琼氏不动杆菌对碳青霉烯类抗生素耐药分子机制研究  

Molecular mechanism of carbapenem resistance in Acinetobacter junii

作　　者：张永[1] 唐英春[1] 张扣兴[1] 陆坚[2] 莫晓能[1] 潘晓娴[1] 席云[1] 

机构地区：[1]中山大学附属第三医院呼吸科,广州510630 [2]深圳市东湖医院传染科

出　　处：《中国抗感染化疗杂志》2005年第2期77-82,共6页Chinese Journal of Infection and Chemotherapy

摘　　要：目的　研究琼氏不动杆菌对亚胺培南耐药的分子机制。方法　对耐亚胺培南琼氏不动杆菌临床分离株ZN3858,采用琼脂对倍稀释法进行药敏试验,协同抑制试验,质粒接合试验,Southern blot等电聚焦电泳,PCR扩增OXA、IMP、VIM型碳青霉烯酶基因及整合子编码序列及其分子克隆和测序,采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS PAGE)方法研究其外膜蛋白表达情况,阐述其对碳青霉烯类分子耐药机制。结果　受试琼氏不动杆菌具有多重耐药性;耐药基因分子克隆、测序并结合等电聚焦分析证实均产OXA 23型碳青霉烯酶,质粒接合试验、Southern blot显示其编码基因定位在染色体上;与敏感株相比,该菌22、35、47kDa外膜蛋白表达不同程度降低,飞行时间质谱显示:它们与一些耐药相关的外膜蛋白有一定的相似性。结论　表达OXA 23型碳青霉烯酶伴外膜蛋白下调是琼氏不动杆菌对碳青霉烯类抗生素耐药重要分子机制。Objective To investigate the molecular mechanism of carbapenem resistance in imipenem resistant Acinetobacter junii.Methods An imipenem reisistant A.junii ZN3858 was isolated.The isolate was characterized by antimicrobial susceptibility testing and synergistic inhibition test.The beta-lactamases produced by this isolate were identified by isoelectric focusing (IEF).Southern blot and PCR techniques were applied to probe genes encoding OXA,IMP,VIM-type carbapenem enzymes,and analyze the sequences of integrons.Plasmid conjugation was performed to study the transfer of carbapenem resistance.The expression of outer membrane protein (OMP) was studied by SDS-PAGE.Results This isolate was highly resistant to imipenem,meropenem and almost all antibiotics tested.The results of molecular cloning,DNA sequencing and IEF showed that this isolate harbored blaOXA-23 gene and was capable of producing OXA-23 type carbapenemase.Plasmid conjugation experiment,DNA hybridization,together with DNA sequencing suggested that OXA-23 gene was located in chromosome.OMP profile was analyzed by ultra-centrifugation of the sonicated bacterial cells and SDS-PAGE,and then by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOFMS).Compared with carbapenem susceptible isolate,the expression of 22-,35-,and 47-kDa OMPs in this carbapenem-resistant isolate reduced to varying degree.MALDI-TOFMS indicated that these OMPs were similar to some antibiotic resistance associated OMPs.Conclusions The increased expression of OXA-23 type carbapenemase,together with diminished production of OMPs,is the important molecular mechanism of imipenem resistance in Acinetobacter junii.

关 键 词：琼氏不动杆菌 耐药 DNA序列分析 细菌外膜蛋白 质谱 

分 类 号：R516[医药卫生—内科学]