Susceptibilidade de Candida albicans resistente a fluconazol ao efeito fotodinâmica e inibidores dos sistemas de efluxo
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Data
2018-08-06
Autores
Vega Chacón, Yuliana del Pilar
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Universidade Estadual Paulista (Unesp)
Resumo
Um dos principais mecanismos de resistência microbiana são os sistemas de efluxo, que transportam medicamentos antimicrobiano para fora da célula. A eficácia de alguns agentes de inibição dos sistemas de efluxo tem sido reportada para reverter a resistência microbiana e também para potencializar as terapias antimicrobianas. Além disso, métodos alternativos aos agentes antimicrobianos convencionais têm sido investigados, como a Terapia Fotodinâmica antimicrobiana (aPDT). O objetivo desse estudo foi avaliar in vitro o efeito da aPDT e de dois inibidores de sistemas de efluxo microbiano (curcumina e verapamil) na resistência à inativação de C. albicans. Foram utilizadas duas cepas de C. albicans, uma susceptível (CaS) e outra resistente (CaR) a fluconazol. Os parâmetros de inativação fúngica foram determinados submetendo-se culturas planctônicas de ambas as cepas à curcumina, ao verapamil, ao fluconazol e também à aPDT (mediada pela curcumina 40 μM (14,73 μg/mL) e luz de LED azul de ≅455 nm a 5,28 J/cm2). As duas cepas foram cultivadas e tratadas associando-se os agentes de inibição do efluxo ao fluconazol em concentrações não letais. Os dados de UFC/mL foram analisados pelos testes paramétricos t de Student, ANOVA/Welch e post-hoc de Games-Howell e pelo teste não paramétrico de Mann-Whitney (α=0,05; n=12). Os resultados demostraram que aPDT promoveu uma redução significativa (p<0,001) de 4,5 e 4,42 log10 para CaS e CaR, respectivamente. Para CaS, os valores de Concentrações Inibitórias Mínimas (CIMs) de fluconazol, curcumina e verapamil foram de 0,5 μg/mL, 20 μM (7,37 μg/mL) e 4 mg/mL (4.000 μg/mL), respectivamente; as quais promoveram reduções ≥ 1,4 log10 nas concentrações ≥ CIM (p≤0,045). Para CaR, fluconazol e verapamil apresentaram valores de CIM de 128 μg/mL e 4 mg/mL (4.000 μg/mL), respectivamente; a curcumina não reduziu a viabilidade de CaR, ao passo que redução significativa ≥ 1,74 log10 foram observadas para verapamil e fluconazol ≥ CIM (p<0,001). A associação sub-CIM de verapamil + fluconazol de 2 mg/mL (2.000 μg/mL) + 0,25 μg/mL, respectivamente, para CaS e 2 mg/mL (2.000 μg/mL) + 64 μg/mL, respectivamente, para CaR mostrou uma redução (p<0,001) maior para CaR de 4,08 log10 em comparação com CaS (p<0,001, 0,59 log10). A associação de curcumina + fluconazol de 10 μM (2,68 μg/mL) + 0,25 μg/mL, respectivamente, para CaS e 40 μM (14,73 μg/mL) + 64 μg/mL, respectivamente, para CaR promoveu redução maior para CaR (p<0,001) de 2,28 log10 do que para CaS (p<0,001) de 1,58 log10. A associação de curcumina, verapamil e fluconazol nessas mesmas concentrações resultou em uma redução (p<0,001) de 0,77 log10 para CaS e de 2,86 log10 e para CaR. Concluiu-se que a aPDT reduziu tanto a viabilidade de CaS como a de CaR. O verapamil e a curcumina reverteram a resistência de C. albicans ao fluconazol em culturas planctônicas, tendo um efeito inibitório maior para CaR do que para CaS.
One of the major mechanisms of microbial resistance is the efflux systems, or efflux pumps, present in the plasma membrane of microorganisms that carry an antimicrobial drug out of the cell. The efficacy of some inhibitors of efflux systems has been reported to reverse microbial resistance, including C. albicans, and also to potentiate antimicrobial therapies. In addition, alternative methods to conventional antimicrobial agents have been investigated, such as antimicrobial Photodynamic Therapy (aPDT). The objective of this study was to evaluate in vitro the effect of aPDT and two inhibitors of microbial efflux systems (curcumina and verapamil) on the resistance to inactivation of C. albicans. For this, two strains of C. albicans, one susceptible (CaS) and another resistant (CaR) to fluconazol were used. Fungal inactivation parameters were determined by subjecting planktonic cultures of both strains to curcumina, verapamil, fluconazol, and also aPDT (mediated by curcumin at 40 μM (14.73 μg/mL) and blue LED light of ≅455 nm and 5.28 J/cm2). These strains were then cultured and treated associating one of the efflux inhibitors with fluconazole using non-lethal concentrations. For the statistical analysis, the normality and the homogeneity of variances were evaluated by the Shapiro-Wilk and Levene tests, respectively. Data were analyzed by Student's t-tests, Welch-corrected ANOVA and Games-Howell post-hoc and Mann-Whitney non-parametric test (α = 0.05) (n = 12). aPDT promoted a significant reduction (p<0.001) of 4.5 log10 for CaS and 4.42 log10 for CaR. For CaS, the values of Minimum Inhibitory Concentrations (MIC) of fluconazole, curcumin and verapamil were 0.5 μg/mL, 20 μM (7.37 μg/mL) and 4 mg/mL (4000 μg/mL), respectively; which promoted reductions of ≥ 1.4 log10 at concentrations ≥ MIC (p≤0,045). For CaR, fluconazole and verapamil showed MIC values of 128 μg/mL and 4 mg/mL (4000 μg/mL), respectively; curcumin did not reduce the viability of CaR, whereas significant reduction ≥ 1.74 log10 were observed for verapamil and fluconazole at ≥ MIC (p<0.001). The association of verapamil + fluconazole at sub-MIC of 2 mg/mL (2000 μg/mL) + 0.25 μg/mL, respectively, for CaS, and 2 mg/mL (2000 μg/mL) + 64 μg/mL, respectively, for CaR showed higher reduction (p<0.001) for CaR of 4.08 log10 compared to CaS (p<0.001; 0.59 log10). The association of curcumin + fluconazole at 10 μM (3.68 μg/mL) + 0.25 μg/mL, respectively, for CaS and at 40 μM (14.73 μg/mL) + 64 μg/mL, respectively, for CaR promoted higher reduction for CaR (p<0.001) of 2.28 log10 than for CaS (p<0.001) of 1.58 log10. The association of curcumin, verapamil, and fluconazole at the same concentrations showed reduction (p<0.001) of 0.77 log10 for CaS and 2.86 log10 for CaR. In conclusion, aPDT significantly reduced the viability of CaS and CaR. Verapamil and curcumin reversed the resistance of C. albicans to fluconazole in planktonic cultures, having a higher inhibitory effect for CaR than CaS.
One of the major mechanisms of microbial resistance is the efflux systems, or efflux pumps, present in the plasma membrane of microorganisms that carry an antimicrobial drug out of the cell. The efficacy of some inhibitors of efflux systems has been reported to reverse microbial resistance, including C. albicans, and also to potentiate antimicrobial therapies. In addition, alternative methods to conventional antimicrobial agents have been investigated, such as antimicrobial Photodynamic Therapy (aPDT). The objective of this study was to evaluate in vitro the effect of aPDT and two inhibitors of microbial efflux systems (curcumina and verapamil) on the resistance to inactivation of C. albicans. For this, two strains of C. albicans, one susceptible (CaS) and another resistant (CaR) to fluconazol were used. Fungal inactivation parameters were determined by subjecting planktonic cultures of both strains to curcumina, verapamil, fluconazol, and also aPDT (mediated by curcumin at 40 μM (14.73 μg/mL) and blue LED light of ≅455 nm and 5.28 J/cm2). These strains were then cultured and treated associating one of the efflux inhibitors with fluconazole using non-lethal concentrations. For the statistical analysis, the normality and the homogeneity of variances were evaluated by the Shapiro-Wilk and Levene tests, respectively. Data were analyzed by Student's t-tests, Welch-corrected ANOVA and Games-Howell post-hoc and Mann-Whitney non-parametric test (α = 0.05) (n = 12). aPDT promoted a significant reduction (p<0.001) of 4.5 log10 for CaS and 4.42 log10 for CaR. For CaS, the values of Minimum Inhibitory Concentrations (MIC) of fluconazole, curcumin and verapamil were 0.5 μg/mL, 20 μM (7.37 μg/mL) and 4 mg/mL (4000 μg/mL), respectively; which promoted reductions of ≥ 1.4 log10 at concentrations ≥ MIC (p≤0,045). For CaR, fluconazole and verapamil showed MIC values of 128 μg/mL and 4 mg/mL (4000 μg/mL), respectively; curcumin did not reduce the viability of CaR, whereas significant reduction ≥ 1.74 log10 were observed for verapamil and fluconazole at ≥ MIC (p<0.001). The association of verapamil + fluconazole at sub-MIC of 2 mg/mL (2000 μg/mL) + 0.25 μg/mL, respectively, for CaS, and 2 mg/mL (2000 μg/mL) + 64 μg/mL, respectively, for CaR showed higher reduction (p<0.001) for CaR of 4.08 log10 compared to CaS (p<0.001; 0.59 log10). The association of curcumin + fluconazole at 10 μM (3.68 μg/mL) + 0.25 μg/mL, respectively, for CaS and at 40 μM (14.73 μg/mL) + 64 μg/mL, respectively, for CaR promoted higher reduction for CaR (p<0.001) of 2.28 log10 than for CaS (p<0.001) of 1.58 log10. The association of curcumin, verapamil, and fluconazole at the same concentrations showed reduction (p<0.001) of 0.77 log10 for CaS and 2.86 log10 for CaR. In conclusion, aPDT significantly reduced the viability of CaS and CaR. Verapamil and curcumin reversed the resistance of C. albicans to fluconazole in planktonic cultures, having a higher inhibitory effect for CaR than CaS.
Descrição
Palavras-chave
Proteínas de membrana transportadoras. Candidíase. Resistência microbiana á medicamentos. Antifúngicos. Fotoquimioterapia., Membrane transport proteins. Candidiasis. Drug resistance. Antifungal agents. Photochemotherapy.