产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌bla_(CTX-M)基因型及耐药性检测

目的运用变性高效液相色谱(DHPLC)技术对已经确认产超广谱β-内酰胺酶(ESBLs)的临床分离株CTX-M型质粒进行基因分型,探讨其敏感性和特异性,建立一种快速检测CTX-M型ESBLs基因型的新方法。同时了解产CTX-M酶菌的耐药情况。方法利用逆转录聚合酶链反应(PCR)技术从大肠埃希菌和肺炎克雷伯菌临床分离株中扩增出CTX-M型质粒的编码序列,扩增产物运用DHPLC技术进行分析和测序确定其基因突变的类型,最后通过对比两者结果后确定其基因型;采用K-B法进行药敏试验。结果从34株临床分离株中扩增出35份CTX-M型质粒的编码序列(其中1株同时扩增到2份CTX-M型编码序列)。对比DHPLC和测序结果,洗脱峰型一致的样本均为同一基因亚型。产CTX-M酶菌对亚胺培南或美洛培南高度敏感,对氨苄西林、头孢唑啉、头孢噻肟、头孢曲松、头孢呋辛钠、哌拉西林高度耐药。结论 DHPLC准确性较高,具有简便快捷、经济等特点,可应用于CTX-M型ESBLs的基因型检测;产CTX-M酶菌耐药表型呈多样性,亚胺培南或美洛培南是临床治疗产CTX-M酶菌感染的首选药物。

Detection of the genotypes of blaCTX-M and antibiotic resistance in extended-spectrum beta-lactamases producing clinical isolates of Escherichia coli and Klebsiella pneumonia

Objective To genotype the plasmid DNA of 34 clinical Escherichia coli and Klebsiella pneumoniae isolates producing blaCTX-M by denaturing high-performance liquid chromatography (DHPLC) and evaluate its sensitivity and speciality,and explore a rapid and convenient method for detecting the genotypes of CTX-M-type extended-spectrum beta-lactamases(blaCTX-M). And investigating the antibiotic resistance of blaCTX-M producing clinical isolates of Escherichia coli and Klebsiella pneumonia. Methods Plasmid DNA from each blaCTX-M producing strain was subjected to PCR amplification. We used DHPLC to analyze the PCR production, and then DNA sequencing was performed to determine the genotypes.Finally we compared their results. And the drug susceptibility was performed by Kirby-Bauer disk diffusion. Results Thirty-five amplicons were amplified from 34 isolates, one of them producing two blaCTX-M. By analyzing the results of DHPLC and DNA sequencing, each peak pattern was corresponded to specific genotype. And the blaCTX-M producing strains were hypersensitive to imipenem and meropenem while had high resistance to ampicillin, cefazolin, cefotaxime, ceftriaxone, cefuroxime-sodium, piperacillin. Conclusion DHPLC not only has high accuratissime, but also is a convenient and rapid technique for detecting genotypes. So we can use DHPLC to detect the genotypes of blaCTX-M. BlaCTX-M producing strains expressed multiple drug resistance phenotypes. Imipenem or Meropenem is the best drug in the treatment of infections caused by blaCTX-M producing strains.