高产AmpC酶大肠埃希菌的分子生物学研究

High AmpC β-Lactamases-producing Escherichia coli Isolated from Hospital Infection Patients:A Molecular Study

目的研究医院临床分离的大肠埃希菌高产AmpC酶的流行情况和分子生物学特性。方法利用药敏试验常规纸片扩散法,以头孢西丁抑菌环直径≤17mm为标准筛选可疑产AmpC酶的大肠埃希菌株;三维实验确证AmpC酶;对AmpC酶阳性的菌株,采用多重PCR技术进行质粒AmpC酶的筛选并测序确定基因型别;对多重PCR阴性的大肠埃希菌进行染色体ampC启动子和衰减子的克隆、测序,分析其基因突变的特点。结果临床分离的586株大肠埃希菌中,头孢西丁抑菌环直径≤17mm的有10株占1.70%,酶粗提取物三维实验全部为阳性;其中4株大肠埃希菌质粒多重PCR阳性(0.07%),为DHA-1型质粒AmpC酶;6株为染色体突变高产AmpC酶(1.02%),启动子、衰减子基因克隆测序与大肠埃希菌K12比较,具有基因多态性的特点。结论分离的大肠埃希菌持续高产AmpC酶的耐药机制是获得DHA-1型质粒AmpC酶或染色体ampC启动子、衰减子基因突变导致。

OBJECTIVE To investigate the occurrence and the genotype character of AmpC β-lactamases-producing Escherichia coli isolated from the Second Hospital affiliated to Harbin Medical University. METHODS K-B disk diffusion test and FOX （≤17mm） test were used as initial screen tests to detect the clinical isolates; AmpC β- lactamases were confirmed by three-dimentional extract tests; multiple-PCR was used for detecting plasmidmediated AmpC gene and their genotypes were determined by DNA sequencing; the regulator genes of high producing AmpC isolates of E. coli were cloned to pUCm vectors and sequenced, the difference among them was analyzed by blast method. RESULTS Among total 586 E. coli clinical isolates, 10 isolates （1.70%） resisted to cefoxitin; three-dimensional extract tests were positive for all 10 isolates; 4 isolates of E. coli were plasmidmediated DHA-1 type AmpC β-lactamases by using multiplex PCR and DNA sequencing; 6 isolates of chromosomal high level AmpC β-lactamases-producing E. coll were sequenced and contrasted to that of E. coli K12, showed that they were gene polymorphism. CONCLUSIONS E. coli clinical isolates producing high level AmpC β-lactamases not only acquire plasmid-mediated DHA-1 AmpC β-lactamases but also cause by chromosomal ampC promotor or attenuator gene mutations in our hostiptal.