Ecg/Egcg与β内酰胺类抗生素对MRSA的协同抗菌机制研究

The mechanism of synergistic antibacterial action between Ecg/Egcg and β-lactam antibiotics against MRSA

目的测定表儿茶素没食子酸酯(Ecg)/表没食子儿茶素没食子酸酯(Egcg)与β内酰胺类抗生素对耐甲氧西林金葡菌(MRSA)的协同抗菌作用,并探讨其作用机制。方法以微量稀释法测定Ecg、Egcg和β内酰胺类抗生素的体外协同抗MRSA作用;以RT-PCR技术检测Ecg和Egcg对自溶酶基因lytM和lgrA表达的影响;以蛋白免疫印迹法测定Egcg对MRSA的PBP2a蛋白表达的影响;测定Ecg和Egcg对β内酰胺酶的抑制情况,并与他唑巴坦的抑酶作用进行比较。结果50mg/L的Ecg/Egcg均能增强苯唑西林、头孢拉啶、头孢呋辛和美罗培南对MRSA的抗菌作用,并以与美罗培南的协同作用最强;且Ecg与β内酰胺类抗生素的协同效果比Egcg明显。随着Ecg和Egcg浓度的增高,MRSA的lytM、lgrA基因表达上调,同时Egcg可使MRSA-2菌PBP2a蛋白表达下调。浓度为256mg/L的他唑巴坦可完全抑制金葡菌的β内酰胺酶,而Egcg/Ecg对金葡菌β内酰胺酶的抑制作用很微弱。结论Ecg/Egcg联合β内酰胺类抗生素对MRSA的协同作用是由于Ecg/Egcg在抑制PBP2a的表达下调的同时使自溶酶lytM和lgrA基因的表达上调,导致自溶酶分泌增多,促使对细菌黏肽水解增加,从而协同β内酰胺类抗生素抑制细菌细胞壁的合成。

Objective To determine the synergistic antibacterial action between epicatechin gallate（Ecg）/epigallocatechin gallate（Egcg）and β-lactam antibiotics against MRSA,and study their synergistic mechanism.Methods The synergistic effect between Ecg/Egcg and β-lactams in vitro against MRSA was determined by microdilution method.Expression of lytM and lgrA genes was studied by RT-PCR.Expression of PBP2a protein was assayed by Western blot.Inhibition of β-lactamase by Ecg,Egcg was analyzed and compared with tazobactam.Results Ecg or Egcg 50 mg/L could enhance the antibacterial activity of oxacillin,cefradine,cefuroxime and meropenem against MRSA.The synergism with meropenem was the strongest.The synergism between Ecg and β-lactams was stronger than Egcg at the same concentration.Expression of lytM and lgrA was up-regulated with increasing concentration of Ecg or Egcg.Egcg could down-regulate the expression of PBP2a protein in MRSA-2.Tazobactam 256 mg/L could completely inhibit the activity of β-lactamase.But Egcg and Ecg had little effect on the β-lactamase of MRSA.Conclusions Ecg/Egcg has synergistic effect with β-lactams against MRSA due to down-regulation of PBP2a expression and up-regulation of lytM and lgrA expression,which results in increased secretion of autolytic enzymes,increased mucopeptide hydrolysis,and synergism with β-lactam antibiotics in inhibiting bacterial cell wall synthesis.