甘草酸对多重耐药大肠杆菌的抗菌增敏作用

多药外排泵转运子AcrB是大肠杆菌产生多重耐药性（MDR）的重要原因和生物学基础。筛选针对大肠杆菌外排泵AcrB的抑制剂有助于解决大肠杆菌多重耐药问题，本试验借助虚拟筛选工具AutoDock Vina以大肠杆菌多药外排泵转运子AcrB为靶点进行筛选，得到结合作用较好的中药单体成分甘草酸，应用DS Visualizer进行相关作用力分析，通过联合抑菌试验验证甘草酸与抗生素的联合抑菌作用，通过尼罗红外排试验、内膜质子梯度影响试验与外膜渗透稳定性试验验证甘草酸抑制外排泵转运子AcrB的作用机制，最后通过实时荧光定量PCR检测甘草酸对AcrB表达相关基因的影响。结果表明，甘草酸与AcrB蛋白多个氨基酸残基形成了疏水作用力，并且还与对接部位形成多个氢键，其结合作用力为47.720 4 kJ·mol^-1；3.125 mg·mL^-1甘草酸与头孢噻肟钠、磷霉素钠对大肠杆菌E320的FICI≤0.5，具有协同作用；甘草酸有明显阻滞尼罗红的外排作用且与已知的AcrB抑制剂PAβN具有相同的趋势；3.125 mg·mL^-1甘草酸对外膜渗透性、内膜质子梯度浓度均无明显影响；甘草酸能够显著提高负调控子AcrR、MarR mRNA的表达。综上表明，甘草酸具有成为降低多重耐药大肠杆菌耐药性外排泵抑制剂的潜质。

Antibacterial and Sensitizing Effects of Glycyrrhizic Acid on Multidrug Resistant Escherichia coli

The multidrug efflux transporter AcrB is an important cause and biological basis of multidrug resistance (MDR) in E. coli. To find an AcrB inhibitor to solve the problem of multidrug resistance, AutoDock Vina was used for screening with AcrB as the target in this experiment, and glycyrrhizic acid was obtained. DS Visualizer was employed to analyze interactions, and the combined drug susceptibility test was used to verify the bacteriostasis of glycyrrhizic acid combined with antibiotics. The inhibitory effect of glycyrrhizic acid on AcrB was verified by Nile red efflux assay, test on proton gradient across the inner membrane and test on outer membrane permeability. Finally, the effect of glycyrrhizic acid on AcrB expression-related genes was tested by real-time fluorescence-based quantitative (RFQ)-PCR. The results showed that glycyrrhizic acid formed hydrophobic force with multiple amino acid residues on AcrB protein, and formed multiple hydrogen bonds with the docking site, the binding force is 47.720 4 kJ·mol^-1；3.125 mg·mL^-1 glycyrrhizic acid, cefotaxime sodium and fosfomycin sodium had synergistic effect on E. coli E320 with fractional inhibitory concentration index (FICI)≤0.5; glycyrrhizic acid could block the efflux of Nile red and has the same tendency as known inhibitor PAβN; 3.125 mg·mL^-1 glycyrrhizic acid had no significant effect on outer membrane permeability and proton gradient across the inner membrane; glycyrrhizic acid could significantly increase the expression level of mRNA in negative regulators AcrR and MarR. This experiment proved that glycyrrhizic acid had the potential to become an efflux pump inhibitor to reduce multidrug resistance in E. coli.