小檗碱和绿原酸对质粒介导的耐药基因接合转移的影响

目的 研究小檗碱和绿原酸对质粒介导的耐药基因接合转移的影响及其可能的机制。方法 采用微量稀释法测定小檗碱和绿原酸对供试菌株的最小抑菌浓度（minimum inhibitory concentrations，MICs），使用临床实验室标准化委员会（Clinical and Laboratory Standards Institution，CLSI）发布的微量肉汤稀释法测定抗生素的MICs，膜接合法测定耐药基因转移水平，S1核酸酶脉冲场凝胶电泳（S1 nuclease pulsed-field gel electrophoresis，S1-PFGE）确定质粒谱，实时荧光定量聚合酶链式反应（Real-time quantitative polymerase chain reaction，RT-qPCR）检测接合转移相关基因的表达水平。结果 小檗碱对供试的6株多重耐药沙门氏菌和1株大肠杆菌的MICs均为1.25 mg/mL，绿原酸对其的MICs均为6.25 mg/mL，小檗碱的抑菌效果强于绿原酸。小檗碱和绿原酸分别连续诱导供试菌后，16种抗生素对原始株和诱导株的MICs变化程度不同，对 庆大霉素和四环素的抗性先增大再减小。小檗碱和绿原酸诱导所得的第2、4、6代子代菌株作为供体，与受体菌大肠杆菌EC600接合时，与原供试菌株相比，绿原酸诱导株的接合频率先增大再减小，小檗碱诱导株的接合频率主要是先减小再略有增大。小檗碱和绿原酸诱导未对原供试菌株携带的质粒产生影响，诱导显著调控了traX和pilL等15个接合转移相关基因的表达水平（P<0.05），与接合频率的变化趋势基本一致。结论 小檗碱和绿原酸对质粒介导的耐药基因接合转移有一定的抑制作用，具有作为耐药基因接合转移抑制剂的潜力。

Effects of berberine and chlorogenic acid on plasmid mediated conjugative transfer of drugresistance genes

Objective To evaluate the effects and possible mechanism of berberine and chlorogenic acid on plasmid mediated conjugative transfer of antibiotic resistance gene. Methods The minimum inhibitory concentrations (MICs) of berberine and chlorogenic acid were detected by microdilution assay. The MICs of antibiotics were tested by broth microdilution method issued by the clinical and laboratory standards institution (CLSI). The level of antibiotic resistance encoding gene horizontal transfer was conducted via conjugation. Plasmid profiles were determined by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), the expression level of genes related to conjugative transfer was detected using real-time quantitative polymerase chain reaction (RT-qPCR) . Results The MICs of berberine against multidrug resistant strains (6 Salmonella and 1 Escherichia coli) were 1.25 mg/mL and that of chlorogenic acid were 6.25 mg/mL, which indicated berberine exhibited a stronger antibacterial effect than that of chlorogenic acid. After the tested strains were continuously induced by berberine and chlorogenic acid, the MICs of 16 tested antibiotics on original strains and their progeny strains changed in varying degrees with firstly increasing and then decreasing for gentamicin and tetracycline. With EC600 used as recipient, the 2nd, 4th, and 6th generational progeny strains induced by berberine and chlorogenic acid used as donors, the conjugation frequency of all progeny strains changed compared with that of the original strain, conjugation frequency variation trend after chlorogenic acid induction was firstly increasing and then decreasing, the most common trend after berberine induction was firstly decreasing and then increasing slightly. Berberine and chlorogenic acid induction did not affect the plasmids carried by the original tested strains, but significantly regulated the expression levels of 15 conjugative transfer-related genes (i.e. traX and pilL) (P < 0.05), whose expression levels are in general agreement with the trend of conjugation frequency. Conclusion Berberine and chlorogenic acid have certain inhibitory effects on plasmid mediated conjugative transfer of antibiotic resistance genes, they have the potential to be acted as inhibitors for antibiotic resistance genes conjugation transfer.