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目的:探讨我院儿科病区分离的碳青霉烯类耐药铜绿假单胞菌(CRPA)流行情况及OprD基因突变及表达在碳青霉烯类耐药中的作用。方法:儿科分离的30株碳青霉烯类耐药的铜绿假单胞菌,随机扩增DNA多态性(RAPD)进行同源性分析;PCR法检测KPC、IMP、VIM、SPM等产酶基因和OprD基因并双向测序及比对;FQ-RT-PCR测定OprD基因的mRNA相对表达量;SDS-PAGE电泳分析OprD外膜蛋白缺失情况。结果:儿科病区30株CRPA中,检出IMP基因阳性菌株1株,接合试验阴性。oprD基因缺失4株(4/30),突变率100%(9/9),碱基突变主要集中在1010 bp~1136 bp和1212 bp~1355 bp区域。CRPA菌株的oprD基因相对表达量与非耐药组差异有显著性(t=6.145,P<0.05),oprD基因突变位点与其表达量之间无相关性。结论:oprD基因发生广泛突变及表达量降低或缺失是本院儿科CRPA菌株对碳青霉烯类抗生素耐药的主要原因之一。
Objective: To investigate the prevalence of carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolated from pediatric ward in our hospital and the role of OprD gene mutation and its expression in carbapenem resistance. Methods: Thirty isolates of carbapenem-resistant Pseudomonas aeruginosa isolated from pediatrics were randomly divided into three groups. Random amplified polymorphic DNA (RAPD) was used to analyze the homology. PCR was used to detect the production of enzymes such as KPC, IMP, VIM and SPM OprD gene and two-way sequencing and comparison; OprD gene mRNA relative expression was measured by FQ-RT-PCR; OprD outer membrane protein was analyzed by SDS-PAGE electrophoresis. Results: One of the 30 strains of CRPA in pediatric ward was detected positive for IMP gene. The joint test was negative. The oprD gene was deleted in 4 isolates (4/30) and the mutation rate was 100% (9/9). The mutations of oprD gene were mainly located in the region of 1010 bp ~ 1136 bp and 1212 bp ~ 1355 bp. The relative expression level of oprD gene in CRPA strains was significantly different from non-drug resistant group (t = 6.145, P <0.05). There was no correlation between the oprD gene mutation and its expression level. CONCLUSION: The extensive mutation of oprD gene and the decrease or loss of its expression level are one of the main reasons for the resistance of pediatric CRPA strains to carbapenem antibiotics.