黄连浸出液对耐药奇异变形菌整合子转录水平的影响研究

Effects of Coptidis Rhizoma extract on transcription of integrons in multi-drug resistant Proteus mirabilis

目的了解黄连对变形菌属整合子转录水平的影响,探讨黄连对细菌耐药基因的作用机制,为临床治疗提供参考依据。方法选取2013年6月试验鉴定携带有Ⅰ、Ⅱ类整合子耐头孢哌酮奇异变形菌1株,利用RT-PCR方法,分析变形菌属Ⅰ、Ⅱ类整合酶基因(intI1和intI2)转录水平检测的合适条件,实时荧光定量PCR技术检测黄连浸出液处理耐药奇异变形菌对其基因转录水平的影响。结果奇异变形菌总RNA提取完整无降解;Ⅰ类整合子的第1对引物、Ⅱ类整合子的第2对引物在不同退火温度下扩增时均出现明显的非特异性条带;随着黄连浓度的升高,奇异变形菌intI1和intI2相对表达量下降,当黄连浓度在50mg/ml以上,可显著抑制奇异变形菌intI1、intI2的转录(P<0.05)。结论初步建立了实时荧光定量PCR技术检测变形菌属intI1、intI2基因转录水平的方法,发现黄连浸出液可抑制变形菌属整合子转录水平,推测与黄连抗耐药机制有关。

OBJECTIVE To investigate the effects of Coptidis Rhizoma on transcription of integrons of Proteus,so as to provide the basis for clinical treatment.METHODS From experiment in Jun.2013,1strain of Proteus mirabilis resistant against cefoperazone,which had been identified with classⅠ and classⅡintegrons（int I1 and int I2）,was selected.Appropriate primers for detecting intI1 and intI2transcriptions were firstly determined by RTPCR.Relative transcription levels of intI1 and intI2in response to Coptidis Rhizoma were measured by quantitative real-time PCR.RESULTS All RNAs of P.mirablilis were extracted completely.Both the first pair of primers of classⅠintegron,and the second pair of primers of classⅡintegrin manifested evident non-specific bands when amplifying in different annealing temperatures.Relative transcription of intI1 and intI2in P.mirabilis was inhibited by the extract of Coptidis Rhizoma in a dose-dependent manner,significant at the concentrations above50mg/ml（P〈0.05）.CONCLUSION Appropriate primers could optimize the RT-PCR detection of intI1 and intI2.Inhibition on the transcription of intI1 and intI2by Coptidis Rhizoma may relate to its action on drug resistant in Proteus.