产CMY型AmpC酶费劳地枸橼酸杆菌的分子学特性研究

Study on molecule characteristics of a novel CMY-type AmpC β-Lactamase from Citrobacter freundii

目的对产CMY型AmpC酶费劳地枸橼酸杆菌进行耐药表型及分子学特性进行研究,探讨研制新的酶抑制剂。方法对临床分离的两株费劳地枸橼酸杆菌所产CMY型AmpC酶用纸片扩散法、三相水解试验进行耐药表型检测,以该菌总基因组DNA和质粒DNA为模板进行PCR扩增、序列分析、质粒接合试验、构建重组表达载体及AmpC酶检测。结果两株菌株对青霉素类、头孢菌素类、喹诺酮类、氨基糖苷类抗生素均表现为耐药,对呋喃类和碳青霉烯类表现为敏感;三相水解试验结果显示该菌能水解头孢西丁;PCR扩增出大小为1 146 bp的基因片段,与GenBank上多种CMY亚型的基因序列同源性为97%;质粒接合试验证实质粒上含CMY基因,为可转移质粒。结论两株菌株所产CMY型AmpC酶为新的CMY型头孢菌素酶,它介导了该菌对青霉素类、头孢菌素类、喹诺酮类等抗生素耐药,其耐药性能水平传播。

Objective To investigate the antibiotic phenotype and moleculelogy characteristics of novel CMY-type AmpC β-Lac- tamase from Citrobacter freundii,in order to triturate bomologue enzyme inhibitors. Methods Slip diffusion method and three phase hydrolyses test were used to analyze antibiotic phenotype of novel CMY-type AmpC β-Lactamase from Citrobaeter freundii that was detached from clinic, The DNA and plasmid of CMY type AmpCβ-Lactamase from Citrobacter freundii Strain was amplified by PCR,sequence analysis,Plasmid Conjugation tests, construction recombinant expression vector and AmpC induce tests. Results Two strains were resistant to penicillins, cephalosporin, quinolone, aminoglycoside, and susceptible to nitrofuran, carbapenem, The results of three dimension test showed,AmpCβ-Lactamase from DNA Strain and recombinant strain could hydrolyz cefoxitin,The 1 146bp DNA fragment of CMY-type AmpC β Lactamase encoding gene sequence shared 97% amino acid identity with CMY type AmpCβ-Lactamase that already registered in GenBank. Plasmid Conjugation tests showed bla CMY located in plasmid and plasmid could conjugate. Conclusion The CMY type AmpC β-Lactamase is a novel CMY-type AmpC cephalosporinase. It is resistant to penicillins, cephalosporin, quinolone, and it＇s drug resistance can be transmissed .