碳青霉烯类抗生素耐药铜绿假单胞菌外膜孔蛋白OprD2的研究

摘要

Objective To study the relation between outer membrane protein OprD2 and carbapenem resistance in P.aeruginosa.Methods Minimal inhibitory concentration (MIC) of 141 strains of P.aeruginosa to imipenem and meropenem was determined by agar dilution method.Outer membrane oprD2 encoding gene was analyzed by PCR and DNA sequencing.Outer membrane proteins were extracted from carbapenem-resistant P.aeruginosa and analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE).The pattern of outer membrane proteins were compared between the IMPR and IMPs strains.Results Three resistant patterns were identified in the 141 strains of carbapenem-resistant P.aeruginosa, including IMPR MEMR (66.7％),IMPR MEMs (32.6 ％ ) and IMPR MEMs (0.7％).DNA sequencing showed various and significant mutations of the oprD2 encoding gene in 136 of the 141 imipenem resistant strains.Of these strains, 34 showed large fragment missing of oprD2 encoding gene; 6 with insertion; 96 with small fragment missing or multiple point mutations.The oprD2 encoding gene was normal in the 4 MBLs-strains and 1 imipenem sensitive (meropenem resistant) strain.The SDS-PAGE profiles of 16 carbapenem-resistant P.aeruginosa showed the loss of membrane protein OprD2 at the band of 46 000 u, including 10 IMPR MEMR strains and 5 IMPR MEMs strains.The OprD2 profile was normal in 1 IMPs MEMR strain.Conclusions The loss or mutation of oprD2 encoding gene is the molecular basis of the loss or reduction of outer membrane protein OprD2, which may contribute to the resistance of P.aeruginosa to imipenem.%目的 研究铜绿假单胞菌外膜孔蛋白OprD2与碳青霉烯类抗生素耐药的关系.方法 琼脂稀释法测定14种抗菌药物对铜绿假单胞菌的MIC.PCR扩增oprD2编码基因并进行序列分析.十二烷基磺酸钠-聚丙酰胺凝胶电泳(SDS-PAGE)观察碳青霉烯类抗生素耐药铜绿假单胞菌外膜蛋白OprD2的变化,比较敏感株和耐药株外膜蛋白的差异,统计分析采用均数t检验.结果 141株耐碳青霉烯类抗生素铜绿假单胞菌对亚胺培南和美罗培南药敏试验结果显示具3种模式,以亚胺培南和美罗培南同时耐药(IMPRMEMR)为主,占66.7%;亚胺培南耐药和美罗培南敏感(IMPRMEMS)占32.6%;亚胺培南敏感和美罗培南耐药(IMPSMEMR)仅占0.7%.PCR扩增141株耐药株oprD2编码基因结果136株耐药株oprD2编码基因发生显著变异,变异位点呈现多样性.其中34株细菌的oprD2编码基因存在大片段缺失,6株的oprD2编码基因中有插入片段,96株有小片段缺失或不同位置的多个点突变.另4株产金属酶菌株及1株亚胺培南敏感(美罗培南耐药)株的oprD2编码基因未发现异常.对其中16株碳青霉烯类抗生素耐药铜绿假单胞菌进行SDS-PAGE分析,结果显示10株IPMRMEMR和5株IPMRMEMS铜绿假单胞菌外膜蛋白均存在分子量46 000 u处的OprD2蛋白减少,而1株IMPSMEMR株OprD2蛋白带未见异常.结论 oprD2编码基因的缺失或突变可能是导致外膜蛋白OprD2缺失或减少的分子基础,并是铜绿假单胞菌对碳青霉烯类抗生素尤其是亚胺培南耐药的主要机制之一.