亚胺培南耐药的琼氏不动杆菌的外膜蛋白飞行时间质谱分析

Matrix assisted laser desorption/ionization time of flight mass spectrometry analysis of the outer membrane proteins of an imipenem resistance Acinetobacter junii isolater

目的:探讨亚胺培南耐药的琼氏不动杆菌外膜蛋白特征及其在不动杆菌耐药中的作用。方法:从住院的慢性阻塞性肺疾病急性加重期患者痰液中分离一株多重耐药(包括耐亚胺培南)琼氏不动杆菌ZN3858、API20NE及ARDRA联合RAPD基因指纹技术鉴定到种。琼氏不动杆菌ATCC17908作为参照,SDS-PAGE方法分离并挑选差异表达的外膜蛋白,基体辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)分析外膜蛋白特性,体外扩增其部分结构基因。结果:ZN3858的35-和47-kDa外膜蛋白低表达,质谱分析检索显示35-和47-kDa外膜蛋白分别匹配耐药相关的外膜蛋白oprD和oprF前体;同时一个40kDa外膜蛋白异常高表达,此蛋白质谱分析及检索显示高度匹配鲍曼不动杆菌中的与碳青霉烯耐药相关的外膜蛋白HMB-AB,其部分结构基因被扩增,并被Genbank收录(序列号:AY626903)。结论:外膜蛋白可能参与琼氏不动杆菌对碳青霉烯类抗生素耐药。omp40可能是琼氏不动杆菌中与鲍曼不动杆菌HMP-AB对应的外膜蛋白,其在细菌耐药中的作用有待进一步阐明。

Objective：To characterize the outer membrane proteins in a Carbapenem resistant Acinetobacter junii（A.junii）clinical isolate and to speculate their roles involved in drug resistance.Methods：A multi-drug,including imipenem,resistant A.junii strain ZN3858 was recovered from the sputum of an acute exacerbation of chronic obstructive pulmonary disease in patient.Species identification was performed by the API 20NE and the combination of ARDRA and RAPD fingerprinting analysis.With the reference of A.junii ATCC 17908,the outer membrane proteins（OMPs）profile was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）and the quantitatively different expressed proteins were selected.Matrix assisted laser desorption/ionization time of flight mass spectrometry（MALDI-TOFMS）and PCR were employed to study the characters of the proteins concerned.Results：This carbapenem-resistant A.junii isolates ZN3858 had reduced expression of 35-,and 47-kDa outer-membrane proteins resembling oprD and oprF precursor respectively,which were OMPs associated with antibiotic resistance.Meanwhile,40 kDa outer membrane protein（omp40）was unexpectedly up-regulated,which highly resemble HMB-AB,an imipenem resistance OMP in imipenem resistant A.baumannii.The partial coding DNA sequence（CDS）of omp40 was obtained and the Genbank Accession Number assigned as AY626903.Conclusions：Outer membrane proteins may be involved in multi-drug resistance in A.junii.omp40 in A.junii is probably the counterpart of HMB-AB in A.baumannii,further research is necessary to elucidate its function in imipenem resistance.