牛源大肠杆菌对喹诺酮类抗生素耐药相关基因的筛选及鉴定

摘要

为在大肠杆菌全基因组范围内筛选和鉴定与喹诺酮类抗生素耐药性相关的基因,通过对临床分离的13株大肠杆菌进行药敏试验,选择耐药谱较广泛的菌株作为研究对象,利用Mariner转座子对其基因组进行随机突变,获得转座子突变株文库,并以亲本菌株为对照筛选文库中对喹诺酮类抗生素敏感的突变体,通过套式PCR、核苷酸测序及序列比对确定突变株中转座子的插入位点及其破坏的基因.结果表明:从突变株文库中筛选得到22株分别对诺氟沙星、环丙沙星、左氧氟沙星、萘啶酸敏感的突变株,其中7株对4种抗生素都敏感;插入位点分析发现,抗生素敏感突变株中被转座子破坏的基因包括ecs4206(磷酸核酮糖激酶)、ecs3959(β-D-半乳糖苷酶β亚基)、ecs3946(假定蛋白)和ecs1857(DNA结合转录调控因子).筛选出的基因可被开发为控制或扭转大肠杆菌耐药性的作用靶点.%To screen and identify quinolones antibiotic resistance genes of Escherichia coli genome,drug sensitivity tests were performed on clinically separated 13 E.coli strains for relatively wide resistance strains.Mariner transposons were used to construct transposon mutant library of E.coli,and quinolones antibiotics were used to screen the antibiotics sensitive mutants from the library.The transposon insertion sites were then determined by nested-PCR,nucleotide sequencing and sequence alignment.The results showed that:Twenty-two strains,which were sensitive to norfloxacin,ciprofloxacin,levofloxacin and nalidixic acid,were selected from the library;Transposon insertion sites analysis results showed that the damaged genes were ecs4206 (Phosphoribulo kinase),ecs3959 (Cryptic beta-D-galactosidase subunit beta),ecs3946 (Hypothetical protein),and ecs1857 (DNA-binding transcriptional regulator).These antibiotic resistance genes identified in this study were potential targets for antimicrobial agents.