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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 the genes related to quinolone antibiotic resistance in the whole genome of Escherichia coli, we selected 13 isolates of Escherichia coli which were clinically isolated for susceptibility testing and selected the strains with broad spectrum of drug resistance as the research object. Using Mariner The transposon randomly mutated its genome to obtain a transposon mutant strain library, and the parent strain was screened for selection of quinolone antibiotic-sensitive mutants in the library, and the results were confirmed by nested PCR, nucleotide sequencing and sequence alignment Transposon insertion sites and their disrupted genes in mutant strains. The results showed that 22 strains were sensitive to norfloxacin, ciprofloxacin, levofloxacin and nalidixic acid, of which 7 were sensitive to 4 kinds of antibiotics. The results of insertion site analysis indicated that, Genes that are damaged by transposons in antibiotic-sensitive mutants include ecs4206 (phosphoribulokinase), ecs3959 (beta-D-galactosidase beta subunit), ecs3946 (putative protein) and ecs1857 (DNA binding transcriptional regulator ). The selected genes can be developed as targets for controlling or reversing E. coli resistance.