Comparação genotípica e fenotípica de diferentes isolados clínicos e ambientais de Rhodotorula spp. / Genotypic and phenotypic comparisons among clinical and environmental isolates of Rhodotorula spp.

Jorge Meneses Nunes

Introduction: Rhodotorula spp. are yeasts widely distributed in the environment and can colonize the human body at different anatomical locations. This emerging pathogen is capable of causing superficial and invasive infections in humans whereas bloodstream infection is the most commonly described in the literature. Mainly three species have been related to human infections: R. mucilaginosa, R. glutinis and R. minuta. Objectives: i) assess the prevalence of Rhodotorula species in clinical and environmental samples by sequencing the ITS region of rDNA; ii) Analysis of interspecific variations in the genus Rhodotorula by the use of Randomly Amplified Polymorphic DNA (RAPD); iii) evaluate the accuracy of the commercial system ID32C in identifying species of the genus Rhodotorula; iv) analysis the capability of biofilm formation of Rhodotorula spp. and v) the susceptibility profile to five antifungal agents. Methods: we analyzed 51 isolates of clinical samples and 8 of environmental samples. All isolates were identified by sequencing the ITS region of rDNA and subjected to RAPD using different primers for analysis of interspecific variability between these isolates. The clinical isolates were still subjected to phenotypic identification using the commercial system ID32C, the macro e and micromorphology analysis and nitrate assimilation. A crystal violet biofilm assay was carried out to assess the ability of biofilm formation of Rhodotorula spp. Finally, the broth microdilution method was performed to evaluate the susceptibility profile of these isolates to amphotericin B, caspofungin, fluconazole, voriconazole and posaconazole. Results: Through the sequencing of the ITS region of rDNA we observed that R. mucilaginosa was the most prevalent species among clinical isolates (86.3%), followed by R. minuta, R. dairenensis, Rhodosporidium diobovatum and Rhodosporidium fluviale, and it was also the most prevalent among environmental isolates (87.5%). RAPD using primer B14 were able to discriminate among species of the genus Rhodotorula and Rhodosporidium and showed increased discriminatory power compared to primer M2. The phenotypic identification using the commercial system ID32C with supplementary tests were concordant with sequencing of ITS region to identify 90.2% of clinical isolates. However, the accuracy of identification was low for 78.4% of these isolates. Regarding testing biofilm formation, 67.8% of the isolates were capable to form biofilm whereas only isolates of R. mucilaginosa and R. minuta presented this feature. It was also observed that the storage time of samples did not affect the ability of R. mucilaginosa biofilm formation (p = 0.927) and that clinical isolates of this species tended to form more biofilm than the environmental isolates (p = 0.074). All clinical Rhodotorula spp. isolates showed in vitro resistance to fluconazole (MIC ≥ 64 μg/ml) and caspofungin (MIC ≥ 4 μg/ml). In contrast, all clinical Rhodotorula spp. isolates were susceptible to amphotericin B (MIC ≤ 1 μg/ml), 86.6% were susceptible to voriconazole and 84.1% to posaconazole (MIC of both were ≤ 2 μg/ml). Conclusions: Molecular methods are appropriate and necessary for the accurate identification of Rhodotorula species whereas RAPD may be useful as screening tool for identifying pathogenic species of this genus. Considering that the clinical isolates of Rhodotorula spp. tended to an increased biofilm formation compared to environmental isolates and that the clinical relevant species R. mucilaginosa and R. minuta were the only ones with the ability to form biofilm, we can suggest that this feature has potential role in the virulence and pathogenesis of these species. The in vitro susceptibility testing suggests that amphotericin B and the new triazoles are the best therapeutic options against infections caused by Rhodotorula species.

Comparação genotípica e fenotípica de diferentes isolados clínicos e ambientais de Rhodotorula spp. / Genotypic and phenotypic comparisons among clinical and environmental isolates of Rhodotorula spp.

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