耐药基因mecA阻断策略逆转MRSA耐药性研究  

Reversal of antibiotics resistance in methicillin-resistant Staphylococcus aureus by blocking mecA by antisense oligodeoxynucleotide

作　　者：祝强强 杨福泰[1] 周诗萌 王宁[2] 贾敏[2] 马雪[2] 孟静茹[2] 

机构地区：[1]第四军医大学药学院学员队,西安710032 [2]第四军医大学药学院药理学教研室,西安710032

出　　处：《中国抗生素杂志》2013年第12期936-941,共6页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(30901813);陕西省自然科学基础研究计划(2009JQ4004)

摘　　要：目的探讨脂质体包裹的靶向耐甲氧西林金黄色葡萄球菌(MRSA)耐药基因mecA的硫代寡聚脱氧核苷酸(PSODN010)能否有效逆转MRSA对β-内酰胺类抗生素的耐药性。方法以mecA mRNA为靶点设计合成PS-ODN010;薄膜分散法制备新型的包裹PS-ODN/PEI纳米微粒脂质体并进行质量评估;体外实验测定细菌生长曲线、菌落数(CFU)等;实时定量PCR法检测PS-ODN010对mecA表达的影响。结果实验结果显示脂质体的包封率为75.9%±3.91%;脂质体包裹的PS-ODN010能浓度依赖性的抑制mecA表达;给予脂质体包裹的PS-ODN010后,苯唑西林明显抑制MRSA的生长(P<0.01),MRSA的菌落数明显低于对照组(P<0.01),苯唑西林等5种β-内酰胺类抗生素对MRSA的MIC显著降低。结论脂质体包裹的抗mecA的PS-ODN010能够选择性抑制MRSA的mecA表达,有效逆转MRSA对β-内酰胺类抗生素的耐药性。Objective To investigate whether antisense phosphothioate oligodeoxynucleotide （PS-ODN010） could reverse the resistance of methicillin-resistant Staphylococcus aureus （MRSA） by anti-mecA PS-ODN010 encapsulated into liposomes. Methods We design and synthesis PS-ODN targeting mecA mRNA of MRSA. The liposomes encapsulating PS-ODN/PEI nanometer particle were prepared by thin film-dispersion technique. And then the amount of PS-ODN was monitored by UV absorbance at 260nm （A260） using an UV/Visible Spectrophotometers to calculate encapsulation efficiency of liposomes. The total colony forming unit （CFU） per sample was determined by correcting the colony count for the dilution and the change of MRSA growth rates in the broth medium was monitored by A630 measurements at different time points. Drug-resistant characters of MRSA were evaluated by measuringminimal inhibitory concentration （MIC） of different ^-lactam antibiotics. To determine whether the expression of mecA was inhibited after anti-mecA PS-ODN treatment, real-time PCR was used. Results The encapsulation efficiencies of liposomes were found to be 75.9% ＋3.91%. Encapsulated PS-ODN010 significantly inhibited the expression of mecA concentration-dependently, and PS-ODN203 showed no effects on the expression of mecA. To test whether oxacillin restored bactericidal effect after WHO-2 strain was treated with encapsulated PS- ODN010, we counted CFU. The results showed the CFU counts of MRSA on the Mueller-Hinton agar containing oxacillin （6mg/L） significantly reduced in all encapsulated PS-ODN010 treated groups in a concentration-dependent manner （P〈0.01）. And oxacillin significantly inhibited the growth of MRSA cells treated with encapsulated PS- ODN010 as compared to the grown of MRSA strains in control. Then we evaluated the change of MICs of some [3-1actam antibiotics used clinically, including oxacillin, flucloxacillin, cefoxitin, cephalothin and cefoperazone. Compared to untreated group, The MICs of four antibiotics for MRSA strai

关 键 词：耐甲氧西林金黄色葡萄球菌 硫代寡聚脱氧核苷酸 耐药性 MECA 纳米微粒 脂质体 

分 类 号：R9[医药卫生—药学]