摘要
目的探讨阴沟肠杆菌ampD基因突变对持续高产型AmpC β-内酰胺酶(AmpC酶)的调控机制,为临床有效控制产持续高产型AmpC酶细菌引起的感染提供理论依据。方法收集分离自临床的阴沟肠杆菌,用改良的头孢西丁三维试验筛选产持续高产型AmpC酶的菌株,运用T载体和pMW219质粒分别对ampD基因进行测序比对,分析并验证其基因突变是否与AmpC酶高产有关。结果 127株阴沟肠杆菌中有4株为持续高产型AmpC酶阳性,阳性率为3.15%;4株阳性菌株中,E290菌株ampD基因扩增未见条带;E101与E539氨基酸序列一致,将其氨基酸序列与野生株进行比对,95.00%一致,有8个位点发生突变,63位由Phe→Tyr、71位由Ala→Arg、72位由Leu→Val、75位由Asp→His、95位Trp→Arg、131位Glu→Gln、141位Ile→Val、143位Arg→Leu;E487为180个氨基酸,与野生型ampD氨基酸187个氨基酸的序列相比,78.00%一致,有40个位点不一致;重组子的表型检测结果显示,4株菌重组后对头孢替坦、头孢他啶、头孢曲松和氨曲南的MIC出现8～64倍的降低。结论 4株阳性菌中3株产生持续高产型AmpC酶与ampD基因突变有关,使其对β-内酰胺类药物MIC明显升高;新发现的突变位点数目多且分散,其中72、75、71、131和143位氨基酸,研究的意义较大。
Abstract： OBJECTIVE To study the regulatory mechanism of overexpression of AmpC β-actamase （AmpC） in En- terobacter cloacae with mutant ampD gene so as to provide a theoretical basis for effective control of the infections caused by the pathogens overexpressing AmpC. METHODS E. cloacae was isolated from the hospital. The modi- fied three dimensional test was used to detect AmpC and extended spectrum beta-lactmases. T vector and pMW219 plasmids were used respectively to analyze the sequences of wild-type and mutant ampD genes of four clinical iso- lates to verify the relationship between the gene mutation and overexpression of AmpC. RESULTS A total of 127 strains of E. cloacae isolates were isolated, four of them were overexpression AmpC. ampD genes were sequenced and the amplification of E290 strain was failed, the remaining three were successful. The amino acid sequence of E101 and E539 were the same. The remaining three isolates had mutations of ampD compared With the wild-type strain, 95.00% were the same containing 8 common mutations, these amino substitutions were as follow： Phe-63 to Tyr, Ala-71 to Arg, Leu-72 to Val Asp-75 to His, Trp-95 to Arg, Glu-131 to Gln, Ile-141 to Val and Arg-143 to Leu in E101 and E539. E487 containing 180 amino acids, compared with wild-type ampD which contained 187 amino acids, 78.00M were the same and 40 sites were inconsistent; transformants of four clinical isolates harboringarnpD-pMW219 exhibited significant reduction （8 to 64-fold） in the MIC of cefotetan, ceftazidime, ceftriaxone, and aztreonam. CONCLUSION Three clinical strain＇s mutations of ampD gene are responsible for their overexpres- sion of AmpC. These mutations are numerous and scattered throughout the entire protein sequence. It is of great significance to study the sites of 72, 75, 71 and 143 amino acids.
出处
《中华医院感染学杂志》
CAS
CSCD
北大核心
2013年第12期2781-2784,共4页
Chinese Journal of Nosocomiology
基金
上海市科学技术委员会资助项目(10ZR1410600)
中央保健委员会资助项目(B2009B042)
