介导对氟喹诺酮类药物耐药的金黄色葡萄球菌norA基因的研究  

Study on drug resistance of Staphylococcus aureus mediated by norA gene to fluoroquinolones

作　　者：陈寒冬[1] 钟利[2] 闫宏钧[1] 彭勋[1] 王守云[1] 杨丽敏[1] 赵培利[1] 苗亮[1] 李丹[1] 房雷[1] 

机构地区：[1]河北省秦皇岛市第三医院,秦皇岛066000 [2]四川省泸州医学院附属医院感染科,泸州646000

出　　处：《中国抗生素杂志》2012年第11期851-855,共5页Chinese Journal of Antibiotics

基　　金：四川省教育厅资助项目

摘　　要：目的研究导致对氟喹诺酮类药物耐药的金葡菌norA基因的介导机制。方法 K-B纸片扩散法测定细菌敏感性(Kirby-Bauer法),研究两种FQNS在菌体内的蓄积量,通过Real-time PCR方法检测金黄色葡萄球菌norA基因的表达。结果两种药物在敏感菌菌体内的稳态蓄积浓度明显高于耐药菌,所有实验菌株中均检测到norA基因的存在,经RealTime PCR扩增后,检测到高度耐药菌株的norA基因表达量较敏感菌菌株平均norA基因表达量高0～8倍;中度耐药菌株的norA基因表达量较敏感菌株高5～17倍。结论推测norA基因表达增加是菌体内药物蓄积量减少的主要原因之一;部分菌株的耐药可能没有norA基因参与,即使有norA基因参与,起作用也不相同;可能有其他外排泵共同参与金葡菌的耐药。Objective To study the mechanism of fluoropuinolone resistance mediated by norA gene in Staphylococcus aureus. Methods The susceptibility test was performed by Kirby-Bauer disk diffusion method. FQNS can be issued fluorescence by laser excitation at a certain wavelength, and the relationship of fluorescence value and the concentration of FQNS was linear. The accumulation concentration of two FQNS in bacteria were measured by fluorescene measured method. Study on norA gene of S. aureus. Total RNA was extracted by Trizol reagent, Reverse transcription was done by TOYOBO＇s reverse transcriptase kit. For RT-PCR, the volume of reaction is 25μL, 10μL of the product of reverse transcription of total RNA, 1 μL of upstream primer, 1 μL of downstream primer, 12.5μL of 2xPCR Master Mix, up to 25μL of double distilled H20. PCR products were visualized on horizontal agarose gels after electrophoresis. Then the PCR products were purified and were sequencing and diluted series of gradient, as a quantitative standard reference. Expression level of norA gene was detected by Fluorescence quantitative RT-PCR of TaqMan probe. The volume of PCR reaction is 25μL, 1μL of probe, 4μL of upstream primer,4pL of downstream primer, 5pL of cDNA or standard reference, 25μL of Realtime PCR Master Mix and 1 μL of double distilled water and carried out as hot start at 95℃ for 60s, 40 cycles of denaturation at 95℃ for 15s, renaturation at 60℃ for 60s, extension at 60℃ for lmin. Results There are MDR and CDR in Four FQNS. The steadystate accumulation concentration of two drugs in the sensitive bacteria was significantly higher than that of the resistant bacteria, nora gene were detected in all experimental strains. The expression level of nora gene in bad resistant strain were 0-8 times more than the average expression in sensitive stains. Conclusion There is CDR to four different quinolones in S. aureus, while there is MDR to other antibacterials.The decrease of accumulation concentration of FQNS is one of the mechanisms of re

关 键 词：氟喹诺酮 金黄色葡萄球菌 耐药 外排泵 norA基因 

分 类 号：R378.1[医药卫生—病原生物学]