Biofilmes mistos de Mycobacterium tuberculosis e Paracoccidioides brasiliensis e ação de chalcona livre e carreada no sistema nanoemulsionado
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Data
2018-03-23
Autores
Medina-Alarcón, Kaila Petronila
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Universidade Estadual Paulista (Unesp)
Resumo
As infecções pulmonares são um problema global e estão classificadas em terceiro lugar entre as principais causas de morte. Entre as infecções pulmonares crônicas estão a tuberculose (TB) e a paracoccidioidomicose (PCM). A TB é uma doença cujo principal agente etiológico é o Mycobacterium tuberculosis e a PCM tem como agente etiológico o complexo Paracoccidioides spp. As duas doenças podem ocorrer na forma de coinfecção, com incidência aproximada de 2,8 a 5,5% dos casos nos países da América do Sul e 10% no Brasil. Ambos os microrganismos já foram descritos na forma de biofilme monoespécie e o tratamento de ambas as doenças tem merecido atenção, devido a casos de resistência e as poucas opções terapêuticas. Diante do exposto, este trabalho visa mostrar, pela primeira vez, a capacidade de M. tuberculosis e P. brasiliensis se associarem por meio do crescimento na forma de um biofilme misto in vitro, a fim de elaborar um novo protótipo de 3’chalcona carreada numa nanoemulsão com atividade para as formas planctônica e de biofilme. Para isso, inicialmente foi padronizada e caracterizada a formação dos biofilmes monoespécie da linhagem ATCC M. tuberculosis (H37Rv), da linhagem clínica (Rv40) e da linhagem de P. brasiliensis (Pb18) e dos biofilmes mistos das associações de H37Rv+Pb18 e Rv40+ Pb18 (nesta ordem de crescimento), ambas as associações mostraram um aparente sinergismo entre elas. Na formação do biofilme misto inverso denominado assim pela ordem de associação, onde primeiramente colocou-se a linhagem de P. brasiliensis (Pb18), seguida da adição de linhagem ATCC M. tuberculosis (H37Rv), formando o biofilme misto (Pb18+H37Rv), foi observada aparente competição entre os microrganismos. Todos os biofilmes, seja monoespécie e mistos se mostraram consolidados entre 30 a 45 dias de formação para todas as situações. Posteriormente avaliou-se a atividade antifúngica e antimicobacteriana dos derivados de chalconas semissintéticas frente às linhagens já relatadas anteriormente na sua forma planctônica. Os melhores resultados foram para a 3’chalcona, a qual foi selecionada para verificação da atividade antibiofilme. Devido à pouca solubilidade e estabilidade da 3’chalcona, foram desenvolvidas três nanoemulsões. Assim, a 3’chalcona foi incorporada na nanoemulsão 1 (SNL+3’chalcona), nanoemulsão 2 sem ergosterol (Me+3’chalcona) e a nanoemulsão 3 com ergosterol (Me+E+3’chalcona), sendo que as duas primeiras possuem colesterol na sua composição e a terceira possui colesterol e ergosterol. Todas as nanoemulsões mostraram uma alta atividade antifúngica e boa atividade antimicobacteriana, melhorando a atividade antifúngica em 4 vezes e em 2 vezes a atividade antimicobacteriana quando comparada com a 3’chalcona livre, além de estas formulações não apresentarem citotoxicidade com altos Índices de seletividade (IS) e alta atividade contra os biofilmes monoespécie, com inibição de até 78%. Também mostraram uma alta inibição dos biofilmes misto, com inibição de até 78% e 76% para H37Rv + Pb18 e Rv40 + Pb18, respectivamente, melhorando a atividade antibiofilme da 3’chalcona livre em até 50%. Além disso, todas as nanoemulsões apresentaram maior atividade antibiofilme do que a rifampicina, fármaco utilizado como controle de tratamento. Portanto, todas as nanoemulsões apresentaram resultados muito promissores; ressalta-se que a nanoemulsão SNL+3’chalcona apresentou os melhores resultados antibiofilme em todas as situações e, poderia se tornar um possível protótipo medicamentoso para o tratamento desta coinfecção.
Pulmonary infections are a global problem and rank third among the leading causes of death. Among the chronic lung infections are tuberculosis (TB) and paracoccidioidomycosis (PCM). The TB is a disease whose main etiologic agent is Mycobacterium tuberculosis and PCM has the etiological agent Paracoccidioides spp. The two diseases may occur in the form of coinfection, with an approximate incidence of 2.8 to 5.5% of the cases in the countries of South America and 10% in Brazil. Both microorganisms have already been described in the form of monospecies biofilm and the treatment of both diseases has deserved attention, due to cases of resistance and the few therapeutic options. In view of the above, this research aims to show, for the first time, the ability of M. tuberculosis and P. brasiliensis to associate by means of growth in the form of a mixed biofilm in vitro, in order to elaborate a new prototype in a nanoemulsion with activity for planktonic and biofilm forms. For this purpose, it was initially standardized and characterized the formation of monospecies biofilms of the ATCC strain M. tuberculosis (H37Rv), the clinical lineage (Rv40) and the P. brasiliensis (Pb18) lineage and the mixed biofilms of the associations of H37Rv + Pb18 and Rv40 + Pb18 (in this order of growth), both associations showed an apparent synergism between them. In order to establish the mixed biofilm (Pb18 + H37Rv), it was first proposed the association of P. brasiliensis (Pb18), followed by ATCC M. tuberculosis (H37Rv) with order of inverse growth, in this association a apparent competition between the microorganisms was observed. All biofilms, monospecies and mixed, were consolidated from 30 to 45 days of training for all situations. Subsequently, the antifungal and antimycobacterin activity of the semi-synthetic chalcone derivatives were evaluated against the strains previously reported in their planktonic form. The best results were for 3'chalcona, which was selected for verification of antibiofilm activity. Due to the poor solubility and stability of 3'chalcone, three nanoemulsions were developed. Thus, 3'chalcona was incorporated into nanoemulsion 1 (SNL + 3'chalcona), nanoemulsion 2 without ergosterol (Me + 3'chalcona) and nanoemulsion 3 with ergosterol (Me + E + 3'chalcona), both of which The first nanoemulsions have cholesterol in their composition and the third nanoemulsion has cholesterol and ergosterol. All the nanoemulsions showed a high antifungal activity and good antimycobacterial activity, improving the antifungal activity in 4 times and in 2 times the antimycobacterial activity when compared with 3'chalcona free. Besides these formulations did not present cytotoxicity with high selectivity indices (IS) and showed high activity against monospecies biofilms, with up to 78% inhibition. They also showed a high inhibition of mixed biofilms, with inhibition of up to 78% and 76% for H37Rv + Pb18 and Rv40 + Pb18, respectively, improving the antibiofilm activity of 3'chalcona free by up to 50%. In addition, all nanoemulsions presented higher antibiofilm activity than rifampicin, a drug used as a treatment control. Therefore, all nanoemulsions presented very promising results; it is noteworthy that the SNL + 3'chalcona nanoemulsion presented the best antibiofilm results in all situations and could become a possible drug prototype for the treatment of this coinfection.
Pulmonary infections are a global problem and rank third among the leading causes of death. Among the chronic lung infections are tuberculosis (TB) and paracoccidioidomycosis (PCM). The TB is a disease whose main etiologic agent is Mycobacterium tuberculosis and PCM has the etiological agent Paracoccidioides spp. The two diseases may occur in the form of coinfection, with an approximate incidence of 2.8 to 5.5% of the cases in the countries of South America and 10% in Brazil. Both microorganisms have already been described in the form of monospecies biofilm and the treatment of both diseases has deserved attention, due to cases of resistance and the few therapeutic options. In view of the above, this research aims to show, for the first time, the ability of M. tuberculosis and P. brasiliensis to associate by means of growth in the form of a mixed biofilm in vitro, in order to elaborate a new prototype in a nanoemulsion with activity for planktonic and biofilm forms. For this purpose, it was initially standardized and characterized the formation of monospecies biofilms of the ATCC strain M. tuberculosis (H37Rv), the clinical lineage (Rv40) and the P. brasiliensis (Pb18) lineage and the mixed biofilms of the associations of H37Rv + Pb18 and Rv40 + Pb18 (in this order of growth), both associations showed an apparent synergism between them. In order to establish the mixed biofilm (Pb18 + H37Rv), it was first proposed the association of P. brasiliensis (Pb18), followed by ATCC M. tuberculosis (H37Rv) with order of inverse growth, in this association a apparent competition between the microorganisms was observed. All biofilms, monospecies and mixed, were consolidated from 30 to 45 days of training for all situations. Subsequently, the antifungal and antimycobacterin activity of the semi-synthetic chalcone derivatives were evaluated against the strains previously reported in their planktonic form. The best results were for 3'chalcona, which was selected for verification of antibiofilm activity. Due to the poor solubility and stability of 3'chalcone, three nanoemulsions were developed. Thus, 3'chalcona was incorporated into nanoemulsion 1 (SNL + 3'chalcona), nanoemulsion 2 without ergosterol (Me + 3'chalcona) and nanoemulsion 3 with ergosterol (Me + E + 3'chalcona), both of which The first nanoemulsions have cholesterol in their composition and the third nanoemulsion has cholesterol and ergosterol. All the nanoemulsions showed a high antifungal activity and good antimycobacterial activity, improving the antifungal activity in 4 times and in 2 times the antimycobacterial activity when compared with 3'chalcona free. Besides these formulations did not present cytotoxicity with high selectivity indices (IS) and showed high activity against monospecies biofilms, with up to 78% inhibition. They also showed a high inhibition of mixed biofilms, with inhibition of up to 78% and 76% for H37Rv + Pb18 and Rv40 + Pb18, respectively, improving the antibiofilm activity of 3'chalcona free by up to 50%. In addition, all nanoemulsions presented higher antibiofilm activity than rifampicin, a drug used as a treatment control. Therefore, all nanoemulsions presented very promising results; it is noteworthy that the SNL + 3'chalcona nanoemulsion presented the best antibiofilm results in all situations and could become a possible drug prototype for the treatment of this coinfection.
Descrição
Palavras-chave
Mycobacterium tuberculosis, Paracoccidioides brasiliensis, Coinfecção, Biofilme misto, chalconas, Nanoemulsão, Mycobacterium tuberculosis, Paracoccidioides brasiliensis, Coinfection, Mixed Biofilm, chalcone, Nanoemulsion