient with chronic renal disease (CKD), as well as to evaluate their phylogeny, antifungal 32 susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible 33 infection. 34 Methodology/Principal Findings: For this study, two isolates of R. mucilaginosa from oral 35 colonization of a CKD patient were isolated, identified and characterized by classical 36 (genotypic and phenotypic) methods. Susceptibility to conventional antifungals was evaluated, 37 followed by biofilm production, measured by different techniques (total biomass, metabolic 38 activity, colony forming units and extracellular matrix quantification). Finally, the 39 pathogenicity of yeast was evaluated by infection of Tenebrio molitor larvae. 40All isolates were resistant to azole and sensitive to polyenes and they were able to adhere and 41 form biofilm on the abiotic surface of polystyrene. In general, similar profiles among isolates 42 were observed over the observed periods (2, 24, 48 and 72 hours). Regarding extracellular 43 matrix components of biofilms at different maturation ages, R. mucilaginosa was able to 44 produce eDNA, eRNA, proteins, and polysaccharides that varied according to time and the 45 strain. The death curve in vivo model showed a large reduction in the survival percentage of 46 the larvae was observed in the first 24 hours, with only 40% survival at the end of the 47 Conclusions/Significance: We infer that colonization of chronic renal patients by R. 49 mucilaginosa offers a high risk of serious infection. And also emphasize that the correct 50 identification of yeast is the main means for an efficient treatment. 51 52 53 3 Author Summary 54The genus Rhodotorula is known to be a common contaminant, however, it has been 55 increasing in the last years, reports of different forms infections by this yeast, reaching mainly 56 individuals with secondary diseases or with low immunity. However, very little is known about 57 the mechanism that triggers the disease. Thus, this study aims to characterize microbiologically 58 clinical isolates of R. mucilaginosa isolated from a patient with chronic renal disease, as well 59 as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order 60 to infer the potential to become a possible infection. It was possible to characterize in general 61 the clinical isolates, to determine that they are resistant to an important class of the antifungal 62 agents which are the azoles. In addition, they are able to adhere and to form biofilm on abiotic 63 surfaces, t...

Few antifungals available today are effective in treating biofi lms. Thus, it is urgent to discover new compounds, such as natural products, that provide improvements to existing treatments or the development of new antifungal therapies. This study aimed to perform a comparative analysis between the green propolis extract (PE) and its by-product, a waste of propolis extract (WPE) through a screening with Candida sp., Fusarium sp. and Trichophyton sp. The antifungal property of PE and WPE was assessed by the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) determination in planktonic cells. The infl uence of both extracts on the inhibition of biofi lm formation in these fungi was also tested. The WPE MIC and MFC values (68.75 to 275.0 µg/mL) were three to twelve times lower than the values obtained for PE (214.06 to 1712.5 µg/mL). PE was more effi cient than WPE in inhibiting the biofi lm initial phase, especially in C. albicans. Meanwhile, WPE had dose-dependent behavior for the three fungi, being more effective on fi lamentous ones. Both PE and WPE showed excellent antifungal activity on planktonic cells and demonstrated great effi cacy for inhibiting biofi lm formation in the three fungi evaluated.

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.

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Biofilms are important to the virulence of human pathogenic fungi, and some molecules have been found to play key roles in the growth and regulation of fungal biofilms. Farnesol, one of these molecules, is well-described for some microorganisms but is still scarcely known for Rhodotorula spp. This study aimed to evaluate the influence of farnesol on the biofilm of R. mucilaginosa. Initially, screening with 0.2 mM to 2.1 mM of farnesol was evaluated against planktonic forms. A concentration of this compound was then chosen and evaluated for its effect on biofilm in formation and on preformed biofilm after 24, 48 and 72 hours. The impact of farnesol was evaluated by colonyforming units (CFU) counts, determination of metabolic activity and quantification of total biomass. In the presence of 0.9 mM, farnesol was able to decrease the CFU number, at 48 hours, when the biofilm was in formation, although it did not affect the preformed biofilms. Thus, our results show that farnesol exerts a modulating activity during biofilm formation for R. mucilaginosa, with this compound reducing the metabolic activity and total biomass of the biofilms.