目的 了解大连2所医院临床分离革兰阴性菌中介导高水平氨基糖苷类抗生素耐药的16S rRNA甲基化酶基因armA、rmtB的流行情况,并研究其耐药机制.方法 收集临床分离的耐阿米卡星的革兰阴性杆菌134株.PCR法筛选2种甲基化酶基因armA及rmtB;PCR产物进行测序.质粒提取、接合试验及转化试验确定armA及rmtB基因定位.琼脂稀释法测定阳性菌株、结合子和转化产物对阿米卡星、庆大霉素、妥布霉素3种氨基糖苷抗生素的MIC值.结果 134株耐药菌株中,21株鲍曼不动杆菌检出armA基因,5株大肠埃希菌和5株肺炎克雷伯菌检出rmtB基因.质粒抽提试验及接合试验rmtB阳性菌获得成功.接合子及转化产物DH5a(pMDarmA)均获得高水平耐氨基糖苷类抗生素的特性. 结论 大连2所医院检测到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中;rmtB基因位于大肠埃希菌及肺炎克雷伯菌的质粒上.armA和rmtB可以导致细菌对氨基糖苷类抗生素的高水平耐药.%Objective To investigate the prevalence of 16S rRNA methylase genes, armA and rmtB, which mediate high level aminoglycoside resistance, in gram-negative bacteria isolated from 2 hospitals in Dalian and study the mechanism of aminoglycoside resistance.Methods A total of 134 amikacin-resistant clinical isolates of gram-negative bacteria were collected. Two 16S rRNA methylase genes, armA and rmtB, were identified by PCR-based assays. PCR products were extracted for DNA sequencing analysis. armA and rmtB gene mapping were conducted by plasmid extraction, conjugation and transformation. The MICs of amikacin, gentamicin and tobramycin were determined for the positive isolates, transconjugants and the resultant strain of transformation using agar dilution technique. Results Overall armA was identified in 21 strains of Acinetobacter baumannii, rmtB in 5 strains of Escherichia coli and 5 strains of Klebsiella pneumoniae. Plasmid extraction and conjugation experiments were only successful for rmtB-positive isolates. Transconjugant and DH5a (pMDarmA) exhibited high-level resistance to aminoglycosides.Conclusions The 16S rRNA methylase genes, armA and rmtB are identified in Dalian. armA gene is identified in A. baumannii. rmtB gene is located on the plasmid of E. coli and K. pneumoniae. armA and rmtB can induce high-level resistance to aminoglycosides.
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