目的 研究鲍曼不动杆菌对碳青霉烯耐药的表型和分子机制.方法 采用KB法及琼脂稀释法从临床分离的84株泛耐药鲍曼不动杆菌中随机选取8株,采用外排泵抑制试验筛查外排泵表型阳性菌株,EDTA协同实验筛选金属β-内酰胺酶表型,改良三维试验检测ESBLs和AmpC酶,改良Hodge试验检测碳青霉烯酶,PCR扩增耐药菌株的KPC酶、碳青霉烯酶和ESBLs基因,并测序分析.提取外膜蛋白行SDS-PAGE.结果 PAβN抑制试验显示亚胺培南MIC值下降均≤4倍,而美罗培南MIC值下降≥4倍者占50%(4/8).EDTA协同试验结果显示阴性.改良Hodge试验检测8株耐药菌结果阳性.耐药基因PCR扩增和测序证实8株耐药菌中7株有blaOXA-23基因,5株含blaTEM-1基因,8株耐药菌均含有KPC-2酶基因;耐药菌株的外膜蛋白电泳条带在25 ku处出现明显缺失.结论 鲍曼不动杆菌对碳青霉烯耐药机制主要是产生KPC-2酶、blaOXA-23酶,外膜蛋白25 ku表达的缺失所致,外排泵过度表达可能参与美罗培南的耐药.%To study the phenotype and molecular mechanism of carbapenem-resistant Acinetobacter bau-mannnii. Methods The minimum inhibitory concentrations of a total of 84 clinical isolates were evaluated by the disk diffusion and agar dilution method, and 8 pan-drug resistant isolates were selected randomly. We used efflux pump inhibitor PAβN to confirm the positive phenotype. Metallo-β-lactamase( MBLs ) were screened by EDTA -disk synergy test. The activities of β-lactamases, such as extended-spectrum β-lactamases( ESBLs ) and cephalos-porinase( AmpC ) were detected by the modified three-dimensional test using enzyme extraction. Carbapenemase were screened with modified Hodge test. The blaKPC, carbapenemase and ESBLs gene were amplified by PCR method and then sequenced. The out membrane proteins ( OMPs ) were extracted and analyzed by using SDS-poly-acry-lamide gel electrophoresis with a carbapenem-susceptible Acinetobacter baumannnii strain as control. Results The efflux activity inhibitor test showed that the MIC of imipenem all decreased less than fourfold, while the MIC of meropenem decreased fourfold or greater accounted for 50% ( 4/8 ). None was positive by EDTA-disk synergy test and efficiency test. Modified three-dimensional test indicated 8 carbapenem-resistant abaumannii produced carbap-enemases and the carbapenem-sensitive strain was negative. And 7 of 8 carbapenem-resistant strains produced blaOXA-23 type carbapenemases; The PCR amplification and sequencing results showed that the 8 resistant Acinetobacter baumannii isolates all carried blaKPC and 5 of which contained blaTEM-1 gene. The deficiency of the 25 ku band on SDS-PAGE was found in carbapenem-resistant abaumannii compared with the control. Conclusion The carbapenem-resistant acinetobacter baumannii strains carried blaKPC gene, blaOXA23 gene and lacked a 25 ku OMP and had a higher expression of efflux pump, which were probably attributed to the carbapenem-resistant of organism.
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