A carregar...
Publicação
Characterization of models of young and mature bacterial biofilms to study the activity and mechanism of action of a designed cyclic analog of gomesin
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 4.08 MB | Adobe PDF |
Autores
Resumo(s)
Os biofilmes bacterianos apresentam alta recalcitrância a agentes antimicrobianos devido
a vários fatores, incluindo a presença de uma matriz extracelular que dificulta a sua
difusão, a reduzida suscetibilidade de bactérias dentro do biofilme e a degradação
enzimática. Neste sentido, o tratamento de infeções associadas a biofilmes enfrenta
severos problemas ao nível do tratamento, exigindo o uso de doses mais elevadas ou a
aplicação de terapias combinadas para obter eficácia, destacando a necessidade urgente
de abordagens inovadoras e alternativas aos antimicrobianos convencionais. O
desenvolvimento de biofilmes tem sido uma área central de pesquisa na busca por novas
terapias, uma vez que a compreensão da estrutura e dos mecanismos de crescimento do
biofilme ainda é mal compreendida. Neste estudo, selecionamos uma estirpe de MRSA
formadora de biofilme (LUH14616) para realizar uma ampla caracterização in vitro de
biofilmes jovens (1 dia) e maduros (5 dias), e posteriormente investigar a atividade e
mecanismo de ação de [G1K, K8R]cGm, um análogo cíclico do péptido antimicrobiano
gomesina. Resultados obtidos do ensaio de violeta de cristal revelaram uma maior
quantidade de biomassa aderida em estágios de maturação posteriores, embora houvesse
menos bactérias por unidade de massa. Notavelmente, evidenciado por microscopias
confocal e de força atómica, biofilmes jovens exibiram uma estrutura mais espessa e
robusta com bactérias revestidas por uma camada espessa de matriz, contrastando com
um biofilme mais fino e rarefeito em estágios de maturação posteriores. Para iniciar o
estudo da atividade e do mecanismo de ação de [G1K, K8R]cGm contra biofilmes jovens,
foram testadas concentrações crescentes do péptido por 4 e 24 h. No ensaio fluorométrico
de redução da resazurina, resultados mostraram que [G1K, K8R]cGm conseguiu reduzir
a atividade metabólica das bactérias de forma dose-dependente, atingindo um decréscimo
máximo de 55% a 50 µM. Por sua vez, estes resultados foram confirmados por confocal,
onde biofilmes tratados com 50 µM [G1K, K8R]cGm apresentavam uma percentagem
significativamente maior de células bacterianas com membranas comprometidas,
revelando a capacidade do péptido em difundir através da espessa camada de
exopolissacárido e permeabilizar a membrana citoplasmática, causando a sua disrupção
e, por fim, levando à morte celular. No entanto, uma predominância de bactérias com
membrana intacta nas camadas mais profundas foi observada, mostrando um mecanismo
dependente da profundidade do biofilme. Estudos de AFM revelaram alterações
significativas na morfologia e topografia da superfície dos biofilmes tratados com [G1K,
K8R]cGm, indicando um aumento na rugosidade da superfície do biofilme e das
bactérias, mostrando uma relação com a capacidade de [G1K,K8R]cGm na disrupção da
membrana da célula bacteriana. Em geral, os nossos resultados reforçam a importância
de aprofundar o conhecimento sobre a fisiologia dos biofilmes e a sua estrutura
tridimensional em termos de distribuição de células e matriz, uma vez que a
heterogeneidade dos biofilmes pode afetar a atividade dos agentes antimicrobianos. São
necessárias investigações adicionais para avaliar o potencial de [G1K, K8R]cGm contra
biofilmes maduros e a sua aplicação no combate a biofilmes bacterianos em diferentes
estágios de maturação.
Bacterial biofilms present high recalcitrance to antimicrobial agents due to various factors, including the presence of an extracellular matrix that hinders their diffusion, reduced susceptibility of biofilm-embedded bacteria, and enzymatic degradation. Therefore, the treatment of biofilm-associated infections faces severe challenges, requiring higher doses or the application of combination therapies to achieve effectiveness. This highlights the urgent need for innovative and alternative approaches to conventional antimicrobials. The development of biofilms has been a central area of research in the search for new therapies, as the understanding of biofilm structure and growth mechanisms is still poorly understood. In this study, we selected a strain of biofilm-forming MRSA (LUH14616) to perform a comprehensive in vitro characterization of young (1-day) and mature (5-day) biofilms, and subsequently investigate the activity and mechanism of action of [G1K, K8R]cGm, a cyclic analog of the antimicrobial peptide gomesin. Results from the crystal violet assay revealed a greater amount of adhered biomass in later maturation stages, although there were fewer bacteria per unit mass. Notably, as evidenced by confocal and atomic force microscopy, young biofilms exhibited a thicker and more robust structure with bacteria coated by a thick layer of matrix, contrasting with a thinner and more sparse biofilm in later maturation stages. To initiate the study of the activity and mechanism of action of [G1K, K8R]cGm against young biofilms, increasing concentrations of the peptide were tested for 4 and 24 h. In the resazurin reduction fluorometric assay, results showed that [G1K, K8R]cGm was able to dose-dependently reduce bacterial metabolic activity, reaching a maximum decrease of 55% at 50 µM. These results were further confirmed by confocal microscopy, where biofilms treated with 50 µM [G1K, K8R]cGm exhibited a significantly higher percentage of bacterial cells with compromised membranes, revealing the peptide's ability to diffuse through the thick exopolysaccharide layer and permeabilize the cytoplasmic membrane, causing its disruption and ultimately leading to cell death. However, a predominance of bacteria with intact membranes in the deeper layers was observed, indicating a depth-dependent mechanism within the biofilm. AFM studies revealed significant changes in the morphology and surface topography of biofilms treated with [G1K, K8R]cGm, indicating an increase in biofilm and bacterial surface roughness, which correlates with the peptide's capacity to disrupt the bacterial cell membrane. Overall, our results reinforce the importance of deepening the knowledge of biofilm physiology and its three-dimensional structure in terms of cell and matrix distribution, as the heterogeneity of biofilms can affect the activity of antimicrobial agents. Further investigations are needed to evaluate the potential of [G1K, K8R]cGm against mature biofilms and its application in combating bacterial biofilms at different stages of maturation.
Bacterial biofilms present high recalcitrance to antimicrobial agents due to various factors, including the presence of an extracellular matrix that hinders their diffusion, reduced susceptibility of biofilm-embedded bacteria, and enzymatic degradation. Therefore, the treatment of biofilm-associated infections faces severe challenges, requiring higher doses or the application of combination therapies to achieve effectiveness. This highlights the urgent need for innovative and alternative approaches to conventional antimicrobials. The development of biofilms has been a central area of research in the search for new therapies, as the understanding of biofilm structure and growth mechanisms is still poorly understood. In this study, we selected a strain of biofilm-forming MRSA (LUH14616) to perform a comprehensive in vitro characterization of young (1-day) and mature (5-day) biofilms, and subsequently investigate the activity and mechanism of action of [G1K, K8R]cGm, a cyclic analog of the antimicrobial peptide gomesin. Results from the crystal violet assay revealed a greater amount of adhered biomass in later maturation stages, although there were fewer bacteria per unit mass. Notably, as evidenced by confocal and atomic force microscopy, young biofilms exhibited a thicker and more robust structure with bacteria coated by a thick layer of matrix, contrasting with a thinner and more sparse biofilm in later maturation stages. To initiate the study of the activity and mechanism of action of [G1K, K8R]cGm against young biofilms, increasing concentrations of the peptide were tested for 4 and 24 h. In the resazurin reduction fluorometric assay, results showed that [G1K, K8R]cGm was able to dose-dependently reduce bacterial metabolic activity, reaching a maximum decrease of 55% at 50 µM. These results were further confirmed by confocal microscopy, where biofilms treated with 50 µM [G1K, K8R]cGm exhibited a significantly higher percentage of bacterial cells with compromised membranes, revealing the peptide's ability to diffuse through the thick exopolysaccharide layer and permeabilize the cytoplasmic membrane, causing its disruption and ultimately leading to cell death. However, a predominance of bacteria with intact membranes in the deeper layers was observed, indicating a depth-dependent mechanism within the biofilm. AFM studies revealed significant changes in the morphology and surface topography of biofilms treated with [G1K, K8R]cGm, indicating an increase in biofilm and bacterial surface roughness, which correlates with the peptide's capacity to disrupt the bacterial cell membrane. Overall, our results reinforce the importance of deepening the knowledge of biofilm physiology and its three-dimensional structure in terms of cell and matrix distribution, as the heterogeneity of biofilms can affect the activity of antimicrobial agents. Further investigations are needed to evaluate the potential of [G1K, K8R]cGm against mature biofilms and its application in combating bacterial biofilms at different stages of maturation.
Descrição
Palavras-chave
Microbiologia Biofilmes bacterianos Biofilmes MRSA Resistência antimicrobiana