临床分离耐药摩氏摩根菌的β-内酰胺酶表型和基因型分析  

Phenotype and genotype analyses of a β-lactamases-producing Morganella morganii clinical isolate

作　　者：余娴[1] 凌保东[1] 谢勇恩[1] 周岐新[2] 雷军[1] 

机构地区：[1]川北医学院药物研究所,南充637007 [2]重庆医科大学药理教研室,重庆400016

出　　处：《中国抗生素杂志》2005年第11期665-668,685,共5页Chinese Journal of Antibiotics

基　　金：四川省重点科技攻关项目(编号02SG022-030)

摘　　要：目的探讨临床分离耐药摩氏摩根菌3-87耐药的分子机制。方法采用常规琼脂二倍稀释法对摩氏摩根菌3-87进行15种抗菌药的M IC测定;超声破碎法提取β-内酰胺酶;并用n itrocefin做定性检测,用紫外分光光度计检测酶活性;超薄层等电聚焦测定β-内酰胺酶等电点和酶抑制实验;ESBL s表型鉴定;Am pC酶的检测;纸片协同法筛选产金属β-内酰胺酶菌株;以摩氏摩根菌3-87质粒DNA为模板扩增blaSHV、blaTEM和blaCTX-M基因,以基因组DNA为模板扩增ampC结构基因;ampC结构基因扩增产物的DNA序列测定及同源性分析。结果摩氏摩根菌3-87对10种β-内酰胺类抗生素、2种氟喹诺酮类抗菌药耐药,对哌拉西林/三唑巴坦和头孢哌酮/舒巴坦、氨基糖苷类抗生素阿米卡星敏感。产酶活性为3727.5U的β-内酰胺酶。产生的pI7.3、8.2及9.6的三种β-内酰胺酶分别能被氯唑西林、克拉维酸、EDTA部分抑制。β-内酰胺酶表型鉴定试验结果显示菌株产ESBL s、Am pC酶及金属β-内酰胺酶三种β-内酰胺酶。blaCTX-M基因引物扩增出阳性扩增条带。ampC结构基因引物扩增阳性扩增带,对扩增产物进行序列分析与已报道的摩氏摩根菌ampC结构基因的核苷酸序列高度同源,推导的氨基酸序列发生了1个氨基酸残基的变异。结论临床分离耐药摩氏摩根菌3-87呈多重耐药,其对β-内酰胺类抗生素耐药的机制是产生了ESBL s、Am pC酶及金属β-内酰胺酶。Objective To investigate antibacterial resistance mechanism of Morganella rnorganii 3-87 clinical isolate. Method MICs of 15 antibacterials were determined by two-fold agar dilution method. Crude β-lactamase preparations were obtained by sonication. The enzyme activity was determined by ultraviolet spectrophotometry. Isoelectric points of β-lactamases were determinded by IEF and the inhibitory activity of inhibitors to β-lactamase was observed. The disc diffusion test according to NCCLS Screening and Confirmatory Test for ESBLs was used. AmpC enzyme was examined by three dimension test. Metallo-β-lactamase was screened by double disk synergy. PCR amplification Of blaSHV, blaTEM and blaCTX-M genes was carried out using plasmid as template. PCR amplification of ampC structure gene was carried out using genomic DNA as template. PCR product of ampC structure gene was sequenced. The nucleotide sequence and deduced amino acid sequence were analyzed by BLAST of NCBI. Result Morganella morganii 3-87 was resistant to 10 β-lactam antibiotics, ciprofloxacin and gatifloxacin, while susceptible to piperacillin/tazobactam, cefoperazone/sulbactam and amikancin. ^-lactamase enzyme activity was 3,727.5U. The strain produced ^-lactamases of pI 7.3, pI8.2 and pI9.6 which could be partly inhibited by cloxacillin,clavulanic acid and EDTA respectively. The phenotype tests of β-lactamase showed that the strain produced ESBLs, AmpC enzyme and metallo - β-lactamase. The amplification of blaCTX-M gene was positive, ampC structure gene was amplified and the product was highly homologous to that in Morganella morganii which had been reported; one amino acid residue mutated in deduced amino acid sequences. Conclusion Morganella morganii 3-87 clinical isolate was multi-drug resistant. The mechanism of resistance to β-lactam antibiotics was that the strain produced three types of β-lactamases, ESBLs, AmpC enzyme and metallo-β-lactamase.

关 键 词：摩氏摩根菌 Β-内酰胺酶 ESBLS AMPC酶 金属Β-内酰胺酶 

分 类 号：R378[医药卫生—病原生物学]