铜绿假单胞菌外膜蛋白OprD2缺失和金属β-内酰胺酶与亚胺培南耐药的关系

Contribution of OprD2 Expression and Metallo-β-lactamases in Imipenem Resistance of Pseudomonas aeruginosa Clinical Isolates

目的了解临床分离耐亚胺培南铜绿假单胞菌中外膜蛋白OprD2的缺失和金属β-内酰胺酶(MBLs)的情况。方法2-巯基丙酸协同试验筛选产MBLs菌株,MBLs序列特异性引物PCR扩增和克隆含MBLs基因,亚胺培南为水解底物测定MBLs活性;Western印迹分析检测亚胺培南耐药株外膜蛋白的表达。结果128株亚胺培南耐药株中有17株(13.3%)产生MBLs,其中有7株(5.4%)和10株(7.8%)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的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Western印迹分析检测显示,106株(83.3%)表现为外膜蛋白OprD2的表达缺失,10株(7.8%)表现为外膜蛋白OprD2的表达降低,其余12株(9.3%)的OprD2的表达正常。结论临床分离对耐亚胺培南铜绿假单胞菌大多存在外膜蛋白OprD2的表达缺失或降低,产生VIM-2和IMP-1型MBLs菌株在本地区有一定的流行。

OBJECTIVE To investigate the possible contribution of OprD expression and metallo-β-lactamases （MBLs） in Pseudornonas aeruginosa clinical stains resistant to imipenem. METHODS Clinical strains resistant to imipenem were screened for MBLs production by a disk diffusion synergy test and subjected to PCR assays with primers specific for MBLs. Sequence analysis was provided to identify the prevalence of MBLs gene. Biochemical properties of MBLs were determined by β-lactamase assays with crude preparations of β-lactamases. Expression of OprD2 was determined by quantitative RT-PCR and Western blot analysis. RESULTS Among 128 imipenem resistant strains, 7（5.4%） and 10（7.8%） isolates were positive for VIM-2 and IMP-1 genes, respectively. Crud extraction of β-lactamases showed imipenem-hydrolyzing activity and could be inhibited by treatment with EDTA. In these imipenem-resistant clinical isolates, OprD2 protein was. low-expressed in 10 isolates （7.8 %） and normally expressed in 12 isolates （9.3%） but not expressed in 106 isolates （83.3%）. However, 17 isolates（13.3%） of MBLs producing strains were all lack of OprD2 expression. CONCLUSIONS Reduced or lack of OprD2 expression is the essential mechanisms for most imipenem-resis＇tant clinical isolates of P. aeruginosa, hlaVIM-2 And blalMP-1 are prevalent in P. aeruginosa clinical resistant strains and may lead to nosocomial infection.