BANDEIRA, S. P. Combinação de técnicas fenotípicas para distinguir Candida albicans de Candida dubliniensis e uma investigação dos mecanismos de resistência a derivados azólicos em isolados de Candida albicans de origem animal. 2017. 77 f. Tese (Doutorado em Microbiologia Médica) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Carolinda Oliveira (ppgmm@ufc.br) on 2017-09-12T15:37:04Z
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Previous issue date: 2017-07-11 / Azole-resistant Candida albicans have been described in human clinical practice and in strains of veterinary origin. The objective of this study was to reevaluate laboratory identification of azole-resistantC. albicansfrom veterinary isolates, proposing a combination of phenotypic methods for the reliable identification of the species. In addition, this studyaimed to investigate the resistance mechanisms of the strains. Thirty-seven veterinary strains of azole-resistant C. albicansand three human strains of C. dubliniensis were submitted to phenotypic identification by germ tube test, micromorphological analysis oncornmeal agar, growth on chromogenic agar, growth on sunflower seed agar, opacification test onTween 80™ agar and PCR. For study of resistance mechanisms, C. albicansstrains were evaluated for efflux of6G rhodamine (n=11), determination of ergosterol content (n=6) and expression of CDR1, CDR2, MDR1 and ERG11 genes (n=30). All studied strains presented positive germ tube test and production of chlamydoconidia on cornmeal agar. Thirty five strains (35/37) showed green colonies on chromogenic agar. On sunflower seed agar, 36 strains (36/37) presented a smooth colony pattern, and on Tween 80™ agar, 34 (34/37) had opacification in less than 5 days. All strains tested showed specific product on PCR, which confirmed their identity as C. albicans.C. dubliniensis strains showed green colonies on ChromoAgar Candida™, a rough appearance on sunflower agar, did not form opacification zone on Tween80™agar, after up to 12 days, and did not present specific products in PCR.In resistance studies, azole resistant strains showed higher relative fluorescence unit difference values than sensitive isolates in efflux of6G rhodamine tests, thus demonstrating increased activity of efflux pumps. There was no significant difference in ergosterol content. It was verified that 73.3% (22/30) of the isolates presented overexpression of one or more genes, of which 40.9%, 18.2%, 59.1% and 54.5% strains overexpressedCDR1, CDR2, MDR1 and ERG11, respectively. No isolate overexpressed simultaneously the four genes. Finally, we conclude that the combination of phenotypic techniques leads to reliable differentiation of C. albicansandC. dubliniensis, reserving molecular techniques for more specific contexts. Resistance mechanisms to azole derivatives ofthe studied strains is multifactorial, including, at a minimum, the enhanced activity of efflux pump activity and the gene overexpression. / Candida albicans resistentes a derivados azólicos tem sido descritos na prática clínica humana e em cepas de origem veterinária. O presente estudo teve como objetivo reabordar a identificação laboratorial deisolados veterinários de C. albicans resistentes a derivados azólicos, propondo combinação de métodos fenotípicos para distinguir as espécies crípticas C. albicans e Candida dubliniensis.Ademais, almejou-se investigar os mecanismos de resistência das cepas estudadas. Para tanto, trinta e sete cepas veterinárias de C. albicans resistentes a derivados azólicos e três cepas humanas deC. dubliniensis foram submetidas à identificação fenotípica por prova do tubo germinativo, microcultivo em ágar cornmeal, semeadura em ágar cromogênico, semeadura em ágar semente de girassol, prova de opacificação em ágar Tween80™e reação em cadeia da polimerase (PCR). Para estudos de resistência, cepas de C. albicans foram estudadas com testes de efluxo de rodamina 6G (n=11), determinação do conteúdo de ergosterol (n=6) e testes de expressão dos genes CDR1, CDR2, MDR1 e ERG11(n=30). As cepas estudadas apresentaram prova do tubo germinativo positiva e produção de clamidoconídios em ágar cornmeal. Trinta e cinco cepas (35/37) apresentaram coloração verde em ágar cromogênico. Em ágar semente de girassol,36 cepas (36/37) apresentaram padrão de colônia lisa, e no ágarTween 80™, 34 (34/37) apresentaram opacificação em menos de 5 dias. Todas as cepas testadas apresentaram produto específico na PCR, sendo confirmadas como C. albicans.As cepas de C. dubliniensisa presentaram colônias de coloração verde no ChromoAgar Candida™, aspecto rugoso no ágar girassol, não opacificaram o meio em ágar Tween80™ em até 12 dias e não apresentaram produto específico na PCR. Nos estudos de resistência, as cepas resistentes aos azólicos apresentaram valores de diferença de UFR maiores que os isolados sensíveis nos estudos com rodamina 6G, demonstrando
| Identifer | oai:union.ndltd.org:IBICT/oai:www.repositorio.ufc.br:riufc/25602 |
| Date | 11 July 2017 |
| Creators | Bandeira, Silviane Praciano |
| Contributors | Rocha, Marcos Fábio Gadelha, Sidrim, José Júlio Costa |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | Portuguese |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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