Characterization of Malassezia spp. isolated from the external ear canal of dogs and cats and evaluation of virulence factors

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2021-10-21T01:27:01Z No. of bitstreams: 1 2019 - M?rio Tatsuo Makita.pdf: 2187775 bytes, checksum: 7b8cf85e0d95bbcd22202fe1bb5710ed (MD5) Made available in DSpace on 2021-10-21T01:27:01Z (GMT). No. of bitstreams: 1 2019 - M?rio Tatsuo Makita.pdf: 2187775 bytes, checksum: 7b8cf85e0d95bbcd22202fe1bb5710ed (MD5) Previous issue date: 2019-04-25 CAPES - Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior The genus Malassezia currently comprises 18 species of lipophilic and lipiddependent yeasts. Although regarded as a normal part of the skin microbiota of warmblooded vertebrates, including humans, these may also become opportunistic agents in dermatitis and otitis. Fungal otitis in dogs has traditionally been associated with M. pachydermatis, even though lipid-dependent species are commonly found frequently resulting in misdiagnosis. In the veterinary clinical routine, microscopy of otological samples is used as an auxiliary tool towards the implementation of adequate drug therapies. The aim of this work was the isolation of Malassezia spp. from otitis samples from dogs and cats, their respective identification to species level, observe the presence of Malassezia in pure and/or co-infections, in vitro assessment of the main virulence factors being produced, which are inherent to the genus pathogenicity and the establishment of a correlation between the detected virulence factors and the nature of the infection. A total of 170 samples from the external ear canal of 94 dogs and 11 cats with clinical otitis were sent to the Laborat?rio de Leveduras Patog?nicas e Ambientais - UFRRJ, where they were inoculated in Modified Dixon Agar and Sabouraud Dextrose 2% Agar supplemented with chloramphenicol. Classical methods allowed for the phenotypic identification, as recommended by Kurtzman et al. (2011) further corroborated through proteomic analysis by MALDI-TOF MS and biochemical analysis by VITEK? 2. For virulence analysis, protease, phospholipase, lipase, esterase and hemolysin production were tested. Of the 105 animals, 63% (66) individuals were positive for Malassezia spp., as one lipid dependent, and 37% (39) were negative for the genus. The positive and negative predictive values of the direct exam were 75.58% and 67.19%, respectively. The sensitivity of the isolation test was 75.58% and the specificity 67.19%. There was no significant difference between yeast recovery in Sabouraud Dextrose 2% Agar and Modified Dixon Agar. The identification by mass spectrophotometry had an 82% accuracy in the identification of the isolates and remaining isolates did not reach the identification score, whereas VITEK? 2 misidentified all the samples. For the enzymatic tests, 100% (96) of the samples were positive for the production of proteases and lipases. Only one sample did not produce neither phospholipase nor esterase. 89% (86) samples were hemolysin producers. It was possible to conclude that the use of Modified Dixon Agar is important in the laboratory routine for microbiological diagnosis of canine and feline otitis, extending the range of Malassezia species diagnosed. Direct microscopic examination proved to be a good indicator of the presence of yeast, but not enlightening regarding its involvement in the infection. In addition, these yeasts have high enzymatic activity in vitro which may be relevant to the pathogenesis of microbial otitis, even when the yeast is not the main pathological agent, as in co-infection otitis, even with low isolation counts. O g?nero Malassezia compreende 18 esp?cies de leveduras lipof?licas e lipodependentes, componentes naturais da microbiota da pele dos animais endot?rmicos, incluindo humanos, e conhecido agente oportunista em dermatites e otites. A maior parte da casu?stica de otite f?ngica em c?es ? atribu?da a M. pachydermatis, mas esp?cies lipodependentes tamb?m podem ser agentes desta afec??o, sendo poss?vel a ocorr?ncia de subdiagn?sticos. Na rotina cl?nica veterin?ria, ? comum o uso do exame direto de amostras otol?gicas como ferramenta auxiliar para direcionar uma melhor conduta terap?utica. Tendo os pontos mencionados em vista, o trabalho objetiva realizar isolamento de Malassezia spp. a partir de amostras de cer?men de c?es e gatos com otite, identificando-as a n?vel de esp?cie, observar a presen?a da Malassezia em infec??es otol?gicas mistas ou puras, verificar in vitro a produ??o dos principais fatores de virul?ncia inerentes ? patogenicidade e correlacionar esses resultados com a natureza da infec??o. Foram utilizadas 170 amostras oriundas dos condutos auditivos externos de 94 c?es e 11 gatos com otite cl?nica, encaminhadas ao Laborat?rio de Leveduras Patog?nicas e Ambientais da UFRRJ, onde foram semeadas nos meios Agar Dixon Modificado e Agar Sabouraud Dextrose 2% com Cloranfenicol. Foram realizados testes cl?ssicos para identifica??o fenot?pica, como preconizado por Kurtzman et al. (2011), an?lise prote?mica por MALDI-TOF MS e bioqu?mico por VITEK? 2 para confirma??o da identifica??o. Para an?lise da virul?ncia foram testadas as produ??es de proteases, fosfolipase, lipases, esterases e hemolisina. Dos 105 animais, 63% (66) animais possuiam Malassezia spp. nas suas amostras, sendo uma delas lipodependente, e 37% (39) n?o tinham a levedura. O valor preditivo positivo e negativo do exame direto foi de 75,58 e 67,19%, respectivamente. A sensibilidade do exame em rela??o ao isolamento foi de 75,58% e 67,19% de especificidade. N?o houve diferen?a significativa entre a recupera??o das leveduras no diagn?stico em Agar Sabouraud Dextrose 2% e Agar Dixon Modificado. Na identifica??o por espectrofotometria de massa obteve-se 82% de acur?cia na identifica??o dos isolados, n?o havendo scores m?nimos de identifica??o para as restantes. O VITEK? 2, identificou erroneamente as amostras analis?das. Para os testes enzim?ticos, 100% (96) das amostras foram positivas para a produ??o de proteases e lipases. Apenas uma amostra n?o foi produtora de fosfolipase e esterase. 89% (86) das amostras foram positivas para a produ??o de hemolisina. Percebe-se que a utiliza??o do Agar Dixon Modificado ? importante na rotina laboratorial de diagn?stico microbiol?gico de otites canina e felina, para ampliar a gama de esp?cies de Malassezia diagnosticadas. O exame direto, realizado no laborat?rio, mostrou ser um bom indicador para a presen?a da levedura, mas n?o necessariamente para indicar seu envolvimento na infec??o. Conclui-se que n?o h? diferen?a entre o uso de ASD e ADM no diagn?stico laboratorial da Malassezia. Al?m disso, essas leveduras possuem alta atividade enzim?tica in vitro, possivelmente integrando a patog?nese da otite microbiana, mesmo quando n?o aparentam ser o principal agente envolvido, como nos casos de otites mistas, incluindo os casos comb baixas contagens no isolamento.