A ação in vitro de pós bióticos da microbiota da pele na inibição do crescimento do Staphylococcus aureus: potencial uso na dermatite atópica
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Data
2023-07-28
Autores
Fuzeti, Deborah Corrêa [UNIFESP]
Orientadores
Lopes, Patricia Santos [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A dermatite atópica (DA) é uma doença inflamatória de pele crônica recidivante que
possui como principais sintomas xerose, prurido e lesões eczematosas, no entanto a
sua fisiopatologia ainda não é completamente elucidada. Um dos principais fatores
para o desenvolvimento da DA é a disbiose da microbiota da pele, onde o
Staphylococcus aureus, é o principal microrganismo relacionado. Um dos principais
fatores que levam a infecção são os seus fatores de virulência como os
superantígenos e o sistema agr. O sistema agr é um gene regulador do sistema
quorum sensing da espécie Staphylococcus, que auxilia em fatores de infecção por
promover o aumento da densidade celular e a expressão de fatores de virulência
secretados. Nesse contexto foram selecionados dois Staphylococcus comensais da
microbiota humana, o Staphylococcus epidermidis, e o Staphylococcus capitis,
visando a utilização de seus pós bióticos como possível tratamento. Para avaliar a
ação dos pós bióticos perante o S. aureus, realizou-se métodos in vitro para a analisar
o perfil de polaridade utilizando a CLAE, inibição de crescimento microbiano com
métodos de inibição de crescimento por difusão em ágar e em meio líquido e análise
da modulação da expressão gênica com PCR em tempo real. Os resultados
mostraram moléculas com comprimento de onda diferente nos três microrganismos
estudados, indicando ali um perfil molecular particular para cada microrganismo. No
método de inibição de crescimento por difusão em ágar, o uso dos derivados de pós
bióticos mostrou ser o mais eficaz, sendo as amostras derivadas do S. capitis com
melhor índice inibitório de 33,33%, e no teste de inibição de crescimento em meio
líquido também houve inibição do S. aureus com o uso de derivados de pós bióticos,
tendo como melhor índice inibitório o extrato de S. capitis acetato de etila com 86,58%.
Na análise da expressão gênica por PCR em tempo real ocorreu down-regulation nas
amostras S. capitis 25% e S. capitis 50% com o gene agrA, e S. capitis 25% com o
gene agrC, nas demais amostras com os genes agrA, agrC e agrD ocorreu upregulation da expressão do agr de S. aureus com o uso dos pós bióticos.
Atopic dermatitis (AD) is a recurrent chronic inflammatory skin disease whose main symptoms are xerosis, pruritus and eczematous lesions, however its pathophysiology is still not completely elucidated. One of the main factors for the development of AD is the dysbiosis of the skin microbiota, where Staphylococcus aureus is the main related microorganism. One of the main factors that lead to infection is the virulence factors such as superantigens and the agr system. The agr system is a regulatory gene of the quorum sensing system of Staphylococcus species, which aids in infection factors by promoting increased cell density and expression of secreted virulence factors. In this context, two commensal Staphylococcus from the human microbiota, Staphylococcus epidermidis and Staphylococcus capitis were selected with a view to using postbiotic as a possible treatment. To evaluate the action of postbiotics against S. aureus, in vitro methods were performed to analyze the polarity profile using HPC, inhibition of microbial growth with inhibition by diffusion in agar and broth growth and analysis of modulation of gene expression with real-time PCR. The results showed molecules with different wavelengths in the three microorganisms, indicating there a particular molecular profile for each microorganism. In the agar diffusion growth inhibition method, the uso of postbiotics derivatives proved to be the most effective, with samples derived from S. capitis having the best inhibitory indication of 33,33%, and in the growth inhibition test in the broth growth there was also inhibition of S. aureus with the use of derivatives of postbiotics, with the inhibition indication being the extract of S. capitis ethyl acetate with 86,58%. In the analysis of gene expression by real-time PCR, downregulation was observed in samples S. capitis 25% and S. capitis 50% with the agrA gene, and S. capitis 25% with the agrC gene, in the other samples with the agrA, agrC and agrD genes there was up-regulation of S. aureus agr expression with the use of postbiotics.
Atopic dermatitis (AD) is a recurrent chronic inflammatory skin disease whose main symptoms are xerosis, pruritus and eczematous lesions, however its pathophysiology is still not completely elucidated. One of the main factors for the development of AD is the dysbiosis of the skin microbiota, where Staphylococcus aureus is the main related microorganism. One of the main factors that lead to infection is the virulence factors such as superantigens and the agr system. The agr system is a regulatory gene of the quorum sensing system of Staphylococcus species, which aids in infection factors by promoting increased cell density and expression of secreted virulence factors. In this context, two commensal Staphylococcus from the human microbiota, Staphylococcus epidermidis and Staphylococcus capitis were selected with a view to using postbiotic as a possible treatment. To evaluate the action of postbiotics against S. aureus, in vitro methods were performed to analyze the polarity profile using HPC, inhibition of microbial growth with inhibition by diffusion in agar and broth growth and analysis of modulation of gene expression with real-time PCR. The results showed molecules with different wavelengths in the three microorganisms, indicating there a particular molecular profile for each microorganism. In the agar diffusion growth inhibition method, the uso of postbiotics derivatives proved to be the most effective, with samples derived from S. capitis having the best inhibitory indication of 33,33%, and in the growth inhibition test in the broth growth there was also inhibition of S. aureus with the use of derivatives of postbiotics, with the inhibition indication being the extract of S. capitis ethyl acetate with 86,58%. In the analysis of gene expression by real-time PCR, downregulation was observed in samples S. capitis 25% and S. capitis 50% with the agrA gene, and S. capitis 25% with the agrC gene, in the other samples with the agrA, agrC and agrD genes there was up-regulation of S. aureus agr expression with the use of postbiotics.