2株不同序列分型耐黏菌素大肠埃希菌耐药机制分析

目的探讨2株黏菌素耐药大肠埃希菌的耐药机制。方法采用Vitek 2 Compact全自动微生物鉴定药敏系统鉴定细菌及检测常见抗菌药物敏感性,并检测菌株是否产超广谱β-内酰胺酶(ESBLs);用三维试验检测菌株是否产AmpC酶;用微量肉汤稀释法测定菌株对黏菌素的最低抑菌浓度(MIC);用PCR及测序检测耐药基因;多位点序列分型(MLST)和脉冲场凝胶电泳(PFGE)分析菌株的分子分型和同源性;接合试验和S1酶切脉冲场凝胶电泳(S1-PFGE)对菌株的质粒特征进行分析。结果2株菌均对碳青霉烯类药物敏感,对黏菌素耐药,其中N408对头孢类药物耐药;PCR扩增和测序分析显示2株菌均携带mcr-1基因,且N408和N433分别携带blaCIT和blaTEM-1基因;2株菌质粒接合试验均成功,接合子均检测到mcr-1基因;MLST分析显示N408为ST453型,N433为ST8900型,PFGE显示为不同条带。S1-PFGE显示N408有2个质粒,N433有1个质粒。结论本地区已出现携带mcr-1基因黏菌素耐药大肠埃希菌,且可通过质粒在不同菌株间水平播散,应引起临床的重视,加强此类菌株的检测。

Study on drug resistance mechanism of two strains of colistin-resistant Escherichia coli with different sequence type

Objective To explore the mechanism of colistin resistance in two Escherichia coli(E.coli)strains.Methods Vitek 2 Compact full-automatic microbial identification and drug sensitivity analysis system was used to identify the bacteria and the sensitivity for common antibacterial drugs,and detect whether the studied E.coli strains produced extended spectrumβ-Lactamase(ESBLs).3D experiment was used to detect whether the E.coli strains produced AmpC enzymes.The minimal inhibitory concentration(MIC)of colistin for the E.coli strains was determined by micro-broth dilution method.The drug-resistance genes were detected by polymerase chain reaction(PCR)and sequencing.The molecular typing and homology of the strains were analyzed by multilocus sequence typing(MLST)and pulsed-field gel electrophoresis(PFGE)assay.The plasmid characteristics of the strains were analyzed by conjugation experiments and S1 digestion pulsed-field gel electrophoresis(S1-PFGE).Results Both the E.coli strains were sensitive to carbapenems and resistant to colistin,of which strain N408 was resistant to cephalosporins.PCR amplification and sequencing analysis revealed that both strains carried mcr-1 gene,and strain N408 carried blaCIT and strain N433 carried blaTEM-1 gene.The plasmids conjugation tests for the two strains were all successful,and the mcr-1 gene was detectable in their zygote.MLST analysis showed that the strain N408 was ST453 type and strain N433 was ST8900 type.The two stains showed different bands in PFGE.S1-PFGE showed that strain N408 contained two plasmids and strain N433 only contained one plasmid.Conclusion The colistin-resistant E.coli strains carrying mcr-1 gene has emerged in our region and can be disseminated horizontally among different strains by plasmids,which should cause attention in clinical practice and strengthen the detection for such strains.