Approaches by Rhodotorula mucilaginosa from a chronic kidney disease patient for elucidating the pathogenicity profile by this emergent species

Background Traditionally known as a common contaminant, Rhodotorula mucilaginosa is among the leading causes of invasive fungal infections by non-candida yeasts. They affect mainly immunocompromised individuals, often mimicking the cryptococcosis infection, despite invasive infections by Rhodotorula are still not well explained. Thus, here we aimed to characterize microbiologically clinical isolates of R. mucilaginosa isolated from colonization of a patient with chronic renal disease (CKD), as well as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible infection. Methodology/Principal Findings For this study, two isolates of R. mucilaginosa from oral colonization of a CKD patient were isolated, identified and characterized by classical (genotypic and phenotypic) methods. Susceptibility to conventional antifungals was evaluated, followed by biofilm production, measured by different techniques (total biomass, metabolic activity, colony forming units and extracellular matrix quantification). Finally, the pathogenicity of yeast was evaluated by infection of Tenebrio molitor larvae. All isolates were resistant to azole and sensitive to polyenes and they were able to adhere and form biofilm on the abiotic surface of polystyrene. In general, similar profiles among isolates were observed over the observed periods (2, 24, 48 and 72 hours). Regarding extracellular matrix components of biofilms at different maturation ages, R. mucilaginosa was able to produce eDNA, eRNA, proteins, and polysaccharides that varied according to time and the strain. The death curve in vivo model showed a large reduction in the survival percentage of the larvae was observed in the first 24 hours, with only 40% survival at the end of the evaluation. Conclusions/Significance We infer that colonization of chronic renal patients by R. mucilaginosa offers a high risk of serious infection. And also emphasize that the correct identification of yeast is the main means for an efficient treatment. Author Summary The genus Rhodotorula is known to be a common contaminant, however, it has been increasing in the last years, reports of different forms infections by this yeast, reaching mainly individuals with secondary diseases or with low immunity. However, very little is known about the mechanism that triggers the disease. Thus, this study aims to characterize microbiologically clinical isolates of R. mucilaginosa isolated from a patient with chronic renal disease, as well as to evaluate their phylogeny, antifungal susceptibility, virulence, and pathogenicity in order to infer the potential to become a possible infection. It was possible to characterize in general the clinical isolates, to determine that they are resistant to an important class of the antifungal agents which are the azoles. In addition, they are able to adhere and to form biofilm on abiotic surfaces, this skill represents an important factor of virulence, which would guarantee their presence in medical devices, such as catheters, surfaces. These biofilm works as true reservoirs of these fungi disseminate and cause serious infections. This pathogenic potential was reinforced by a great reduction of survival in the larvae infected with this yeast. Therefore our results infer a high risk of infection to patients who are colonized by R. mucilaginosa.


Studied group and isolation
For the purpose of this study, a patient was aleatorily selected from a bigger project voluntary is a man, 55 years old, diabetic (Diabetes Melittus type II), confirmed with chronic 144 kidney diseases (stage 5) at 2 years before, and he was under hemodialysis for 6 months, no using antifungals and absence of oral lesions. The data collection, the oral mucosa examination, 146 were performed according to Pieralisi et al., 2016 biological samples and the cultivation method were performed as described previously [24]. 155 Briefly, yeasts were sub cultured in chromogenic medium CHROMagar™ Candida (Difco,156 USA), to check the culture purity. After, the isolates were identified by classical tests, including 157 macro and micro morphologies, fermentation tests and assimilation of carbohydrate and 158 nitrogen sources [25,26]. To confirm the identification, mass spectrometry assisted by flight 159 time desorption/ionization matrix (MALDI TOF-MS) was performed. For the MALDI TOF-160 MS method, the yeasts were prepared according to specific protocols [27,28] with a Vitek MS 161 mass spectrometer using the Myla or Saramis software for data interpretation. glycerol at -80 °C. All samples were cultured on SDA with additional chloramphenicol (0.1%) 168 and incubated at 25 °C for up to 3 days, after all tests [29]. 171 The morphology was assessed with by optical microscopy (EVOS™ FL,Life 172 Technologies) and by Scanning Electron Microscopy (SEM; Quanta 250™, ThermoFisher). 173 The colony, cell morphology and the polysaccharide capsule were observed by light 174 microscopy at 40x magnification. The colony was observed after microcultive and analyzed 175 directly by light microscopy [26]. To analyze capsule, a suspension of 500 µg/mL phosphate-  187 The DNA extraction of isolates was performed according Vicente et al., (2008) [31], 188 using a silica: celite mixture (silica gel H, Merck 7736, Darmstadt, Germany/Kieselguhr Celite 189 545, Machery, Düren, Germany, 2:1, w/w). The internal transcribed region (ITS) was 190 amplified using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 191 (5'-TCCTCCGCTTATTGATATGC-3') [31,32]. PCR was performed in a 12,5 μL volume of a 192 reaction mixture containing 4,3 μL of mix solution containing 0,3 mM dNTPs, 2,5 mM MgCl2, 193 1,25 μL reaction buffer, 0,5 μL of each primer (10 pmol) and 1 μL rDNA (20 ng/μL). The 194 sequencing was performed by sanger method in automated sequencer ABI3730 (Applied Biosystems Foster City, U.S.A). Consensus sequences of the ITS region were inspected using identification of specie was determined by phylogenetic analysis, using type strains established 198 by Wang et al., 2015 andNunes et al., 2013. Phylogenetic tree was performed MEGA v.7 199 software with 1,000 bootstrap replicates using the maximum likelihood function and the best 200 evolutionary model corresponding to the data set used. Bootstrap values equal to or greater 201 than 80% were considered statistically significant.

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The Minimum Inhibitory Concentration (MIC) was determined according to CLSI, M27-A.

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Cut-off points were: S ≤8 g/mL; SDD = 16-32 g/mL; and R ≥64 g/mL for fluconazole, S 217 ≤ 1 g/mL; SDD = 2 g/mL; R ≥16 g/mL for voriconazole, S ≤0.125 g/mL; SDD 0.25-0.5 218 g/mL; R ≥32 g/mL for itraconazole, S≤ 4 g/mL; SDD = 8-32 g/mL; R ≥ 64 g/mL for  223 The biofilm formation assay was adapted from previously described method [19]. The   All tests were performed in triplicate, on three independent days. Data with a non-296 normal distribution were expressed as the mean ± standard deviation (SD) of at least three 297 independent experiments. Significant differences among means were identified using the      (Table 01).   Figure 03). There was a significant increase of biofilm in number of cells until 48 hours of 356 biofilm age, after this period there is a decrease of viability cells (Fig 03 A). On the other hand, 357 metabolic activity and total biofilm biomass were different among the isolates, decreasing of 358 24 to 48 hours biofilm age (CMRP3463 and ATCC 64684) to metabolic activity (Fig 03 B) 359 and increasing from 24 to 48 hours biofilm age (CMRP3462 and ATCC 64684) to total biofilm biomass (Fig 03 C). It is important to highlight that there was a decrease for all parameters 361 analyzed after 48 hours of biofilm age.

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Analyzing each isolate, regard to adhesion (2 hours), ATCC 64684 strain showed the 363 lowest cell viability by CFU with p<0.01 (Fig 03 A), the metabolic activity (XTT) was similar 364 among the isolates (p>0.05) (Fig 03 B). However, in the evaluation of total biofilm biomass 365 (CV), according to Fig 03 C, the CMRP3463 was the lowest total biofilm biomass (p<0.01).

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From 24 hours of biofilm formation, clinical isolates (CMRP3462 and CMRP3463) were 367 significantly (p<0.01) higher than ATCC 64684 to viable cells in biofilm (Fig 03 A). All 368 isolates presented a significant increase in relation 2 to 24 hours, with no statistical difference 369 among isolates (Fig 03 B) to metabolic activity. The clinical isolates (CMRP3462 and 370 CMRP3463) increase significantly (p<0.01) in the total biofilm biomass at 2 to 24 hours, 371 mainly CMRP3463 (Fig 03 C). Finally, in the period of 48 to 72 hours, the clinical isolates    for standardization. Groups of 10 larvae were infected with three fungal concentration.

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Negative control group the T. molitor larvae were injected just with PBS (without yeasts). understand what would be the mechanism of pathogenicity. Biofilm production ability is one This lack of knowledge deserves concern, since serious and fatal infections due this specie have 511 been related to the formation of biofilms on medical devices [11,12].

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The first study to evaluate the biofilm formation capacity of different Rhodotorula formation, adopted by the authors, which is in agreement to our results on biomass 517 quantification.
The present study is the first one aiming to characterize the biofilm production by R. 519 mucilaginosa on different ages (24, 48 and 72 hours), then we employed the classic methods 520 used in studies with Candida sp. biofilms [35,48], that are CFU, CV, XTT and microscope, 521 addressing to R. mucilaginosa. These results are presented on Fig 03 and Table 2, and 522 complemented by the quantification of the extracellular matrix components determined on the 523 same times (Table 02). It was possible to observe a formation and organization of the biofilm  In addition, we saw a peak of metabolic activity in 24 hours, and soon afterwards a 552 decrease of this activity, being that in 72 hours, these cells were probably "dormant" [35].

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Metabolically "dormant" yeast cells are also known as persistent cells, which originate 554 stochastically as phenotypic variants within biofilms [20]. According to Kojic et al., 2004, 555 persistent biofilm cells represent an important mechanism of resistance, and the eradication of albicans, C. parapsilosis or C. tropicalis., we did not find filaments that give the structure for 562 a complex biofilm, however we found several layers of cells, as well as described by Nunes et 563 al., 2013 [19,54]. With these variables, we infer that the maturity of R. mucilaginosa biofilm 564 occurs in 48 hours due to stability and uniformity, confirmed mainly by microscopy images 565 (Fig 04).

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In view   for standardization. Groups of 10 larvae were infected with three fungal concentration.

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Negative control group the T. molitor larvae were injected just with PBS (without yeasts).