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目的研究插入序列ISEcp1B基因在革兰阴性杆菌中的分布及多态性分析。方法用K-B药敏法分析143株革兰阴性杆菌的耐药性;用多重聚合酶链反应(PCR)技术扩增ESBLs、AmpC及ISEcp1B基因,并进行DNA测序。结果CTX-M-G1及ISEcp1B基因的总检出率分别为41.26%及16.08%;ISEcp1B插入序列的在各菌种的检出率分别为大肠埃希菌9株占20.93%、肺炎克雷伯菌6株占11.32%、铜绿假单胞菌5株占13.89%、阴沟肠杆菌2株占25.00%、弗氏柠檬酸杆菌1株占25.00%;插入序列ISEcp1B阳性株对头孢菌素的耐药率显著高于阴性株(P<0.01),在体外对亚胺培南有较好的抗菌活性;对ISEcp1BPCR产物进行测序,发现其下游均连接CTX-M型ESBLs,暂未见其他类型的β-内酰胺酶;ISEcp1B右端均连接一个反向重复序列,为转位酶提供作用位点;提供-35及-10两个启动子,对下游不同的CTX-M型ESBLs的高水平表达和传播起重要的调控作用,启动子与CTX-M型ESBLs编码起始距离长短不一,并有不同程度的突变,呈多态性。结论插入序列ISEcp1B较广泛分布于革兰阴性杆菌,增加了CTX-M型ESBLs水平播散的危险性,并对其表达起调控作用。
Objective To study the distribution and polymorphism of ISEcp1B gene in gram-negative bacilli. Methods The drug resistance of 143 Gram-negative bacilli was analyzed by K-B method. The ESBLs, AmpC and ISEcp1B genes were amplified by polymerase chain reaction (PCR) and sequenced. Results The overall detection rates of CTX-M-G1 and ISEcp1B genes were 41.26% and 16.08%, respectively. The detection rates of ISEcp1B insertions in all strains were 20.93% for Escherichia coli, 20.93% for Klebsiella pneumoniae, 6 strains of Escherichia coli accounted for 11.32%, 5 strains of Pseudomonas aeruginosa accounted for 13.89%, 2 strains of Enterobacter cloacae accounted for 25.00% and 1 strain of Citrobacter freundii accounted for 25.00%. The ISEcp1B positive strains were resistant to cephalosporins (P <0.01), and had good antibacterial activity against imipenem in vitro. The products of ISEcp1BPCR were sequenced and found to be connected with CTX-M type ESBLs downstream, and no other type of β -lactamase; the right end of ISEcp1B is connected with an inverted repeat sequence, providing the site of transposition enzyme; providing -35 and -10 two promoters, high-level expression and transmission of downstream CTX-M type ESBLs Play an important regulatory role, the promoter and CTX-M-type ESBLs encoding the starting distance varies, and have varying degrees of mutation, was polymorphic. Conclusion The inserted sequence ISEcp1B is more widely distributed in Gram-negative bacilli, which increases the risk of disseminated CTX-M type ESBLs and regulates its expression.