革兰阴性杆菌16S rRNA甲基化酶基因检测及作用研究

目的分析上海中医药大学附属普陀医院临床分离的革兰阴性杆菌中16S rRNA甲基化酶基因的流行情况及革兰阴性杆菌的氨基糖苷类耐药机制。方法收集临床分离的对阿米卡星和/或庆大霉素耐药的革兰阴性杆菌53株。采用聚合酶链反应（PCR）法检测16S rRNA甲基化酶基因：armA、rmtA、rmtB、rmtC、rmtD、npmA;PCR阳性产物测序分析。构建PET32-armA和PET32-rmtB的重组质粒并转化入宿主菌BL21中。纸片扩散法（K-B）检测临床分离16S rRNA甲基化酶基因阳性菌株和重组菌对5种氨基糖苷类药物的敏感性。结果 53株耐药菌中5株鲍曼不动杆菌、2株肺炎克雷伯和1株阴沟肠杆菌检出armA基因,2株大肠埃希菌检出rmtB基因。获得PET32-armA和PET32-rmtB的重组BL21菌株。PET32-armA和PET32-rmtB的重组菌均获得氨基糖苷类抗菌药物耐药性。结论本院临床样本检测到16S rRNA甲基化酶基因armA和rmtB阳性菌株,armA基因存在于鲍曼不动杆菌、肺炎克雷伯菌和阴沟肠杆菌中;rmtB基因位于大肠埃希菌中。将armA和rmtB基因转入非耐药的宿主菌中可以诱导其对氨基糖苷类抗菌药物的耐药。

Study on the determination and its influence of 16S rRNA methylase gene in gram negative bacteria

Objective To analyze the prevalence of the 16S rRNA methylase gene isolated from gram negative bacteria in Putuo Hospital,and study the aminoglycoside resistance mechanisms.Methods 53 amikacin-resistant and/or gentamicin-resistant isolates of gram negative bacteria were collected.The 16S rRNA methylase genes armA,rmtA,rmtB,rmtC,rmtD and npmA were determined by polymerase chain reaction（PCR）,and the sequence of positive products was analyzed.The recombinant plasmids of PET32-armA and PET32-rmtB were constructed and transformed into host strain BL21.The sensitivities to 5 kinds of aminoglycoside of 16S rRNA methylase gene positive strains and recombinant strains were detected by disc diffusion（K-B） method.Results The armA was identified in 5 strains of Acinetobacter baumannii,2 strains of Klebsiella pneumoniae and 1 strain of Enterobacter cloacae.The rmtB was identified in 2 strains of Escherichia coli.PET32-armA and PET32-rmtB recombinant bacteria BL21 were obtained.The recombinant bacteria of PET32-armA and PET32-rmtB received aminoglycoside antibiotic resistance.Conclusions The 16S rRNA methylase genes armA and rmtB are identified in clinical specimens.The armA gene is identified in Acinetobacter baumannii,Klebsiella pneumoniae and Enterobacter cloacae,and the rmtB gene is identified in Escherichia coli.Transforming armA and rmtB genes into the host strains of non-resistant bacterium can lead to their resistance to aminoglycoside antibiotics.