运用多重PCR技术检测CTX-M型耐药基因的研究

Detection of blaCTX-M genes by multiplex-PCR assay in Enterobacteriaceae bacteria

目的了解产CTX-M型超广谱β-内酰胺酶(ESBLs)肠杆菌科细菌的阳性率、耐药性以及基因型分布。方法采用K-B琼脂扩散法,检测临床分离的85株大肠埃希菌和41株肺炎克雷伯菌对18种抗生素的耐药性,用双纸片增效试验检测产ES-BLs菌株,用多重PCR技术检测相关耐药基因,用DNA序列分析确认基因亚型。结果检测菌株中,ESBLs阳性56株,阳性率为44.4%。所有检测菌株对亚胺培南敏感。产ESBLs的菌株对头孢唑啉、头孢噻肟、头孢曲松、头孢哌酮、头孢他啶等的耐药率为100.0%;质粒接合试验证实,CTX-M型ESBLs介导的耐药可以水平转移;ESBLs阳性菌株中有48株扩增到CTX M型耐药基因,其中CTX-M-3型11株,CTX-M-9型7株,CTX-M-14型25株。结论产CTX-M型ESBLs大肠埃希菌和肺炎克雷伯菌检出率较高,以CTX-M-14型为多。碳青霉烯类抗生素是目前治疗产ESBLs最有效药物。

Objective To understand the positive rate,drug resistance and genotype distribution of Enterobaeteriaceae bacteria which producing CTX-M type extended-Spectrum 13 lactamases（ESBLs）. Methods Assay for the resistance of 136 clinical isolates of Escherichia coli and Klebsiella pneumoniae to 18 kinds of antibiotics with K-B agar diffusion, using double disk synergy test to assay strains producing ESBLs, using multiplex PCR to detect the relevant resistance gene, and using DNA sequence analysis confirmed that gene subtypes. Results There were 56 of 76 stains Escherichia coli and Klebsiella pneumoniae had ESBLs positive phenotype,the positive rate was 44.4 %. All detected strains were imipenem-sensitive,the strains producing ESBLs were all resist ant to cefazolin, cefotaxime, ceftriaxone, cefoperazone, ceftazidime and other cephalosporins; Plasmid bonding test confirmed that cTx-M type ESBLs-mediated resistance was transferable; phenotype positive strains have amplified 48 to CTX-M type resistance genes,ll strains were CTX-M-3 type,7 strains were CTX M-9 type and 25 strains were CTX-M-14 type. Conclusion The detection rate of Escherichia coli and Klebsiella pneumoniae producing CTX-M type ESBLs is high,most of them are CTX-M-14 type; cai-bapenem is the most effective drugs to treat the bacteria producing ESBLs currently.