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ResistÃncia a azÃlicos em candida spp. de origem veterinÃria: um fenÃmeno mediado por bombas de efluxo / AZOLE RESISTANCE IN CANDIDA SPP. FROM VETERINARY SOURCES: AN EFFLUX-MEDIATED PHENOMENONDÃbora Castelo Branco de Souza Collares Maia 15 December 2011 (has links)
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O monitoramento da sensibilidade antifÃngica em espÃcies de Candida de origem veterinÃria à uma prÃtica recente e os mecanismos envolvidos ainda nÃo foram completamente elucidados. Considerando que o arsenal de drogas antifÃngicas à limitado e que o fenÃmeno de resistÃncia tem se tornado mais freqÃente, a compreensÃo deste fenÃmeno e a busca por alternativas terapÃuticas se fazem necessÃrias. Dessa forma, o presente trabalho teve como objetivo monitorar a sensibilidade in vitro de Candida spp. oriundas de animais, com Ãnfase na resistÃncia aos azÃlicos mediada por bombas de efluxo e na avaliaÃÃo da atividade do imidazÃlico levamisol sobre o crescimento destas leveduras. Para tanto, em um primeiro momento, foram avaliados 126 isolados de Candida, sendo 19 C. albicans, 17 C. famata, 5 C. guilliermondii, 8 C. krusei, 29 C. parapsilosis, 48 C. tropicalis, dos quais 22 foram isolados de rapinantes, 32 de periquitos do sertÃo, 56 de papagaios, 7 de araras canindà e 3 de um ouriÃo-cacheiro. Todas as cepas foram submetidas ao teste de microdiluiÃÃo em caldo, ante a anfotericina B, itraconazol, fluconazol, segundo metodologia padronizada pelo Clinical Laboratory Standards Institute (documento M27-A3). As concentraÃÃes inibitÃrias mÃnimas (CIMs) variaram de 0,03125 a 2 Âg/mL, 0,125 a 250 Âg/mL e de 0,03125 a 125 Âg/mL para anfotericina B, fluconazol e itraconazol, respectivamente. Dos 126 isolados avaliados por microdiluiÃÃo, 33 (26,2%) foram resistentes aos azÃlicos, sendo 7 (5,6%) resistentes somente a fluconazol, 1 (0.8%) resistente somente ao itraconazol e 24 (19%) resistentes a ambas as drogas. Em um segundo momento, todas estas cepas resistentes aos derivados azÃlicos, mais 20 C. albicans e 3 C. tropicalis resgatadas da nossa coleÃÃo de leveduras resistentes de origem veterinÃria, foram submetidas ao teste de inibiÃÃo de bomba de efluxo com prometazina, perfazendo um total de 56 cepas resistentes a azÃlicos. Dessa forma, as CIMs de fluconazol e itraconazol sofreram reduÃÃes significativas de 2 a 250 e de 16 a 4000 vezes, respectivamente. Investigou-se, ainda, a atividade antifÃngica do levamisol contra 12 C. albicans, 12 C. krusei, 12 C. parapsilosis e 12 C. tropicalis, sendo obtidas CIMs e concentraÃÃes fungicidas mÃnimas que variaram de 0,58 a 2,34 mg/mL e de 2,34 a 9,37 mg/mL, respectivamente. Paralelamente, foi demonstrado que o levamisol inibe a formaÃÃo do biofilme de C. albicans e C. tropicalis, bem como interfere na manutenÃÃo do biofilme maduro. Os dados apontam que a resistÃncia aos derivados azÃlicos Ã, pelo menos em parte, mediada por bombas de efluxo, bem como demonstram o potencial antifÃngico do imidazÃlico levamisol e sua capacidade de inibir o biofilme de cepas de Candida spp. oriundas de animais. / Monitoring of the antifungal susceptibility of Candida species from veterinary sources is a recent practice and the mechanisms involved in antifungal resistance have not been completely elucidated. Considering that the antifungal arsenal is limited and that antifungal resistance has become more frequent, the comprehension of this phenomenon and the pursuit for therapeutic alternatives are necessary. Thus, the present work aimed at monitoring the in vitro susceptibility of Candida spp. isolated from animals, with emphasis on efflux pump-mediated azole resistance and on the evaluation of the effect of the imidazole levamisole on the growth of these yeasts. For such, in a first approach, 126 Candida isolates (19 C. albicans, 17 C. famata, 5 C. guilliermondii, 8 C. krusei, 29 C. parapsilosis, 48 C. tropicalis), out of which 22 were recovered from raptors, 32 from cactus parakeets, 56 from Amazon parrots, 7 from blue-and-gold macaws and 3 from a Brazilian porcupine. All isolates were submitted to broth microdilution test against amphotericin B, itraconazole and fluconazole, according to the methodology recommended by the Clinical Laboratory Standards Institute (document M27-A3). The MICs ranged from 0.03125 to 2 Âg/mL, 0.125 to 250 Âg/mL and 0.03125 to 125 Âg/mL for amphotericin B, fluconazole and itraconazole, respectively. Out of 126 evaluated isolates, 33 (26.2%) were resistant to azoles, with 7 (5.6%) isolates resistant to fluconazole, 1 (0.8%) isolate resistant to itraconazole and 24 (19%) resistant to both drugs. In a second approach, all these azole resistant isolates, plus 20 C. albicans and 3 C. tropicalis that were recovered from our collection of resistant yeasts from veterinary sources, were submitted to the efflux pump inhibition assay with promethazine, with a total of 56 azole resistant isolates. Thus, MICs for fluconazole and itraconazole significantly reduced from 2 to 250 times for fluconazole and from 16 to 4000 times for itraconazole. The antifungal activity of levamisole against 12 C. albicans, 12 C. krusei, 12 C. parapsilosis and 12 C. tropicalis, was also evaluated, and MICs and minimum fungicidal concentrations varying from 0.58 to 2.34 mg/mL and from 2.34 to 9.37 mg/mL were obtained, respectively. Parallelly, it was demonstrated that levamisole significantly inhibits biofilm formation and interferes with the maintenance of mature biofilms. These data show that azole resistance is partially mediated by efflux-pumps and demonstrate the antifungal potential of the imidazole levamisole and its capacity of inhibiting the biofilm of strains of Candida spp. from animals.
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