产KPC酶超耐药阿斯肠杆菌EaH7的发现及其耐药伴生质粒的遗传特征

 目的  分析1株亚胺培南抗性阿斯肠杆菌(Enterobacter asburiae)的耐药机制及其遗传特征。方法  Vitek-2 Compact系统初步鉴定菌株并测定抗生素最小抑菌浓度,对16s rRNA基因测序以确定菌株;PCR法扩增β-内酰胺类、喹诺酮类和氨基糖苷类等17种耐药基因并测序确认；以CaCl₂诱导的化学法转化质粒;构建伴生质粒DNA文库并测序,以Glimmer 3.02 和BLASTP软件注释并预测伴生质粒序列的编码基因功能;采用接合试验验证伴生质粒对耐药性质粒转移的作用。结果  该菌株为阿斯肠杆菌,对亚胺培南等15种β-内酰胺及氨基糖苷类抗生素耐药,仅对左氧氟沙星和环丙沙星敏感;该菌株同时含有2种质粒,其中耐药性质粒pEa-1携带耐药基因blaKPC-2、blaCTX-M-15和blaTEM-1,而伴生质粒pEa-2则携带4种转移蛋白基因mobA、mobB、mobC和mobD;pEa-2可促进耐药质粒pEa-1接合转移。结论  在国内首次报道了产KPC-2酶的阿斯肠杆菌,该菌携带的质粒pEa-2具有促进耐药性质粒pEa-1接合转移的作用。

Detection of a strain of ultra-drug-resistant Enterobacter asburiae called EaH7 producing KPC enzyme and the genetic characteristics of its resistant-associated plasmid

Objective  To analyze the drug-resistant mechanism and genetic characteristics of a strain of imipenem-resistant Enterobacter asburiae. Methods  The strain and its antibiotics minimum inhibitory concentration was identified by Vitek-2 Compact System, then it was determined by sequencing its 16s rRNA gene. Seventeen genes including β-lactamase resistance gene, quinolone resistance gene and aminoglycoside resistance gene were detected by PCR method and confirmed by sequencing. The plasmid of Enterobacter asburiae was transformed by CaCl₂-induced chemical method. Associated plasmid DNA library was constructed and sequenced. We annotated and predicted encoding function of the associated plasmid by Glimmer 3.02 and BLASTP. The role of the associated small plasmid on transferring to the drug-resistant plasmid was verified by conjugation. Results  This strain belonged to Enterobacter asburiae.It was resistant to 15 kinds of β-lactam antibiotics, such as imipenem and aminoglycoside antibiotics, but it was sensitive to levofloxacin and ciprofloxacin. This strain contained kinds of plasmids.The drug-resistant plasmid pEa-1 carried resistance genes blaKPC-2, blaCTX-M-15 and blaTEM-1, while the associated plasmid pEa-2 carried 4 mobilization proteins genes called mob A, mob B, mob C and mob D.pEa-2 might promote the conjugation of drug-resistant plasmid pEa-1. Conclusions  It is the first time to report that a strain of Enterobacter asburiae can produce KPC-2 enzyme in China, and plasmid pEa-2 carried by the strain might promote the conjugation of drug-resistant plasmid pEa-1.