β-内酰胺抗生素诱导大肠埃希菌ESBLs基因表达及其抑制机制

The expression of ESBLs genes in Escherichia coli isolates induced by β-lactam antibiotics and inhibi- ted by histidine kinase inhibitoes

目的了解大肠埃希菌临床菌株超广谱β内酰胺酶（ESBLs）基因及其携带模式、β-内酰胺类抗生素诱导以及组氨酸激酶抑制剂抑制细菌蛋白磷酸化和ESBLs基因表达的作用机制。方法采用PCR和测序法确定大肠埃希菌主要ESBLs基因（KPC、TEM、SHV、CTX-M）及其携带模式。采用E-test和微量试管稀释法分别测定最低抑菌浓度（MIC）和最低杀菌浓度（MBC）。采用固定化金属螯合层析法、细菌蛋白磷酸化检测试剂盒及实时荧光定量RT-PCR分别检测1／4MIC青霉素和头孢噻肟诱导或组氨酸激酶抑制剂（氯氰碘柳胺、自行设计的溴化或碘化甲基咪唑）抑制大肠埃希菌耐药菌株蛋白磷酸化和ESBLs基因mRNA水平升高的作用。结果183株β-内酰胺类抗生素耐药大肠埃希菌临床菌株中，TEM和CTX-M基因检出率（83．1％和77．1％）高于其他基因且以两者同时携带模式为主（65．0％）（P〈0．05）。1／4MIC青霉素和头孢噻肟钠能诱导大肠埃希菌耐药菌株蛋白磷酸化及TEM、SHV、CTX—M基因mRNA水平显著升高（P〈0．05）。200μmol氯氰碘柳胺、2或10μmol溴化甲基咪唑、20或50μmol碘化甲基咪唑无抑制或杀灭大肠埃希菌的作用，但能抑制上述抗生素诱导的细菌蛋白磷酸化和ESBLs基因mRNA水平升高（P〈0．05），也能提高大肠埃希菌耐药菌株对青霉素和头孢噻肟的敏感性（P〈0．05）。结论TEM和CTX—M是本地区β-内酰胺类抗生素耐药大肠埃希菌临床菌株优势ESBLs基因，TEM＋CTX-M是ESBLs基因优势携带模式。亚致死量β内酰胺类抗生素可经TCSS上调大肠埃希菌ESBLs基因表达水平，但可被组氨酸激酶抑制剂所抑制。

Objective To investigate the genotypes of extended spectrum β-1actamases （ESBLs） and their carrying modes in Escherichia coli （E. coli） isolates, and to analyze the mechanism of protein phosphorylation and ESBLs gene expression induced by β-1actam antibiotics or inhibited by histidine kinase inhibitors. Methods The predominant genotypes of ESBLs （KPC, TEM, SHV and CTX-M） and their car- rying modes were identified by PCR and sequencing analysis. E-test and micro-tube dilution method were ap- plied to measure minimal inhibitory concentrations （MICs） and minimum bactericidal concentrations （MBCs）. Immobilized metal ion affinity chromatography, bacterial protein phosphorylation detection kit and real-time fluorescent quantitation RT-PCR were performed to analyze the enhancing effects of 1/4 MIC peni- cillin or cefotaxime or the inhibitory effects of histidine kinase inhibitors （ closantel, bromized or iodized methylimidazol） on protein phosphorylation and the expression of ESBLs at mRNA level in E. coli isolates. Results In 183 ^-lactam antibiotics-resistant E. coli isolates, TEM and CTX-M genes （83. 1% and 77.1% ） were highly expressed than other two ESBLs genes with a prevalent carrying mode of coexisting （65.0%） （P〈0.05）. Penicillin or cefotaxime at 1/4 MIC induced the protein phosphorylation and promo- ted the expression of TEM, SHV and CTX-M at mRNA level （P〈0.05）. Closantel （200 I^mol）, bromized methylimidazol （2 or 10 txmol） or iodized methylimidazol （20 or 50 ttmol） could neither kill E. coli isolates nor inhibit their growth, but could inhibit the protein phosphorylation induced by above mentioned antibioticsand enhance the expression of ESBLs at mRNA level （ P〈0.05 ）. Moreover, the susceptibility of antibiotic- resistant E. coli strains to penicillin and cefotaxime were increased （P〈0. 05）. Conclusion TEM and CTX-M were the predominant genotypes of ESBLs carried by β-1actam antibiotics-resistant E. coli strains iso- lated from Zhejiang province, which were mostly found in a TEM plus CTX-M carrying mode. Sublethal dose of β-1actam antibiotics could up-regulate the expression of ESBLs genes in E. coli isolates via TCSS, but it could be inhibited by histidine kinase inhibitors.