Associação da quitosana à terapia fotodinâmica para controle de Candida albicans: estudo em culturas planctônicas, biofilmes e células persistentes
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Data
2021-10-08
Autores
Lapena, Simone Aparecida Biazzi De
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Universidade Estadual Paulista (Unesp)
Resumo
A terapia fotodinâmica (TFD) vem se mostrando como um método eficaz no controle de micro-organismos patogênicos, sendo investigada para o tratamento de diversas doenças infecciosas, como a candidose bucal. Recentemente, alguns agentes potencializadores dessa terapia têm sido estudados, incluindo a Quitosana (QT), um polímero natural extraído do exoesqueleto de crustáceos. O objetivo desse estudo foi avaliar o efeito potencializador da QT na TFD mediada pelo fotossensibilizador Azul de Metileno (AM) sobre cepas de Candida albicans, investigando sua ação em culturas planctônicas, biofilmes e células persistentes ao fluconazol. Além disso, foi avaliado a capacidade da QT em interferir na absorção do AM pelas células de Candida. Foram utilizadas duas cepas de C. albicans, sendo uma padrão (ATCC 18804) e uma clínica isolada de candidose orofaríngea (70). Para os ensaios em culturas planctônicas, as cepas de C. albicans foram cultivadas em caldo Yeast Nitrogen Base (YNB) por 24 horas. Os biofilmes foram formados por 48 horas no fundo das placas de 96 poços. Para indução de células persistentes, C. albicans foi cultivada com altas concentrações de fluconazol por 48 horas. A seguir, foram realizados os tratamentos com QT a 5 mg/mL (pH de 6,5), AM nas concentrações de 300 ou 600 μM, e irradiação com LED (660 nm) na densidade de energia de 30 J/cm2. Foram incluídos oito grupos experimentais: TFD com AM e QT na presença de Luz (AM+QT+L+), AM e QT sem Luz (AM+QT+L-), QT e Luz (QT+L+), QT sem Luz (QT+L-), TFD com AM e Luz (AM+L+), AM sem Luz (AM+L-), Solução Fisiológica com Luz (F-L+) e apenas Solução Fisiológica (F-L-). Após os tratamentos, foi realizada contagem de unidades formadoras de colônias (UFC/mL). Para determinar a absorção do fotossensibilizador pelas células de C. albicans, as células foram lisadas e centrifugadas para leitura da densidade óptica. Os dados foram analisados por ANOVA e teste de Tukey (p<0,05%). Na TFD em culturas planctônicas, o AM (300 μm) reduziu as células de Candida em 1,6 log (UFC/mL), enquanto que a associação AM+QT levou à redução de 4,7 log. Na TFD em biofilmes, ocorreu redução microbiana de 2,9 log para o tratamento com AM (600 μm) e de 5,3 log para AM+QT. Em relação às células persistentes, a redução encontrada foi de 0,8 log para AM e 1,5 log para AM+QT. No teste de absorção, a penetração do AM nas células de Candida (DO 0,02) foi aumentada na presença da QT (DO 0,39). Concluiu-se que a QT potencializou o efeito antimicrobiano da TFD em culturas planctônicas, biofilmes e células persistentes de C. albicans, provavelmente por facilitar a penetração do AM nas células fúngicas.
Photodynamic therapy (PDT) has been shown to be an effective method to control pathogenic microorganisms, being investigated for the treatment of several infectious diseases, such as oral candidiasis. Recently, some agents that enhance this therapy have been studied, including Chitosan (CS), a natural polymer extracted from the exoskeleton of crustaceans. The aim of this study was to evaluate the potentiating effect of CS on Methylene Blue (MB) photosensitizer-mediated PDT on Candida albicans strains, investigating its action on planktonic cultures, biofilms and persister cells to fluconazole. In addition, the ability of CS to interfere with MB absorption by Candida cells was evaluated. Two strains of C. albicans were used, one standard (ATCC 18804) and one isolated clinical of oropharyngeal candidosis (70). For assays in planktonic cultures, C. albicans strains were cultivated in Yeast Nitrogen Base broth (YNB) for 24 hours. Biofilms were formed for 48 hours at the bottom of 96-well plates. For the induction of persister cells, C. albicans was cultivated with high concentrations of fluconazole for 48 hours. Next, treatments were performed with CS at 5 mg/mL (pH 6.5), MB at concentrations of 300 or 600 μM, and irradiation with LED (660 nm) at an energy density of 30 J/cm2. eight experimental groups were included: PDT with MB and CS in the presence of Light (MB+CS+L+), MB and CS without Light (MB+CS+L-), CS and Light (CS+L+), CS without Light (CS +L-), PDT with MB and Light (MB+L+), MB without Light (MB+L-), Physiological Solution with Light (F-L+) and Physiological Solution only (FL-). After the treatments, colony forming units (CFU/mL) were counted. To determine the absorption of the photosensitizer by C. albicans cells, the cells were lysed and centrifuged for optical density reading. Data were analyzed by ANOVA and Tukey test (p<0.05%). In PDT in planktonic cultures, MB (300 μm) reduced Candida cells by 1.6 log (CFU/mL), while the MB+CS association led to a 4.7 log reduction. In PDT in biofilms, there was a microbial reduction of 2.9 log for the treatment with MB (600 μm) and of 5.3 log for MB+CS. Regarding persister cells, the reduction found was 0.8 log for MB and 1.5 log for MB+CS. In the absorption test, the penetration of MB into Candida cells (OD 0.02) was increased in the presence of CS (OD 0.39). It was concluded that CS potentiated the antimicrobial effect of PDT in planktonic cultures, biofilms and persister cells of C. albicans, probably by facilitating the penetration of MB into fungal cells .
Photodynamic therapy (PDT) has been shown to be an effective method to control pathogenic microorganisms, being investigated for the treatment of several infectious diseases, such as oral candidiasis. Recently, some agents that enhance this therapy have been studied, including Chitosan (CS), a natural polymer extracted from the exoskeleton of crustaceans. The aim of this study was to evaluate the potentiating effect of CS on Methylene Blue (MB) photosensitizer-mediated PDT on Candida albicans strains, investigating its action on planktonic cultures, biofilms and persister cells to fluconazole. In addition, the ability of CS to interfere with MB absorption by Candida cells was evaluated. Two strains of C. albicans were used, one standard (ATCC 18804) and one isolated clinical of oropharyngeal candidosis (70). For assays in planktonic cultures, C. albicans strains were cultivated in Yeast Nitrogen Base broth (YNB) for 24 hours. Biofilms were formed for 48 hours at the bottom of 96-well plates. For the induction of persister cells, C. albicans was cultivated with high concentrations of fluconazole for 48 hours. Next, treatments were performed with CS at 5 mg/mL (pH 6.5), MB at concentrations of 300 or 600 μM, and irradiation with LED (660 nm) at an energy density of 30 J/cm2. eight experimental groups were included: PDT with MB and CS in the presence of Light (MB+CS+L+), MB and CS without Light (MB+CS+L-), CS and Light (CS+L+), CS without Light (CS +L-), PDT with MB and Light (MB+L+), MB without Light (MB+L-), Physiological Solution with Light (F-L+) and Physiological Solution only (FL-). After the treatments, colony forming units (CFU/mL) were counted. To determine the absorption of the photosensitizer by C. albicans cells, the cells were lysed and centrifuged for optical density reading. Data were analyzed by ANOVA and Tukey test (p<0.05%). In PDT in planktonic cultures, MB (300 μm) reduced Candida cells by 1.6 log (CFU/mL), while the MB+CS association led to a 4.7 log reduction. In PDT in biofilms, there was a microbial reduction of 2.9 log for the treatment with MB (600 μm) and of 5.3 log for MB+CS. Regarding persister cells, the reduction found was 0.8 log for MB and 1.5 log for MB+CS. In the absorption test, the penetration of MB into Candida cells (OD 0.02) was increased in the presence of CS (OD 0.39). It was concluded that CS potentiated the antimicrobial effect of PDT in planktonic cultures, biofilms and persister cells of C. albicans, probably by facilitating the penetration of MB into fungal cells .
Descrição
Palavras-chave
Terapia fotodinamica, Quitosana, Candida albicans, Células persistentes, Azul de metileno, Photodynamic therapy, Candida albicans, Chitosan, Methylene blue, Persister cells