【摘要】： 目的:初步探討耐氟喹諾酮類藥物的表皮葡萄球菌與其主動外排蛋白的相關(guān)性,以揭示表皮葡萄球菌對氟喹諾酮類藥物的耐藥機制,為臨床耐藥性表皮葡萄球菌的防治提供實驗依據(jù)。 方法:(1)收集臨床分離表皮葡萄球菌菌株,用紙片擴散法(Kirby-Bauer)檢測50株表皮葡萄球菌對氟喹諾酮藥物的敏感性,篩選出耐藥菌株和敏感菌株,以標準菌株ATCC12228作為對照;(2)PCR法擴增norA基因并測序。(3)提取表皮葡萄球菌的總RNA,應(yīng)用實時熒光定量PCR檢測表皮葡萄球菌多藥轉(zhuǎn)運蛋白norA的mRNA表達水平,分析norA在敏感菌株和耐藥菌株中的表達情況。(4)應(yīng)用熒光法檢測氧氟沙星在表皮葡萄球菌內(nèi)的累積量,以及加入疊氮化鈉后對氧氟沙星累積量的影響。 結(jié)果:(1)表皮葡萄球菌耐藥率較高,80%以上對一種或一種以上氟喹諾酮類藥物產(chǎn)生耐藥。(2)50株臨床分離表皮葡萄球菌有48株檢測到NorA基因,陽性率為98%;對norA基因啟動子區(qū)測序分析發(fā)現(xiàn),5株耐藥菌株中有3株在386位點有堿基C的缺失。(3)臨床分離耐藥菌株norA基因mRNA表達水平明顯高于敏感菌株,差異有顯著性(P＜0.05)。(4)氧氟沙星在表皮葡萄球菌內(nèi)的累積量耐藥組低于敏感組,差異有統(tǒng)計學(xué)意義(P＜0.05);加入能量抑制劑疊氮化鈉后氧氟沙星在菌體內(nèi)的累積量有所增加,菌株SE40尤為明顯。 結(jié)論:表皮葡萄球菌對氟喹諾酮類藥物存在明顯的交叉耐藥,臨床醫(yī)生在應(yīng)用該類藥物時應(yīng)予以注意,減少耐藥菌株的產(chǎn)生;表皮葡萄球菌nora基因的轉(zhuǎn)錄水平的增加使norA基因介導(dǎo)的主動外排增強使氟喹諾酮類藥物在表皮葡萄球菌菌體內(nèi)蓄積量減少,可能是表皮葡萄球菌對氟喹諾酮類耐藥的重要原因;norA基因啟動子區(qū)的堿基C的缺失與norA基因轉(zhuǎn)錄水平的增高相關(guān)。
[Abstract]:Objective: to explore the relationship between Staphylococcus epidermidis resistant to fluoroquinolones and its active efflux protein in order to reveal the mechanism of resistance of Staphylococcus epidermidis to fluoroquinolones. To provide experimental basis for the prevention and treatment of Staphylococcus epidermidis. Methods: (1) Clinical isolates of Staphylococcus epidermidis were collected. The susceptibility of 50 strains of Staphylococcus epidermidis to fluoroquinolone was detected by disk diffusion method (Kirby-Bauer). The standard strain ATCC12228 was used as control. (2) norA gene was amplified by PCR and sequenced. (3) Total RNA, extracted from Staphylococcus epidermidis was used to detect the mRNA expression of multidrug transporter norA in Staphylococcus epidermidis by real-time fluorescence quantitative PCR. The expression of norA in sensitive and resistant strains was analyzed. (4) the accumulation of ofloxacin in Staphylococcus epidermidis and the effect of sodium azide on the accumulation of ofloxacin were detected by fluorescence method. Results: (1) the resistance rate of Staphylococcus epidermidis to one or more fluoroquinolones was higher than 80%. (2) NorA gene was detected in 48 strains of 50 clinical isolates of Staphylococcus epidermidis and the positive rate was 98%. The sequence analysis of promoter region of norA gene showed that 3 of the 5 drug-resistant strains had base C deletion at 386.3.The mRNA expression level of norA gene in clinical isolates was significantly higher than that in sensitive strains. The cumulative dose of ofloxacin in Staphylococcus epidermidis group was significantly lower than that in sensitive group (P < 0. 05). The accumulation of ofloxacin in the bacteria was increased with the addition of energy inhibitor sodium azide, especially the strain SE40. Conclusion: Staphylococcus epidermidis has obvious cross resistance to fluoroquinolones. The increase of transcription level of nora gene in Staphylococcus epidermidis makes the active efflux mediated by norA gene enhance the accumulation of fluoroquinolones in Staphylococcus epidermidis, which may be an important reason for the resistance of Staphylococcus epidermidis to fluoroquinolones. The deletion of base C in the promoter of norA gene was associated with the increase of transcription level of norA gene.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R378

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