Avaliação da eficácia de agentes antifúngicos e do tratamento fotodinâmico antimicrobiano em conídios, micélios e biofilmes de Aspergillus fumigatus e Aspergillus flavus
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Data
2020-03-20
Autores
Francisco, Camila Guedes
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Universidade Estadual Paulista (Unesp)
Resumo
Os fungos filamentosos são microrganismos encontrados em diferentes ambientes atuando como decompositores nos ecossistemas. Contudo, algumas espécies como Aspergillus fumigatus e Aspergillus flavus podem ser agentes causadores de problemas para outros organismos, como plantas e animais, por causarem doenças ou pela produção de metabólitos secundários tóxicos. Assim como a maioria dos microrganismos, fungos filamentosos são hábeis em se desenvolver sobre superfícies, formando biofilmes. Tais biofilmes são estruturas com características particulares como, por exemplo, a tolerância aos agentes antimicrobianos rotineiramente utilizados, dificultando o controle microbiano. Neste contexto, a busca por estratégias alternativas de controle microbiano, como o Tratamento Fotodinâmico Antimicrobiano (TFDA), principalmente no que se refere aos biofilmes, é um desafio. Embora seja apontada como uma possibilidade promissora para o controle microbiano, até o momento não foram desenvolvidos estudos para avaliar a eficácia do TFDA em biofilmes de fungos filamentosos desenvolvidos sobre suportes inertes. O TFDA consiste na aplicação de uma molécula fotossensibilizadora sobre o microrganismo alvo, que após ser excitada por um comprimento de onda específico, leva à formação de espécies reativas de oxigênio (EROs), promovendo a morte do microrganismo. Assim sendo, o objetivo do presente estudo foi avaliar a eficácia de diferentes agentes antifúngicos convencionais (Anfotericina B, Miconazol e hipoclorito de sódio) e do TFDA em conídios, em micélios livres e biofilmes de A. flavus e A. fumigatus. As linhagens de A. fumigatus e A. flavus foram resistentes aos antifúngicos convencionais analisados, sendo mais susceptíveis ao hipoclorito de sódio. A concentração fungicida mínima (CFM) de anfotericina B (>5000 μg/mL), miconazol (>7500 μg/mL) e hipoclorito de sódio (5 mg/mL) para os biofilmes de A. flavus evidenciou que os biofilmes são igualmente susceptíveis, quando comparados aos micélios livres, aos antifúngicos usados. O mesmo ocorreu ao utilizar o miconazol e o hipoclorito de sódio no tratamento de biofilmes e micélios livres de A. fumigatus, cujo valores de CFM foram respectivamente >7500 μg/mL e 5000 μg/mL. No entanto, os biofilmes de A. fumigatus foram mais resistentes a anfotericina B se comparados aos micélios. Os biofilmes das linhagens de A. fumigatus e A. flavus avaliadas foram resistentes ao TFDA utilizando azul de metileno e novo azul de metileno como fotossenssibilizadores, tanto na presença como na ausência de iodeto de potássio. Houve um aumento na atividade catalásica dos biofilmes de A. fumigatus após o TFDA.
Filamentous fungi are microorganisms found in different environments acting as decomposers in ecosystems. However, some species such as Aspergillus fumigatus and Aspergillus flavus, may be troublemaking agents for other organisms, such as plants and animals, either by causing disease or by the production of toxic secondary metabolites. Like most microorganisms, filamentous fungi are able to grow on surfaces, forming biofilms. Biofilms are structures with particular characteristics as tolerance to routinely used antimicrobial agents, hindering microbial control. In this context, the search for alternative strategies for microbial control, especially on biofilms, such as the Antimicrobial Photodynamic Therapy (APDT), is a challenge. Although it is pointed out as a promising possibility for microbial control, to date, no studies have been developed to evaluate the effectiveness of APDT in biofilms of filamentous fungi developed on inert supports. The APDT consists of the application of a photosensitizing molecule on the target microorganism, which after being excited by a specific wavelength, leads to the formation of reactive oxygen species (ROS), promoting the death of the microorganism. Therefore, the aim of the present study was to evaluate the efficacy of different conven-tional antifungal agents (Amphotericin B, Miconazole and sodium hypochlorite) and APDT in the conidia, in the free mycelia and biofilms of A. flavus and A. fumigatus. The strains of A. fumigatus and A. flavus were resistant to the conventional antifungals ana-lyzed, being more susceptible to sodium hypochlorite. The minimum fungicidal concen-tration (MFC) of A. flavus biofilms for amphotericin B (> 5000 μg/mL), miconazole (> 7500 μg/mL) and sodium hypochlorite (5000 μg/mL) showed that biofilms were suscep-tible, as the mycelia, to the antifungals used. The same occurred using miconazole and sodium hypochloride for the treatment of biofilms and mycelia of A. fumigatus, whose MFC values were respectively >7500 μg/mL and 5000 μg/mL. However, A. fumigatus biofilms were more resistant to amphotericin B compared to mycelia. The biofilms of A. fumigatus and A. flavus strains evaluated were resistant to APDT using methylene blue and new methylene blue as photosensitizers, both in the presence and in the absence of potassium iodide. Increase in the catalytic activity of the catalase of A. fumigatus biofilms after APDT was observed.
Filamentous fungi are microorganisms found in different environments acting as decomposers in ecosystems. However, some species such as Aspergillus fumigatus and Aspergillus flavus, may be troublemaking agents for other organisms, such as plants and animals, either by causing disease or by the production of toxic secondary metabolites. Like most microorganisms, filamentous fungi are able to grow on surfaces, forming biofilms. Biofilms are structures with particular characteristics as tolerance to routinely used antimicrobial agents, hindering microbial control. In this context, the search for alternative strategies for microbial control, especially on biofilms, such as the Antimicrobial Photodynamic Therapy (APDT), is a challenge. Although it is pointed out as a promising possibility for microbial control, to date, no studies have been developed to evaluate the effectiveness of APDT in biofilms of filamentous fungi developed on inert supports. The APDT consists of the application of a photosensitizing molecule on the target microorganism, which after being excited by a specific wavelength, leads to the formation of reactive oxygen species (ROS), promoting the death of the microorganism. Therefore, the aim of the present study was to evaluate the efficacy of different conven-tional antifungal agents (Amphotericin B, Miconazole and sodium hypochlorite) and APDT in the conidia, in the free mycelia and biofilms of A. flavus and A. fumigatus. The strains of A. fumigatus and A. flavus were resistant to the conventional antifungals ana-lyzed, being more susceptible to sodium hypochlorite. The minimum fungicidal concen-tration (MFC) of A. flavus biofilms for amphotericin B (> 5000 μg/mL), miconazole (> 7500 μg/mL) and sodium hypochlorite (5000 μg/mL) showed that biofilms were suscep-tible, as the mycelia, to the antifungals used. The same occurred using miconazole and sodium hypochloride for the treatment of biofilms and mycelia of A. fumigatus, whose MFC values were respectively >7500 μg/mL and 5000 μg/mL. However, A. fumigatus biofilms were more resistant to amphotericin B compared to mycelia. The biofilms of A. fumigatus and A. flavus strains evaluated were resistant to APDT using methylene blue and new methylene blue as photosensitizers, both in the presence and in the absence of potassium iodide. Increase in the catalytic activity of the catalase of A. fumigatus biofilms after APDT was observed.
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Palavras-chave
Biofilmes, Fungos filamentosos, Aspergillus