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【目的】铜绿假单胞菌是一种重要的条件致病菌,临床上常引起难治性和顽固性感染,随着各种抗生素的广泛使用,该菌对多种抗生素呈现耐药性,研究其耐药性机理有着重要意义。【方法】以一株临床分离株Pseudomonas aeruginosa PA68作为出发菌株,应用人工Mu转座技术构建突变文库并从中筛选得到一株对链霉素抗性明显增强的菌株M122,并对突变株M122进行测序分析及表型检测。通过Southern杂交实验证实转座子是否为单拷贝插入,对突变株M122的基因表达谱与野生型PA68菌株进行对比分析。【结果】确定了Mu转座子在M122基因组上为单拷贝插入,插入位点为基因PA0058的第214 bp处。对M122进行表型检测,发现其对多种氨基糖苷类抗生素的耐药性均得到增强,通过转入携带完整基因PA0058的表达质粒可以使突变株M122的耐药性有所降低,利用同源重组的方法,在模式菌株P.aeruginosa PAK中进行PA0058基因敲除,得到的敲除株具有链霉素耐药性升高的表型。基因PA0058的缺失引起多种基因表达水平改变,尤其是katB、ahpC、ahpF等抗氧化酶基因转录表达显著增高。【结论】首次发现铜绿假单胞菌PA0058基因的插入失活提高了细菌对氨基糖苷类抗生素的耐药性,且导致突变株M122中抗氧化酶基因转录表达水平的上调。
【Objective】 Pseudomonas aeruginosa is an important opportunistic pathogen, which often causes refractory and intractable infection clinically. With the widespread use of various antibiotics, the bacterium shows resistance to many kinds of antibiotics, To study the mechanism of drug resistance is of great significance. 【Method】 A strain of clinical isolates Pseudomonas aeruginosa PA68 was used as the starting strain. Mutant library was constructed by artificial Mu transposon technique and a strain M122 with significantly enhanced streptomycin resistance was screened out. Mutant M122 was sequenced Analysis and phenotypic testing. Southern hybridization experiments confirmed that the transposon is a single copy insert, the mutant M122 gene expression profile and wild-type PA68 strains were analyzed. 【Result】 The results showed that Mu transposon was inserted into M122 genome as a single copy, inserted at the 214th position of gene PA0058. M122 phenotype detection and found that its resistance to a variety of aminoglycoside antibiotics were enhanced by carrying the expression plasmid carrying the complete gene PA0058 mutant M122 resistance can be reduced, the use of homologous Recombinant method, PA0058 gene knockout in the model strain P. aeruginosa PAK, the resulting knockout strain has the phenotype of increased streptomycin resistance. The deletion of PA0058 gene caused a variety of gene expression changes, especially katB, ahpC, ahpF and other antioxidant enzyme gene transcription was significantly increased. 【Conclusion】 The first inactivation of PA0058 gene in Pseudomonas aeruginosa increased the bacterial resistance to aminoglycoside antibiotics, and led to the up-regulation of the transcriptional expression of antioxidant enzymes in mutant M122.