Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Diversos sÃo os relatos de resistÃncia in vitro de cepas de Candida spp. a fÃrmacos antifÃngicos, em especial a derivados azÃlicos. Deste modo, a prospecÃÃo de novos compostos com propriedade antifÃngica se faz necessÃria. Assim, o objetivo deste estudo foi avaliar a atividade antifÃngica do sesquiterpeno farnesol, sua aÃÃo sinÃrgica com antifÃngicos fluconazol (FLC), itraconazol (ITC), voriconazol (VRZ), anfotericina B (AMB) e caspofungina (CASP), bem como avaliar sua aÃÃo sob a atividade enzimÃtica de fosfolipases e proteases, consideradas importantes fatores de virulÃncia em Candida spp. Para tanto, foram avaliadas cepas de C. albicans (n=23), C. parapsilosis (n=16) e C. tropicalis (n=6). A concentraÃÃo inibitÃria mÃnima (CIM) foi determinada por meio da tÃcnica de microdiluiÃÃo, preconizada pelo Clinical Laboratory Standards Institute (CLSI). Em adiÃÃo, foi investigado o efeito do farnesol sobre a atividade de fosfolipase utilizando Ãgar Sabouraud suplementado com gema de ovo e a atividade de protease por tÃcnica espetrofotomÃtica. Paralelamente 14 cepas escolhidas aleatoriamente, foram prÃ-incubadas em trÃs concentraÃÃes sub-CIM de farnesol, afim de avaliar se o farnesol era capaz de causar algum dano na sensibilidade da cepa. Para as 45 cepas avaliadas, os valores de CIM para farnesol variaram de 9,37 a 150 ÂM para Candida spp. Para os derivados azÃlicos, o intervalo dos valores de CIM para fluconazol foi de 0,03125 a > 64 Âg/ml, observando-se a presenÃa de 18 cepas resistentes (CIM > 64 Âg/mL). Em relaÃÃo ao itraconazol, os valores de CIM variaram de 0,03125 a >16 Âg/mL e 35 cepas apresentaram resistÃncia a esse antifÃngico (CIM ≥1 Âg/mL). Constatou-se que das 45 cepas em estudo, 19 apresentaram resistÃncia a anfotericina B, com CIM > 1 Âg/mL. Quanto a caspofungina, os valores de CIM variaram de 0,0625 a 2 Âg/mL, com apenas seis cepas exibido resistÃncia, com CIM de 2 Âg/mL. A combinaÃÃo anfotericina B e farnesol apresentou sinergismo em 94,7% (18/19) das cepas de Candida spp. resistentes, evidenciado pelos valores de FICI ≤ 0,5. Na associaÃÃo fluconazol e farnesol, observou-se uma interaÃÃo sinÃrgica frente a todas as cepas de Candida spp. avaliadas, apresentando FICI ≤ 0,5. Trinta e seis cepas (18 C. albicans, 12 C. parapsilosis e 6 C. tropicalis) foram testadas frente à associaÃÃo de itraconazol e farnesol, observando a ocorrÃncia de sinergismo (FICI ≤ 0,5) em 94,4% dos isolados. Quanto à caspofungina todas as cepas testadas (C. parapsilosis, n=4; C. albicans, n=2) tiveram FICI ≤ 0,5, caracterizando associaÃÃo sinÃrgica com o farnesol. ApÃs constataÃÃo de que 17 das 45 cepas expressavam a enzima fosfolipase, as mesmas foram incubadas em concentraÃÃes sub-CIM de farnesol, por 24 horas, quando observouâse uma medida de Pz mÃdia de 0,73 para as cepas testadas sem contato com farnesol e valores de 0,71, 0,61, e 0,54 para aquelas incubadas em doses sub-inibitÃria de farnesol. Em relaÃÃo à produÃÃo de protease, observou-se que todas as 14 cepas produziam a enzima, em baixas concentraÃÃes, variando de 0,002 a 0,02 U/mL, e que a prÃ-incubaÃÃo com farnesol nÃo interferiu na produÃÃo enzimÃtica. No tocante a prÃ-incubaÃÃo das cepas com farnesol constatou-se que o mesmo interferi direta e indiretamente no fenÃmeno de resistÃncia das cepas. Estes resultados demonstram, em especial, o efeito do composto farnesol sobre a sensibilidade antifÃngica de Candida spp., abrindo a perspectiva de novos estudos com o intuito de determinar os mecanismos de aÃÃo desses compostos no metabolismo celular dos fungos. / There are several reports of in vitro resistance to antifungal drugs, especially azole derivatives, in strains of Candida spp. For this reason, the pursuit for new compounds that present antifungal properties is necessary. Thus, this study aimed at evaluating the antifungal activity of farnesol and its interaction with classical antifungal drugs against Candida spp., as well as evaluating its effect on the production of phospholipase and protease, which are important virulence factors for Candida species. Fourty-five strains of Candida spp. (23 C. albicans, 16 C. parapsilosis and 6 C. tropicalis) were tested through broth microdilution as described by the Clinical Laboratory Standards Institute (CLSI), document M27A2, and the minimum inhibitory concentrations (MICs) for amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC), caspofungin (CAS) and farnesol (FAR) were individually determined. In addition, the effect of FAR on phospholipase activity was assessed by growing the strains on 2% Sabouraud agar, supplemented with egg yolk, and protease activity was determined through spectrophotometry. Then, 13 strains were randomly chosen, pre-incubated at three sub-inhibitory concentrations of FAR for 24 hours and re-submitted to microdilution assay. For the 45 evaluated Candida spp. strains the MIC values for FAR varied from 9.37 to 150 ÂM. For the classical antifungal drugs, MICs ranged from 0.0625 to 4 Âg/mL for AMB, with 19 resistant strains (MIC>1 Âg/mL); from 0.0125 to >64 for FLC, with 18 resistant strains (MIC> 64 Âg/mL); from 0.03125 to >16 Âg/mL for ITC, with 35 resistant strains (MIC ≥1 Âg/mL), and from 0.0625 to 2 Âg/mL for CAS, with six resistant strains (MIC≥2 Âg/mL). All resistant strains were tested against the combination of FAR with the drug to which they presented resistance. The combination of AMB and FAR was synergistic against 94.7% (18/19) of the Candida spp. isolates, as shown through the obtention of FICI≤0.5. FAR and FLC interacted synergistically against all tested strains (n=18), exhibiting FICI values of ≤0.5. The combination of FAR and ITC presented synergistic interactions (FICI≤0.5) against 94.4% of the tested isolates (33/35). Finally, FAR and CAS showed to interact synergistically (FICI≤0.5) against all tested strains. Concerning virulence factors, it was observed that 17/45 isolates produced phospholipase, with a mean Pz of 0.71. After incubating these strains at three different concentrations of FAR the mean Pz values of 0.71, 0.61 and 0.54 were obtained, after incubation at the lowest, the intermediate and the highest concentrations of FAR, respectively. In addition, it was observed that the 14 randomly chosen strains that were screened for protease activity produced low concentrations of these enzymes, varying from 0.002 to 0.02 U/mL and that FAR presented no effect on enzymatic production. Finally, it was observed that pre-incubating strains at the highest sub-inhibitory concentration of FAR reduced the MIC range of the tested antifungal drugs. These results especially show the effects of FAR on the susceptibility of Candida species to classical antifungal drugs, providing perspectives for the development of researches on the mechanisms of this compound on fungal cell metabolism
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:6398 |
| Date | 13 January 2011 |
| Creators | Carlos Eduardo Cordeiro Teixeira |
| Contributors | Marcos FÃbio Gadelha Rocha, Maria Fatima da Silva Teixeira, Raimunda SÃmia Nogueira Brilhante, Eveline Pipolo Milan |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em Microbiologia MÃdica, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
Page generated in 0.0024 seconds