Atividade antibacteriana e mecanismos de ação da melitina e sua associação com oxacilina frente ao Staphylococcus aureus meticilina resistente (MRSA)
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Data
2022-08-25
Autores
Pereira, Ana Flávia Marques [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A resistência bacteriana é um problema de saúde pública mundial e isolados resistentes como o Staphylococcus aureus resistente à meticilina (MRSA), frequentemente causam infecções graves e de difícil tratamento. A pesquisa por novos agentes antimicrobianos é necessária e produtos naturais como os peptídeos antimicrobianos têm sido evidenciados, como é o caso da melitina, uma fração do veneno da abelha Apis mellifera, que já possui ação antimicrobiana reportada. A melitina pode apresentar atividade hemolítica, sendo ideais novas estratégias como um sinergismo com antibióticos ou associada a sistemas nanocarreados, como nanopartículas ou nanoemulsões. Os objetivos desse trabalho foram sintetizar uma nanopartícula de ouro conjugada com melitina (AuNPs melitina) e duas formulações diferentes de nanoemulsões contendo melitina, avaliar a atividade antibacteriana da melitina, da sua associação com oxacilina, dos antibióticos oxacilina e cefalotina, da AuNPs melitina e das nanoemulsões sobre MRSA ATCC 33591 e um MRSA isolado clínico, avaliar atividade antibiofilme dos produtos que apresentaram atividade antibacterina e investigar os seus mecanismos de ação sobre MRSA. As nanopartículas de ouro (AuNPs) foram funcionalizadas com citrato de sódio e ativadas com 1-etil-3-(3- dimetilaminopropil)carbodiimida (EDC) e N-hidroxisuccinimida (NHS) e posterior conjugação da melitina; também foi realizada a síntese de duas formulações diferentes de nanoemulsões contendo melitina. A atividade antibacteriana sobre os isolados foi realizada de acordo com a metodologia Resazurin Microtiter Assay (REMA), para determinar a concentração inibitória mínima (CIM) da melitina, antibióticos, AuNPs melitina e nanoemulsões com melitina. O sinergismo da melitina com antibióticos (oxacilina e cefalotina) foi testado por meio da curva de sobrevivência (time kill curve). A ação antibiofilme de melitina, oxacilina e da associação de melitina com oxacilina (mel+oxa) e a ação desses produtos sobre a membrana celular dos isolados foram verificadas. Ensaios de atividade hemolítica e de citometria de fluxo, citotoxicidade em células HaCaT, foram realizados. Os mecanismos de ação dos produtos testados com atividade antibacteriana foram investigados por meio da análise proteômica, sendo que a cepa MRSA ATCC foi tratada com concentrações subinibitórias dos tratamentos. As AuNPs melitina e as duas nanoemulsões diferentes contendo melitina foram sintetizadas, obtendo uma concentração final de 250 μg/mL de melitina em cada nanoestruturado, sendo que nenhum nanoestruturado apresentou ação antibacteriana frente à MRSA. A melitina apresentou uma CIM de 5,3 µg/mL sobre MRSA ATCC e de 4,0 µg/mL para MRSA isolado clínico; já a oxacilina apresentou CIM de 16 µg/mL para MRSA ATCC e de 8 µg/mL para MRSA isolado. A associação mel+oxa mostrou ser sinérgica e bactericida para ambos MRSA. Concentrações subinibitórias da mel+oxa foram capazes de reduzir a formação de biofilme de MRSA ATCC e concentrações suprainibitórias foram capazes de erradicar biofilmes estabelecidos. A melitina e mel+oxa mostraram ter ação sobre a permeabilidade da membrana celular pelo aumento na condutividade elétrica relativa, extravasamento de proteínas, liberação de ácidos nucleicos e efluxo de íons potássio e fosfato. A taxa de hemólise da melitina em sua CIM foi de 0,2%, já a combinação de mel+oxa, em sua CIM não apresentou hemólise em eritrócitos humanos. A melitina e mel+oxa não causaram apoptose ou morte tardia em células de queratinócitos HaCaT. A análise proteômica sobre MRSA ATCC mostrou que a melitina, oxacilina e mel+oxa causaram uma expressão diferencial de proteínas em relação ao controle, indicando ação sobre a síntese proteica e metabolismo energético. Os nanoestruturados contendo a melitina não apresentaram atividade antibacteriana nas concentrações e formulações testadas sobre MRSA. O tratamento mel+oxa mostrou atividade antibacteriana e antibiofilme sobre MRSA, não apresentou hemólise no valor da CIM e nem citotoxicidade em células HaCaT e mostrou ação sobre a membrana celular de MRSA e sobre diferentes alvos intracelulares, afetando principalmente o metabolismo energético e a síntese proteica de MRSA. A associação de melitina e oxacilina apresenta considerável potencial para o desenvolvimento de agentes antibacterianos ou de novos tratamentos contra MRSA.
Bacterial resistance is a global public health problem and resistant isolates such as methicillin-resistant Staphylococcus aureus (MRSA) often cause serious and difficult-to-treat infections. Research for new antimicrobial agents is necessary and natural products such as antimicrobial peptides have been evidenced, such as melittin, a fraction of the venom of Apis mellifera bee, which already has reported antimicrobial action. Melittin may have hemolytic activity and new strategies are ideal such as synergism with antibiotics or associated with nanocarried systems, such as nanoparticles or nanoemulsions. The objectives were to synthesize a gold nanoparticle conjugated with melittin (melittin AuNPs) and two different formulations of nanoemulsions containing melittin, to evaluate the antibacterial activity of melittin, its association with oxacillin, antibiotics oxacillin and cephalothin, AuNPs melittin and nanoemulsions on ATCC 33591 MRSA and a clinical isolate MRSA, to evaluate the antibiofilm activity of the producys that showed antibacterial activity and investigate its mechanisms of action on MRSA. Gold nanoparticles (AuNPs) were functionalized with sodium citrate and activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and subsequent conjugation of melittin; the synthesis of two different formulations of nanoemulsions containing melittin was also carried out. Antibacterial activity against the bacterial isolates was performed according to the Resazurin Microtiter Assay (REMA) to determine the minimum inhibitory concentration (MIC) of melittin, antibiotics, melittin AuNPs and melittin nanoemulsions. The synergism of melittin with antibiotics (oxacillin and cephalothin) was tested using the time kill curve. The antibiofilm action of melittin, oxacillin and the association of melittin with oxacillin (mel+oxa) were tested and the mechanisms of action of these products on the bacterial isolates cell membrane were verified. Hemolytic activity and flow cytometry assays, cytotoxicity in HaCaT cells, were performed. The proteomics analysis investigated the mechanisms of action of the tested products with antibacterial activity; the MRSA ATCC was treated with subinhibitory concentrations of the treatments. The synthesized AuNPs melittin and the two different nanoemulsions containing melittin, with a final concentration of 250 μg/mL of melittin in each nanostructure, showed no antibacterial action against MRSA. Melittin had a MIC of 5.3 µg/mL for MRSA ATCC and 4.0 µg/mL for isolate MRSA; oxacillin presented a MIC of 16 µg/mL for MRSA ATCC and 8 µg/mL for isolate MRSA. The mel+oxa association proved to be synergistic and bactericidal for both MRSA. Subinhibitory concentrations of mel+oxa reduced the biofilm formation of both MRSA and suprainhibitory concentrations eradicate established biofilms. Melittin and mel+oxa showed action on the permeability of the cell membrane, increased the relative electrical conductivity, promoted protein extravasation, released nucleic acids and caused efflux of potassium and phosphate ions.The hemolysis rate of melittin in its MIC was 0.2%, whereas the combination of mel+oxa, in its MIC, did not present hemolysis in human erythrocytes. Melittin and mel+oxa did not cause apoptosis or necrosis in HaCaT keratinocyte cells. Proteomic analysis on MRSA ATCC showed that melittin, oxacillin and mel+oxa caused a differential expression of proteins in relation to the control, indicating action on protein synthesis and energy metabolism. The mel+oxa treatment showed antibacterial and antibiofilm activity on MRSA, showing no hemolysis in its MIC value and no cytotoxicity in HaCaT cells and presented action on the MRSA cell membrane and on different targets, mainly affecting energy metabolism and protein synthesis. The association of melittin and oxacillin presents considerable potential for the development of antibacterial agents or new treatments against MRSA.
Bacterial resistance is a global public health problem and resistant isolates such as methicillin-resistant Staphylococcus aureus (MRSA) often cause serious and difficult-to-treat infections. Research for new antimicrobial agents is necessary and natural products such as antimicrobial peptides have been evidenced, such as melittin, a fraction of the venom of Apis mellifera bee, which already has reported antimicrobial action. Melittin may have hemolytic activity and new strategies are ideal such as synergism with antibiotics or associated with nanocarried systems, such as nanoparticles or nanoemulsions. The objectives were to synthesize a gold nanoparticle conjugated with melittin (melittin AuNPs) and two different formulations of nanoemulsions containing melittin, to evaluate the antibacterial activity of melittin, its association with oxacillin, antibiotics oxacillin and cephalothin, AuNPs melittin and nanoemulsions on ATCC 33591 MRSA and a clinical isolate MRSA, to evaluate the antibiofilm activity of the producys that showed antibacterial activity and investigate its mechanisms of action on MRSA. Gold nanoparticles (AuNPs) were functionalized with sodium citrate and activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and subsequent conjugation of melittin; the synthesis of two different formulations of nanoemulsions containing melittin was also carried out. Antibacterial activity against the bacterial isolates was performed according to the Resazurin Microtiter Assay (REMA) to determine the minimum inhibitory concentration (MIC) of melittin, antibiotics, melittin AuNPs and melittin nanoemulsions. The synergism of melittin with antibiotics (oxacillin and cephalothin) was tested using the time kill curve. The antibiofilm action of melittin, oxacillin and the association of melittin with oxacillin (mel+oxa) were tested and the mechanisms of action of these products on the bacterial isolates cell membrane were verified. Hemolytic activity and flow cytometry assays, cytotoxicity in HaCaT cells, were performed. The proteomics analysis investigated the mechanisms of action of the tested products with antibacterial activity; the MRSA ATCC was treated with subinhibitory concentrations of the treatments. The synthesized AuNPs melittin and the two different nanoemulsions containing melittin, with a final concentration of 250 μg/mL of melittin in each nanostructure, showed no antibacterial action against MRSA. Melittin had a MIC of 5.3 µg/mL for MRSA ATCC and 4.0 µg/mL for isolate MRSA; oxacillin presented a MIC of 16 µg/mL for MRSA ATCC and 8 µg/mL for isolate MRSA. The mel+oxa association proved to be synergistic and bactericidal for both MRSA. Subinhibitory concentrations of mel+oxa reduced the biofilm formation of both MRSA and suprainhibitory concentrations eradicate established biofilms. Melittin and mel+oxa showed action on the permeability of the cell membrane, increased the relative electrical conductivity, promoted protein extravasation, released nucleic acids and caused efflux of potassium and phosphate ions.The hemolysis rate of melittin in its MIC was 0.2%, whereas the combination of mel+oxa, in its MIC, did not present hemolysis in human erythrocytes. Melittin and mel+oxa did not cause apoptosis or necrosis in HaCaT keratinocyte cells. Proteomic analysis on MRSA ATCC showed that melittin, oxacillin and mel+oxa caused a differential expression of proteins in relation to the control, indicating action on protein synthesis and energy metabolism. The mel+oxa treatment showed antibacterial and antibiofilm activity on MRSA, showing no hemolysis in its MIC value and no cytotoxicity in HaCaT cells and presented action on the MRSA cell membrane and on different targets, mainly affecting energy metabolism and protein synthesis. The association of melittin and oxacillin presents considerable potential for the development of antibacterial agents or new treatments against MRSA.
Descrição
Palavras-chave
Análise proteômica, Atividade hemolítica, Biofilme, Citometria de fluxo, Sinergismo, Biofilm, Flow cytometry, Hemolytic activity, Proteomic analysis, Synergism