Atividade antimicrobiana e antibiofilme de diferentes extratos naturais sobre cepas clínicas multirresistentes de Acinetobacter baumannii
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2020-10-29
Autores
Yu, Amanda Romagnoli
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Universidade Estadual Paulista (Unesp)
Resumo
O presente trabalho teve como objetivos: a) avaliar a atividade antimicrobiana dos extratos naturais de alecrim, bardana, romã e cavalinha, sobre oito cepas de micro-organismos multirresistentes de Acinetobacter baumanii em cultura planctônica, verificando a concentração inibitória mínima e concentração microbicida mínima (CIM e CMM); b) avaliar a atividade antibiofilme dos extratos que apresentaram atividade antimicrobiana em cultura planctônica sobre cepas multirresistentes de A. baumanii no tempo de 5 minutos; c) avaliar a citotoxicidade das concentrações mais efetivas dos extratos que apresentaram atividade antimicrobiana nos testes em cultura planctônica sobre queratinócitos, em ensaio da atividade mitocondrial celular pelo método MTT, no tempo de 5 minutos. Para a determinação da CIM e CMM dos extratos utilizou-se o método de microdiluição em caldo, segundo Clinical and Laboratory Standards Institute (CLSI), norma M27-A21 (CLSI, 2002) suplementada por M27-S4 (CLSI, 2012). Este teste foi realizado sobre 8 cepas clínicas de Acinetobacter baumanii e 4 extratos, perfazendo 32 grupos experimentais (n=8). Os extratos que apresentaram ação bacteriostática ou bactericida nos testes anteriores foram avaliados sobre biofilmes monomicrobianos (8 cepas clínicas de Acinetobacter baumanii), no tempo de contato de cinco minutos. As concentrações dos extratos, que apresentaram ação anti-biofilme sobre as cepas bacterianas analisadas, foram submetidas à análise de citotoxicidade em queratinócitos humanos. A avaliação foi realizada por meio do teste colorimétrico MTT, que analisou a atividade mitocondrial celular, após contato dos extratos por 5 min. Os resultados foram analisados estatisticamente por ANOVA e Tukey Test, sendo considerada diferença estatística significativa quando p ≤ 0,05. O extrato de romã apresentou CIM e CMM para todas as cepas analisadas, em quanto o extrato de alecrim apresentou CIM e CMM para 6 cepas das 8 analisadas, ambos apresentaram redução de biofilme, entretanto, romã foi o que apresentou as maiores reduções para a maioria das cepas. O extrato de romã apresentou viabilidade celular superior a 80% na maioria das concentrações e o alecrim apresentou viabilidade celular para 5 concentrações das 8 analisadas. Com isso pode-se concluir que o extrato de romã apresentou um melhor resultado comparado aos outros extratos, pois demonstrou uma significativa atividade antimicrobiana e antibiofilme e ausência de toxicidade conforme tempo de aplicação e concentração utilizada contra as cepas clínicas multirresistentes de Acinetobacter baumanni, podendo ser considerado potencial agente terapêutico para o combate destes patógenos.
The objective of the present work was: a) to evaluate the antimicrobial activity of the natural extracts of rosemary, burdock, pomegranate and horsetail on eight strains of multidrug resistant Acinetobacter baumanii microorganisms in planktonic culture, verifying the minimum inhibitory concentration and minimum microbicidal concentration (MIC) and CMM); b) evaluate the antibiofilm activity of extracts that showed antimicrobial activity in planktonic culture on multi-resistant strains of A. baumanii within 5 minutes; c) to evaluate the cytotoxicity of the most effective concentrations of the extracts that showed antimicrobial activity in the tests in planktonic culture on keratinocytes, in an assay of cellular mitochondrial activity by the MTT method, in 5 minutes. For the determination of the minimum inhibitory (MIC) and minimum microbicidal (CMM) concentrations of the extracts, the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI), standard M27-A21 (CLSI, 2002) supplemented by M27 was used. -S4 (CLSI, 2012). This test was performed on 8 clinical strains of Acinetobacter baumanii and 4 extracts, making 32 experimental groups (n = 8). The extracts that showed bacteriostatic or bactericidal action in the previous tests were evaluated on monomicrobial biofilms (8 clinical strains of Acinetobacter baumanii) at contact time of 5 minutes. The concentrations of the extracts, which showed anti-biofilm action on the bacterial strains analyzed, were submitted to cytotoxicity analysis in human kerotinocytes. The evaluation was performed by the MTT colorimetric test, which analyzed the mitochondrial cellular activity, after contact of the extracts for 5 min. The results were statistically analyzed by ANOVA and Tukey Test, being considered statistically significant difference when p ≤ 0.05. Pomegranate extract showed MIC and CMM for all strains analyzed, while rosemary extract showed MIC and CMM for 6 strains of the 8 analyzed, both showed reduced biofilm, however, pomegranate was the one that showed the greatest reductions for most of the strains. Pomegranate extract showed cell viability greater than 80% in most concentrations and rosemary showed cell viability for 5 concentrations of the 8 analyzed.Therefore it can be concluded that the pomegranate extract presented a better result compared to the other extracts, since it demonstrated a significant antimicrobial and antibiofilm activity and absence of toxicity according to the time of application and concentration used against the multidrug-resistant clinical strains of Acinetobacter baumanni, which can be considered a potential therapeutic agent to combat these pathogens.
The objective of the present work was: a) to evaluate the antimicrobial activity of the natural extracts of rosemary, burdock, pomegranate and horsetail on eight strains of multidrug resistant Acinetobacter baumanii microorganisms in planktonic culture, verifying the minimum inhibitory concentration and minimum microbicidal concentration (MIC) and CMM); b) evaluate the antibiofilm activity of extracts that showed antimicrobial activity in planktonic culture on multi-resistant strains of A. baumanii within 5 minutes; c) to evaluate the cytotoxicity of the most effective concentrations of the extracts that showed antimicrobial activity in the tests in planktonic culture on keratinocytes, in an assay of cellular mitochondrial activity by the MTT method, in 5 minutes. For the determination of the minimum inhibitory (MIC) and minimum microbicidal (CMM) concentrations of the extracts, the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI), standard M27-A21 (CLSI, 2002) supplemented by M27 was used. -S4 (CLSI, 2012). This test was performed on 8 clinical strains of Acinetobacter baumanii and 4 extracts, making 32 experimental groups (n = 8). The extracts that showed bacteriostatic or bactericidal action in the previous tests were evaluated on monomicrobial biofilms (8 clinical strains of Acinetobacter baumanii) at contact time of 5 minutes. The concentrations of the extracts, which showed anti-biofilm action on the bacterial strains analyzed, were submitted to cytotoxicity analysis in human kerotinocytes. The evaluation was performed by the MTT colorimetric test, which analyzed the mitochondrial cellular activity, after contact of the extracts for 5 min. The results were statistically analyzed by ANOVA and Tukey Test, being considered statistically significant difference when p ≤ 0.05. Pomegranate extract showed MIC and CMM for all strains analyzed, while rosemary extract showed MIC and CMM for 6 strains of the 8 analyzed, both showed reduced biofilm, however, pomegranate was the one that showed the greatest reductions for most of the strains. Pomegranate extract showed cell viability greater than 80% in most concentrations and rosemary showed cell viability for 5 concentrations of the 8 analyzed.Therefore it can be concluded that the pomegranate extract presented a better result compared to the other extracts, since it demonstrated a significant antimicrobial and antibiofilm activity and absence of toxicity according to the time of application and concentration used against the multidrug-resistant clinical strains of Acinetobacter baumanni, which can be considered a potential therapeutic agent to combat these pathogens.
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Palavras-chave
Micro-organismos multirresistentes, Extratos de plantas, Acinetobacter baumanni, Multiresistant microorganisms, Plant extracts., Acinetobacter baumannii