Cai Jia-Chang, Zhou Hong-Wei, Chen Gong-Xiang, Zhang Rong
Clinical Laboratory Center, Second Affiliated Hospital of Zhejiang University, Hangzhou 310009, China.
Zhonghua Yi Xue Za Zhi. 2008 Jan 8;88(2):135-8.
To investigate the mechanism of carbapenem resistance in Enterobacter cloacae.
A carbapenem-resistant strain of E. cloacae (strain ZY1465) was isolated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments were carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkaline lysis technique and were digested by various endonucleases; The crude beta-lactamase extracts of E. cloacae and E. coli transconjugant were subjected to analytical isoelectric focusing (IEF). Specific PCR amplification and DNA sequence analysis were preformed to confirm the beta-lactamase type. Outer membrane proteins (OMPs) were isolated and examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The E. cloacae isolate showed resistance against carbapenems. The MICs of imipenem and meropenem were both 32 microg/ml. The isolate was also resistant strongly against penicillins, cephalosporins, cefoxitin, aztreonam, quinolones, and aminoglycosides. Conjugation studies with E. coli resulted in the transfer of reduced carbapenem susceptibility from E. cloacae isolate. Plasmid restriction analysis showed identical restriction profiles between the transconjugants of E. cloacae ZY1465 and Serratia marcescens ZN008. Isoelectric focusing demonstrated six beta-lactamases, with the isoelectric points (pIs) of 5.4, 6.7, 7.3, 7.8, 7.9, and 8.6, in E. cloacae ZY1465, and only one beta-lactamase with the pI of 6.7 in transconjugant. Specific PCR amplification and DNA sequence analysis confirmed that E. cloacae ZY1465 harbored TEM-1, KPC-2, DHA-1, CTX-M-14, CTX-M-3 and chromosomal AmpC (not detected in IEF) genes. Urea-SDS-PAGE analysis of OMPs showed that E. cloacae ZY1465 lacked an OMP of approximately 38 000 Da which was present in E. cloacae ATCC13047.
It is the first detection of plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC-2 in a clinical isolate of E. cloacae from China. Production of multiple beta-lactamases, especially KPC-2 and loss of an OMP in E. cloacae ZY1465 result in resistance to carbapenems.
研究阴沟肠杆菌对碳青霉烯类抗生素耐药的机制。
分离出一株对碳青霉烯类抗生素耐药的阴沟肠杆菌（菌株ZY1465）。采用琼脂稀释法测定抗生素敏感性。在混合肉汤培养物中进行接合试验。使用碱性裂解技术制备质粒DNA，并使用各种核酸内切酶进行消化；对阴沟肠杆菌和大肠埃希菌转接合子的粗β-内酰胺酶提取物进行分析性等电聚焦（IEF）。进行特异性PCR扩增和DNA序列分析以确认β-内酰胺酶类型。分离外膜蛋白（OMPs），并通过尿素-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行检测。
阴沟肠杆菌分离株对碳青霉烯类抗生素耐药。亚胺培南和美罗培南的MIC均为32μg/ml。该分离株还对青霉素类、头孢菌素类、头孢西丁、氨曲南、喹诺酮类和氨基糖苷类有较强耐药性。与大肠埃希菌的接合研究导致阴沟肠杆菌分离株对碳青霉烯类抗生素的敏感性降低得以转移。质粒限制性分析显示阴沟肠杆菌ZY1465和粘质沙雷菌ZN008的转接合子之间的限制性图谱相同。等电聚焦显示阴沟肠杆菌ZY1465中有六种β-内酰胺酶，等电点（pIs）分别为5.4、6.7、7.3、7.8、7.9和8.6，而转接合子中只有一种等电点为6.7的β-内酰胺酶。特异性PCR扩增和DNA序列分析证实阴沟肠杆菌ZY1465携带TEM-1、KPC-2、DHA-1、CTX-M-14、CTX-M-3和染色体AmpC（IEF未检测到）基因。OMPs的尿素-SDS-PAGE分析显示阴沟肠杆菌ZY1465缺乏阴沟肠杆菌ATCC13047中存在的约38000Da的一种OMP。
这是首次在中国临床分离的阴沟肠杆菌中检测到质粒介导的碳青霉烯水解β-内酰胺酶KPC-2。阴沟肠杆菌ZY1465中多种β-内酰胺酶的产生，尤其是KPC-2以及一种OMP的缺失导致对碳青霉烯类抗生素耐药。
Zotero 插件