Ação antibacteriana da própolis vermelha sobre Staphylococcus aureus resistente à meticilina (MRSA) e imunomoduladora sobre monócitos humanos in vitro
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2023-07-07
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Universidade Estadual Paulista (Unesp)
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O objetivo desta tese de doutoramento foi analisar a atividade antibacteriana da própolis vermelha em associação ou não com antimicrobianos sobre Staphylococcus aureus resistente à meticilina (MRSA), a ação imunomoduladora em monócitos humanos (CD14+ ) isolados por beads magnéticas, e a capacidade de killing bacteriano dos monócitos sobre MRSA. Inicialmente, foi realizada uma revisão de literatura sobre a possibilidade de a própolis e seus constituintes exercerem ação contra MRSA. Em seguida, foi investigada a ação da própolis vermelha em combinação ou não com β-lactâmicos sobre MRSA e mecanismos envolvidos. Finalmente, as concentrações de própolis vermelha e de antimicrobiano que melhor exerceram ação antibacteriana in vitro foram avaliadas em monócitos, para observar um possível efeito imunomodulador. A revisão inicial demonstrou que os diversos tipos de própolis encontrados no globo e seus principais constituintes podem agir de forma inibitória contra diversas cepas e isolados de MRSA bem como em suas principais moléculas relacionadas à sua resistência à drogas. No capítulo 2, apresentamos o manuscrito proveniente da primeira fase da realização deste projeto, onde demonstramos que a própolis vermelha apresentou baixas concentrações inibitórias (MIC) para diversos tipos de isolados MRSA (MIC90 = 125 µg/mL). As combinações de própolis com imipenem apresentaram sinergismo pelo método de Ckeckerboard (FICI = 0,5) e de curva de sobrevivência bacteriana com diferença estatística entre o grupo controle (p ≤ 0,0001). Esta mesma diferença estatística foi observada quando própolis e imipenem foram combinados com 25% de suas MICs nos testes de formação de biofilme, apresentando porcentagem de inibição do crescimento de biofilme menor que somente própolis ou imipenem à 75% de suas MICs sobre a maioria dos isolados (~ 47% - 64%). A porcentagem de condutividade de membrana dos isolados chegou a 100% também para esta combinação. Predições de molecular docking indicaram que o índice de energia livre de ligação dos compostos da própolis vermelha sobre a proteína ligante de penicilina 2a (PBP2a) foi parecido com ceftarolina (kcal/ mol = -7,1 a 7,8). A última fase foi avaliar a ação imunomoduladora da própolis e imipenem nas mesmas concentrações da fase I, em monócitos humanos. Observamos uma down-regulação de TLR-2 e exercida pela própolis vermelha e aumento na produção de IL-6 exercido pela combinação nos monócitos tratados. A capacidade de killing bacteriano dos monócitos, após tratamento com própolis, chegou próximo do controle LPS (~90%). A própolis vermelha em combinação com imipenem pode ser uma alternativa para o tratamento de MRSA por impedir sua multiplicação, formação de biofilme e modular ação bactericida de monócitos, provavelmente por aumento na produção de IL-6.
The goal of this doctoral thesis was to analyze the red propolis antibacterial activity in association or not with antimicrobials on methicillin-resistant Staphylococcus aureus (MRSA), the immunomodulatory action on human monocytes (CD14+) isolated by magnetic beads, and the ability to kill bacteria of monocytes on MRSA. Initially, a literature review was carried out to search the possibility that propolis and its main constituents exert action against MRSA. Next, the action of red propolis in combination or not with β-lactams on MRSA and this mode-action was investigated. Finally, the concentrations of red propolis and antimicrobial that best exerted an in vitro antibacterial were evaluated on monocytes, to observe a possible immunomodulatory effect. The initial review demonstrated that the different types of propolis found on the globe and its main constituents can act in an inhibitory way against several MRSA strains and isolates as well as its main resistant-related molecules. In chapter 2, we present the manuscript from the first phase of this project. We demonstrate that red propolis had low inhibitory concentrations (MIC) for several types of MRSA isolates (MIC90 = 125 µg/mL). The combinations of propolis with imipenem showed synergism by the Ckeckerboard method (FICI = 0.5) and bacterial survival curve with statistical difference between the control group (p ≤ 0.0001). This same statistical difference was observed when propolis and imipenem were combined with 25% of their MICs in the biofilm formation tests, showing a lower percentage of biofilm growth inhibition than only propolis or imipenem at 75% of their MICs (~47% - 64%). The membrane conductivity percentage of the isolates reached 100% also for this combination. Molecular docking predictions indicated that the free binding energy index of red propolis compounds to penicillin-binding protein 2a (PBP2a) was similar to ceftaroline (kcal/mol = -7.1 to 7.8). The last phase was to evaluate the immunomodulatory action of propolis and imipenem at the same concentrations as in phase I, in human monocytes on MRSA. We observed a down-regulation of TLR-2 excerced by red propolis and an increase in IL-6 production excerced by the combination in treated monocytes. The killling ability of monocytes after treatment with propolis or combination was close to the LPS (~90%). Red propolis in combination with imipenem may be an alternative for the treatment of MRSA by preventing its multiplication, biofilm formation and modulating the killing action of monocytes, probably due to IL-6 up-regulation.
The goal of this doctoral thesis was to analyze the red propolis antibacterial activity in association or not with antimicrobials on methicillin-resistant Staphylococcus aureus (MRSA), the immunomodulatory action on human monocytes (CD14+) isolated by magnetic beads, and the ability to kill bacteria of monocytes on MRSA. Initially, a literature review was carried out to search the possibility that propolis and its main constituents exert action against MRSA. Next, the action of red propolis in combination or not with β-lactams on MRSA and this mode-action was investigated. Finally, the concentrations of red propolis and antimicrobial that best exerted an in vitro antibacterial were evaluated on monocytes, to observe a possible immunomodulatory effect. The initial review demonstrated that the different types of propolis found on the globe and its main constituents can act in an inhibitory way against several MRSA strains and isolates as well as its main resistant-related molecules. In chapter 2, we present the manuscript from the first phase of this project. We demonstrate that red propolis had low inhibitory concentrations (MIC) for several types of MRSA isolates (MIC90 = 125 µg/mL). The combinations of propolis with imipenem showed synergism by the Ckeckerboard method (FICI = 0.5) and bacterial survival curve with statistical difference between the control group (p ≤ 0.0001). This same statistical difference was observed when propolis and imipenem were combined with 25% of their MICs in the biofilm formation tests, showing a lower percentage of biofilm growth inhibition than only propolis or imipenem at 75% of their MICs (~47% - 64%). The membrane conductivity percentage of the isolates reached 100% also for this combination. Molecular docking predictions indicated that the free binding energy index of red propolis compounds to penicillin-binding protein 2a (PBP2a) was similar to ceftaroline (kcal/mol = -7.1 to 7.8). The last phase was to evaluate the immunomodulatory action of propolis and imipenem at the same concentrations as in phase I, in human monocytes on MRSA. We observed a down-regulation of TLR-2 excerced by red propolis and an increase in IL-6 production excerced by the combination in treated monocytes. The killling ability of monocytes after treatment with propolis or combination was close to the LPS (~90%). Red propolis in combination with imipenem may be an alternative for the treatment of MRSA by preventing its multiplication, biofilm formation and modulating the killing action of monocytes, probably due to IL-6 up-regulation.
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Palavras-chave
Própolis, Própolis vermelha, Methicillin Resistant Staphylococcus aureus (MRSA), Flavonoides, Molecular docking, Monócitos, Imunomodulação