耐头孢他啶大肠埃希氏菌产β-内酰胺酶的特性研究

Study on the Characteristics of Ceftazidime-resistant β-Lactamase Produced by Escherichia Coli

目的:研究耐头孢他啶大肠埃希氏菌产β-内酰胺酶的特性。方法:采用K-B纸片扩散法和等电聚焦对2株耐药大肠埃希氏菌产β-内酰胺酶类型进行初步判断;采用碱裂解法提取质粒并进行PCR扩增测序;β-内酰胺酶经饱和硫酸铵反抽提及SephadexG-75凝胶过滤和DE-52阴离子交换层析法纯化;以SDS-PAGE法测定其分子量及紫外分光光度法测定其酶动力学参数。结果:2株菌产生一种等电点为8.7的超广谱β-内酰胺酶(CTX-M-1V),分子量为29kDa,能水解头孢噻肟和氨曲南,不能水解亚胺培南,对舒巴坦(IC50=94nmol/L)和他唑巴坦(IC50=5nmol/L)敏感。结论:CTX-M-1V为对抑制剂敏感的CTX-M型超广谱-内酰胺酶。

OBJECTIVE ： To study the characteristics of ceftazidime - resistant β-lactamase produced by Escherichia coll.METHODS： The types of 2 strains of drug fast β-lactamase produced by Escherichia coil were determined initially by K - B slip diffusion method and ampholine electrophoresis method; The plasmid was extracted by alkaline lysis and the PCR amplification and sequencing were conducted; β-Lactamase was counter- extracted by saturated ammonium sulfate, filtrated by Sephadex G-75 gel and purified by DE- 52 anion exchange chromatography; The molecular weight of which was determined by SDS - PAGE and the enzyme kinetics parameters of β- lactamas were determined by ultraviolet spectrophotometry. RESULTS： The 2 strains produced a super - broad spectrum β- lactamase（CTX - M - 1V） with isoionic point at 8.7 and the molecular weight at 29kDa, which can hydrolyze cefotaxim and aztreonam but imipenem and which was sensitive to sulbactam （IC50= 94nmol/L） and tazobactam（ IC50 = 5nmol/L） . CONCLUSION ： CTX - M - 1V is a CTX - M type super - broad spectrum β-lactamase sensitive to suppressants。