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Phenotypic, genotypic, proteomic analysis and virulence factors of yeasts of the genus Trichosporon: isolated human, animal and environmental.

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Bruna Rossini Lara
Total Authors: 1
Document type: Master's Dissertation
Press: Botucatu. 2021-08-13.
Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. Botucatu
Defense date:
Advisor: Luciana da Silva Ruiz Menezes

Trichosporon spp. are widely distributed in nature, comprehending countless species that inhabit different ecological niches, they can be found in water, soil and animals’ body surfaces. In humans, this fungal genus is occasionally found as a member of the gastrointestinal microbiota and oral cavity; and, transiently, it can colonize the respiratory tract and skin. This microorganism has been classically associated with superficial infections, however, during the last decades, it has also been linked to infections’ spread in immunocompromised patients, behaving as an opportunistic agent. Aiming to increase knowledge about this pathogenic agent, and assist in the understanding of aspects involving early diagnosis and proper therapy, this study aims to: re-identify phenotypically, genotypically and through mass spectrometry MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) (MALDIT-TOF MS) Trichosporon genus yeasts of human, animal and environmental origin kept in a fungi collection (mycotheca); it also aims to determine and compare factors related to virulence: production of the phospholipase and proteinase enzymes, and the production of biofilm; evaluate the biofilms’ susceptibility patterns to certain antifungals; evaluate the isolated fungi’s susceptibility patterns against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin, by comparing broth microdilution and Etest techniques. The level of agreement between the traditional phenotypic methodology and the molecular technique, among the identification of the 59 samples of Trichosporon, was 54.2%. The agreement on the identification between the MALDI-TOF and the molecular technique was 67.8%. No environmental isolate was identifiable by the means of MALDI-TOF MS, while 100% of these isolates were not in agreement in the IGS region’s sequencing identification with the phenotypic characterization. Out of the total studied samples, the species that showed the highest frequency was T. asahii (52.6% - 31/59), followed by T. debeurmannianum (18,6% - 11/59), T. inkin (13.5 % - 08/59) and T. dermatis (5.1% - 03/59). Among the total isolates, 89.8% and 96.6% did not present any activity for proteinase and phospholipase, respectively. The ability to form biofilm under the polystyrene plates’ surface was observed in all isolates, with 54.3% of the samples considered to be highly productive. Regarding the antifungal susceptibility, voriconazole presented the lowest MIC values for human and animal samples in both studied methodologies and for environmental samples in the broth microdilution methodology. When the environmental samples were tested against the commercial Etest method, lower MIC values were observed against the antifungal amphotericin B. Caspofungin and fluconazole were the antifungals with the highest MICs. When comparing the two antifungal susceptibility techniques, the Etest method showed higher MIC values than the microdilution method. Regarding the sensibility of the biofilms, all of the species presented high values of MIC50 and MIC90 against the tested antifungals, however some of these species presented lower values of MIC. T. coremiiforme presented lower values of MIC for fluconazole, T. montevideense for itraconazole, T. insectorum for voriconazole and T. veenhuisii for both fluconazole and voriconazole. A genotypical analysis of 31 isolates of T. asahii was made, where 83,88% were identified as type 1 genotype, while genotypes 4 and 7 were also indentified in 12,90% and 3,22% of the isolates, respectively. As for the geographic location, samples of the T. asahii of genotypes 1 e 4 were isolated in São Paulo-SP and Cuiabá-MT, and in BauruSP, isolates with the genotypes 1 and 7 were found. There was no evidence of a correlation between the genotypes and the results obtained in the virulence (proteinase, phospholipase and the production of biofilm) and antifungal susceptibility (EUCAST and Etest) tests. The phylogenetic analysis of the samples 17 (genotype 7), 47 (genotype 4), 32 (genotype 4) and 33 (genotype 4) presented a distinction from the other isolates, which is justified by the fact that they had strains with different genotypes than the others (genotype 1). (AU)

FAPESP's process: 19/08419-7 - Clinical and environmental isolates of Trichosporon spp.: characterization, virulence research and profile of susceptibility to antifungals
Grantee:Bruna Rossini Lara
Support Opportunities: Scholarships in Brazil - Master