Mecanismos de ação da atividade antibacteriana da nisina e em combinações com antimicrobianos tradicionais sobre Staphylococcus aureus resistente a meticilina (MRSA) e Pseudomonas aeruginosa
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Data
2018-02-23
Autores
Alves, Fernanda Cristina Bergamo
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Universidade Estadual Paulista (Unesp)
Resumo
Combinações entre antimicrobianos, a exemplo de nisina (bacteriocina) e fármacos antibacterianos tradicionais, podem amenizar o problema da resistência bacteriana, pois o possível efeito sinérgico se torna estratégico, possibilitando o uso de doses menores no tratamento de doenças infecciosas com redução nos custos e na toxicidade, além de ter potencial na prevenção do surgimento das linhagens resistentes. No entanto, os mecanismos envolvidos na ação antibacteriana são importantes para pesquisas de novos fármacos. O objetivo do estudo foi investigar como a nisina, alguns fármacos antibacterianos e respectivas combinações interferem no metabolismo de Staphylococcus aureus Meticilina Resistente (MRSA) e Pseudomonas aeruginosa, através de ensaios de estresse oxidativo bacteriano, análises morfológicas por microscopia eletrônica de transmissão (MET) e análise do perfil de proteínas expressas nas bactérias quando expostas aos antimicrobianos e suas combinações em concentrações subletais. Inicialmente foram realizados ensaios visando obter os valores de concentração inibitória mínima (CIM) e concentração subletal máxima (CSM) para nisina e fármacos como tetraciclina, ciprofloxacina, vancomicina, polimixina B, oxacilina e cefalotina para ambas bactérias. Na sequencia, foram realizados ensaios para verificação de sinergismo entre nisina e antibacterianos utilizando metodologia da curva de sobrevivência, sendo escolhidas para ensaios posteriores, as combinações com demostração de sinergismo (redução na contagem bacteriana final acima de 2 logs de UFC/mL em relação ao inóculo inicial). As combinações entre ¼ CIM de nisina e ¼ CIM de oxacilina e ¼ CIM de nisina e ¼ CIM ciprofloxacina foram escolhidas para o estudo com MRSA e P. aeruginosa respectivamente. Nos ensaios sobre estresse oxidativo, a combinação nisina/oxacilina foi capaz de aumentar o estresse oxidativo em MRSA notado pelo aumento significativo das atividades de enzimas antioxidantes (catalase, superoxido dismutase e glutationa peroxidase) e de hidroperóxido de lipídeo em comparação aos tratamentos realizados individualmente e ao controle. Quanto a P.aeruginosa, a combinação nisina/ciprofloxacina aumentou atividades das enzimas antioxidantes e de hidroperóxido de lipídeo, porém não foi superior ao tratamento com a ciprofloxacina individualmente. As imagens obtidas por MET revelaram os danos na estrutura celular, especialmente na parede bacteriana e membrana plasmática, com conseqüente lise e efluxo dos constituintes citoplasmáticos em ambas bactérias, especialmente quando utilizadas no tratamento com as combinações. A análise do perfil proteico de MRSA mostrou alterações na expressão de 85 proteínas quando comparado controle com os ensaios dos antibacterianos individualmente e na combinação nisina/oxacilina, sendo que nesta combinação destacou-se a modificação na expressão de proteínas relacionadas a síntese de proteínas, diminuição na expressão de proteínas relacionadas ao estresse e na indução de proteína de resistência do S.aureus como a beta-lactamase. Para a P. aeruginosa, foram expressas 63 proteínas diferentes entre os ensaios controle, tratamentos individuais e na combinação nisina/ciprofloxacina, sendo que na combinação houve maior alteração para as proteínas relacionadas ao metabolismo energético da bactéria, bem como na diminuição significativa de expressão das proteínas presentes na membrana externa como as porinas. De acordo com esses estudos dos efeitos de antibacterianos sobre metabolismo de patógenos importantes como MRSA e Pseudomonas aeruginosa, pôde-se afirmar que as alterações causadas pelos tratamentos utilizando combinações de antibacterianos foram causadas por diferentes processos biológicos nas células bacteriana, o que pode ser uma resposta global aos tratamentos com combinações de antimicrobianos, que apresentam mecanismos de ação distintos. As respostas bacterianas obtidas neste estudo representam um ponto inicial para desenvolvimento de novas opções farmacêuticas, além de contribuir para redução dos problemas decorrentes da resistência bacteriana.
Combinations of antimicrobials, such as nisin (bacteriocin) and traditional antibacterial drugs, may assuage the problem of bacterial resistance because the possible synergistic effect becomes interesting, allowing the use of smaller doses in the treatment of infectious diseases and reduction in costs and toxicity , besides having potential in the prevention of the emergence of resistant strains. However, the mechanisms involved in antibacterial action are important for research on new drugs. The aim of this study was to investigate how nisin, antibacterial drugs and their combinations interfere in the metabolism of Methicillin Resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, verified in bacterial oxidative stress assays, morphological analyzes by transmission electron microscopy (TEM) and analysis of the protein profile expressed in bacteria when exposed to antimicrobials and combinations in sublethal concentrations. Initially, assays were performed to obtain the minimum inhibitory concentration (MIC) and maximum sublethal concentration (MSC) for nisin and drugs such as tetracycline, ciprofloxacin, vancomycin, polymyxin B, oxacillin and cephalothin for both bacteria. Subsequently, assays were performed to verify the synergism between nisin and antibacterials using time kill curve methodology and the combinations with demonstration of synergism (reduction in final bacterial count above 2 logs of CFU / mL in relation to initial inoculum) were chosen for later experiments. The combinations between ¼ nisin MIC and ¼ MIC of oxacillin and ¼ MIC of nisin and ¼ MIC of ciprofloxacin were chosen for the study with MRSA and P. aeruginosa respectively. In the oxidative stress assays, the nisin / oxacillin combination was able to increase oxidative stress in MRSA, noted by a significant increase in the activities of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) and lipid hydroperoxide compared to treatments individually and the bacterial without treatment (control). As to P.aeruginosa, the combination of nisin / ciprofloxacin increased activities of antioxidant enzymes and lipid hydroperoxide, but not superior to treatment with ciprofloxacin individually. The images obtained by MET revealed the damages in the cellular structure, especially in the bacterial wall and plasma membrane, with consequent lysis and efflux of the cytoplasmic constituents in both bacteria, especially when using the combinations. The analysis of the protein profile of MRSA showed alterations in the expression of 85 proteins when compared the control with antibacterial assays individually and the nisin /oxacillin combination. In the combination highlighted the expression of proteins related to protein synthesis, decrease the expression of stress-related proteins and induction the of S. aureus resistance protein such as beta-lactamase. For P. aeruginosa, 63 different proteins were expressed between the control, individual treatments and nisin / ciprofloxacin combination. There was a greater alteration in the combination proteins related to the energetic metabolism of the bacteria, as well as in the significant decrease of expression of the proteins present in the outer membrane such as porins. According to these studies of the effects of antibacterials on metabolism of important pathogens such as MRSA and Pseudomonas aeruginosa, it could be claim that the changes caused by treatments using antibacterial combinations were caused by different biological processes in bacterial cells, which may be a response treatment with combinations of antimicrobials, which have different mechanisms of action. These bacterial responses obtained in this study are important to researches for the development of new antibacterial drugs and contribute to the reduction of problems due to bacterial resistance.
Combinations of antimicrobials, such as nisin (bacteriocin) and traditional antibacterial drugs, may assuage the problem of bacterial resistance because the possible synergistic effect becomes interesting, allowing the use of smaller doses in the treatment of infectious diseases and reduction in costs and toxicity , besides having potential in the prevention of the emergence of resistant strains. However, the mechanisms involved in antibacterial action are important for research on new drugs. The aim of this study was to investigate how nisin, antibacterial drugs and their combinations interfere in the metabolism of Methicillin Resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, verified in bacterial oxidative stress assays, morphological analyzes by transmission electron microscopy (TEM) and analysis of the protein profile expressed in bacteria when exposed to antimicrobials and combinations in sublethal concentrations. Initially, assays were performed to obtain the minimum inhibitory concentration (MIC) and maximum sublethal concentration (MSC) for nisin and drugs such as tetracycline, ciprofloxacin, vancomycin, polymyxin B, oxacillin and cephalothin for both bacteria. Subsequently, assays were performed to verify the synergism between nisin and antibacterials using time kill curve methodology and the combinations with demonstration of synergism (reduction in final bacterial count above 2 logs of CFU / mL in relation to initial inoculum) were chosen for later experiments. The combinations between ¼ nisin MIC and ¼ MIC of oxacillin and ¼ MIC of nisin and ¼ MIC of ciprofloxacin were chosen for the study with MRSA and P. aeruginosa respectively. In the oxidative stress assays, the nisin / oxacillin combination was able to increase oxidative stress in MRSA, noted by a significant increase in the activities of antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) and lipid hydroperoxide compared to treatments individually and the bacterial without treatment (control). As to P.aeruginosa, the combination of nisin / ciprofloxacin increased activities of antioxidant enzymes and lipid hydroperoxide, but not superior to treatment with ciprofloxacin individually. The images obtained by MET revealed the damages in the cellular structure, especially in the bacterial wall and plasma membrane, with consequent lysis and efflux of the cytoplasmic constituents in both bacteria, especially when using the combinations. The analysis of the protein profile of MRSA showed alterations in the expression of 85 proteins when compared the control with antibacterial assays individually and the nisin /oxacillin combination. In the combination highlighted the expression of proteins related to protein synthesis, decrease the expression of stress-related proteins and induction the of S. aureus resistance protein such as beta-lactamase. For P. aeruginosa, 63 different proteins were expressed between the control, individual treatments and nisin / ciprofloxacin combination. There was a greater alteration in the combination proteins related to the energetic metabolism of the bacteria, as well as in the significant decrease of expression of the proteins present in the outer membrane such as porins. According to these studies of the effects of antibacterials on metabolism of important pathogens such as MRSA and Pseudomonas aeruginosa, it could be claim that the changes caused by treatments using antibacterial combinations were caused by different biological processes in bacterial cells, which may be a response treatment with combinations of antimicrobials, which have different mechanisms of action. These bacterial responses obtained in this study are important to researches for the development of new antibacterial drugs and contribute to the reduction of problems due to bacterial resistance.
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Palavras-chave
Antibacterianos, Análise proteômica, Nisina, Pseudomonas aeruginosa, Staphylococcus aureus resistente a meticilina (MRSA), Antibacterials, nisin, proteome analysis, Pseudomonas aeruginosa, Staphylococcus aureus (MRSA)