摘要
目的了解亚胺培南耐药铜绿假单胞菌外膜蛋白(Opr)D2的缺失和金属β内酰胺酶(MBLs)的产生。方法2-巯基丙酸协同试验筛检产MBLs菌株,MBLs序列特异性引物PCR扩增MBLs基因,以及DNA测序以亚胺培南为水解底物测定MBLs活性;蛋白印迹法检测亚胺培南耐药株Opr的表达。结果34株亚胺培南耐药株中有5株(14.7%)产MBLs,其中有2株(5.9%)和3株(8.8%)分别产VIM-2和IMP-1型MBLs,β内酰胺酶活性测定显示这些菌株产生β内酰胺酶可水解亚胺培南,其水解活性可被EDTA所抑制。实时定量RT-PCR和蛋白印迹检测显示,28株(82.4%)表现为OprD2表达缺失,3株(8.8%)表现为OprD2表达降低,其余3株(8.8%)OprD2表达正常。其中5株(14.7%)产MBLs菌同时存在OprD2表达缺失。结论临床分离铜绿假单胞菌对亚胺培南耐药大多存在OprD2表达缺失或降低,在本地区存在产生VIM-2和IMP-1型的MBLs菌株。
Objective To investigate the possible contribution of OprD expression and metallo-β-lactamases （MBLs） to imipenem resistance in Pseudomonas aeruginosa. Methods Clinical imipenem-resistant strains were screened for MBLs production by a disk diffusion synergy test and subjected to PCR assays with primers specific for MBLs. Sequence analysis was conducted to identify the prevalence of MBLs gene. Biochemical properties of MBLs were determined by β-1actamase assays with crude preparation of β-1actamases. Expression of OprD2 was analyzed by quantitative RT-PCR and Western blot. Results Among the 34 imipenem-resistant P. aeruginosa strains, 2 （5.9%） and 3 （8.8%） were positive for VIM-2 and IMP-1 gene respectively. Crud extraction of β-1actamases showed imipenem-hydrolyzing activity. The activity was inhibited after treatment with EDTA. In these imipenem-resistant clinical isolates, OprD2 protein was low expressed in 3 isolates （8.8 %） and normally expressed in 3 isolates （8.8 % ）, but not expressed in 28 isolates （82.4 % ）. However, the 5 MBLs producing isolates （ 14.7 % ） were all lack of OprD2 expression. Conclusions Reduced or lack of OprD2 expression is the essential mechanism for imipenem-resistance in most P. aeruginosa isolates. VIM-2 and IMP-1 type MBLs are prevalent in P. aeruginosa clinical isolates in our institution.
出处
《中国感染与化疗杂志》
CAS
2007年第6期400-403,共4页
Chinese Journal of Infection and Chemotherapy
