Análise fenotípica, genotípica, proteômica e fatores de virulência de cepas de leveduras do gênero Trichosporon: isolados humanos, animais e ambientais
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Data
2021-07-29
Autores
Lara, Bruna Rossini
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Universidade Estadual Paulista (Unesp)
Resumo
Trichosporon spp. são amplamente distribuídos na natureza, compreendendo inúmeras espécies que habitam diferentes nichos ecológicos, podendo ser encontrados em água, solo e superfície corpórea de animais. Em humanos, este gênero fúngico ocasionalmente encontra-se como membro da microbiota gastrintestinal e cavidade oral e, transitoriamente, pode colonizar o trato respiratório e pele. Este microrganismo tem sido classicamente associado a infecções superficiais, no entanto, nas últimas décadas, também se apresenta relacionado às infecções disseminadas em pacientes imunocomprometidos, comportando-se como agente oportunista. Visando aumentar os conhecimentos acerca deste agente patogênico e auxiliar na compreensão sobre os aspectos que envolvem diagnóstico precoce e terapia adequada este trabalho teve como objetivos: re-identificar fenotipicamente, genotipicamente e por meio de espectrometria de massa MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) (MALDI-TOF MS) leveduras do gênero Trichosporon de origem humana, animal e ambiental mantidas em micoteca; determinar e comparar os fatores relacionados a virulência: secreção das enzimas fosfolipase e proteinase, bem como a produção de biofilme; avaliar os padrões de susceptibilidade dos biofilmes a determinados antifúngicos; avaliar os padrões de susceptibilidade dos isolados frente aos antifúngicos fluconazol, itraconazol, anfotericina-B, voriconazol e caspofungina, comparando as técnicas de microdiluição em caldo e Etest. O nível de concordância entre a metodologia fenotípica tradicional e a técnica molecular, na identificação das 59 amostras de Trichosporon, foi de 54,2%. A concordância na identificação entre o MALDI-TOF MS e a técnica molecular foi de 67,8%. Nenhum isolado ambiental foi passível de identificação por meio do MALDI-TOF MS e 100% destes isolados não foram concordantes na identificação de sequenciamento da região IGS com a caracterização fenotípica. Do total de amostras estudadas, a espécie que apresentou maior frequência foi T. asahii (52,6% - 31/59), seguida por T. debeurmannianum (18,6% - 11/59), T. inkin (13,5% - 08/59) e T. dermatis (5,1% - 03/59). Do total de isolados, 89.8% e 96.6% não apresentaram atividade para proteinase e fosfolipase, respectivamente. A habilidade de formar biofilme sob a superfície de placas de poliestireno foi observada em todos os isolados, sendo 54,3% das amostras consideradas como fortemente produtoras. Em relação a susceptibilidade antifúngica, voriconazol apresentou os menores valores de CIM para as amostras humanas e animais em ambas as metodologias estudadas e para as amostras ambientais na metodologia de microdiluição em caldo. Quando as amostras ambientais foram testadas frente ao método comercial Etest, foi observado valores menos elevados de CIM frente ao antifúngico anfotericina B. A caspofungina e o fluconazol foram os antifúngicos com os maiores CIMs. Quando comparadas as duas técnicas de susceptibilidade antifúngica, o método Etest apresentou valores de CIMs mais elevados em relação ao método de microdiluição. Quanto a sensibilidade dos biofilmes, todas as espécies apresentaram valores elevados de CIM50 e CIM90 frente aos antifúngicos testados, entretanto algumas espécies apresentaram menores valores de CIM. T. coremiiforme apresentou valores menores de CIM para fluconazol, T. montevideense para itraconazol, T. insectorum para vorizonazol e T. veenhuisii para fluconazol e vorizonazol. Foi realizado análise genotípica de 31 isolados de T. asahii, 83,88% foram identificados como genótipo 1, genótipos 4 e 7 também foram identificados com 12,90% e 3,22%, respectivamente. Quanto a localização geográfica, foram isoladas em São Paulo-SP e Cuiabá-MT amostras de T. asahii de genótipos 1 e 4 e na cidade de Bauru-SP foram encontrados isolados com genótipos 1 e 7. Não houve associação que relacione os genótipos aos resultados obtidos nos testes de virulência (proteinase, fosfolipase e produção de biofilme) e susceptibilidade antifúngica (EUCAST e Etest). A análise filogenética das amostras 17 (genótipo 7), 47 (genótipo 4), 32 (genótipo 4) e 33 (genótipo 4), apresenta sua distinção com os demais isolados, o que se justifica devido ao fato de se tratar de cepas com genótipos diferentes das demais (genótipo 1).
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).
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).
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Palavras-chave
Trichosporon spp., Identificação fúngica, Fatores de virulência, Susceptibilidade antifúngica