本文關(guān)鍵詞：銅綠假單胞菌PA2800在耐藥性和致病性中的作用研究　出處：《西北大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 銅綠假單胞菌 PA2800 耐藥性 致病性 毒性因子

【摘要】： 銅綠假單胞菌(Pseudomonas aeruginosa)是一種革蘭氏陰性致病菌,在自然界中分布廣泛并且致病率很高,是引起院內(nèi)感染的三大病原菌之一。隨著抗生素藥物的廣泛使用,銅綠假單胞菌多重耐藥菌株和耐藥率不斷上升,多種抗生素失去其療效,使得臨床的抗感染治療越來越困難。研究銅綠假單胞菌中的耐藥基因及耐藥機(jī)理對于控制銅綠假單胞菌感染具有重要的意義。 在實驗室的前期研究中,通過對銅綠假單胞菌轉(zhuǎn)座突變體文庫進(jìn)行藥物敏感性篩選,發(fā)現(xiàn)基因PA2800突變體對四環(huán)素和環(huán)丙沙星敏感性升高。本論文對PA2800基因進(jìn)行了進(jìn)一步深入研究。 首先通過基因敲除的手段構(gòu)建PA2800的基因敲除突變體,并與野生型銅綠假單胞菌比較,測定了其在固體和液體LB中抗生素抗性的變化,結(jié)果發(fā)現(xiàn)在固體和液體LB中PA2800突變體對四環(huán)素、環(huán)丙沙星和氯霉素敏感性都升高,證實PA2800基因與銅綠假單胞菌耐藥性相關(guān)。研究抗生素對這一基因的調(diào)節(jié)發(fā)現(xiàn),低于抑制濃度的四環(huán)素和紅霉素對PA2800的表達(dá)具有激活作用,暗示這一基因有可能在抗生素存在下誘導(dǎo)銅綠假單胞菌產(chǎn)生抗性。同時,致病力比較研究發(fā)現(xiàn)PA2800和銅綠假單胞菌致病有關(guān),用野生型PAO和PA2800突變體感染果蠅,突變體對果蠅的致死率更高。相關(guān)毒性因子在突變體和野生型中的表達(dá)測定表明,rhlR和znuA在PA2800突變體中的表達(dá)增強(qiáng),這可能是導(dǎo)致突變體較野生型致死率高的因素。同時,我們還發(fā)現(xiàn)oprH在PA2800突變體中的表達(dá)增強(qiáng)。OprH是一個外膜孔蛋白,它的過量表達(dá)可以增強(qiáng)銅綠假單胞菌對喹諾酮類和氯霉素的攝取,所以oprH在突變體中表達(dá)得到增強(qiáng)也可能是導(dǎo)致突變體對壞丙沙星和氯霉素敏感性升高的原因之一。通過實驗證實PA2800實突變體和野生型在運(yùn)動性和生物被膜方面沒有明顯區(qū)別,說明PA2800基因與運(yùn)動性和生物被膜生成沒有直接關(guān)系。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a gram negative pathogen, widely distributed and very high morbidity in nature, which is one of three major pathogens of nosocomial infection. With the extensive use of antibiotics and multi drug resistant Pseudomonas aeruginosa strains and drug resistance rate is rising, many antibiotics lose its efficacy the anti infection treatment, more and more difficult. The clinical drug resistance gene and drug resistance mechanism of Pseudomonas aeruginosa is of great significance for the control of Pseudomonas aeruginosa infection.
In the preliminary research of the laboratory, drug sensitivity screening of Pseudomonas aeruginosa transposition mutant library showed that PA2800 gene mutants increased sensitivity to tetracycline and ciprofloxacin. The PA2800 gene was further studied in this paper.
First of all by knockout methods to construct PA2800 gene knockout mutant, and compared with the wild type Pseudomonas aeruginosa, the changes of antibiotics in solid and liquid LB resistance were determined. The results showed that in the solid and liquid LB in PA2800 mutant of tetracycline, ciprofloxacin and chloramphenicol sensitivity are increased, and confirmed that the PA2800 gene of Pseudomonas Pseudomonas resistance. Regulation of antibiotics for this gene discovery, subinhibitory concentrations of tetracycline and erythromycin on the expression of PA2800 with activation, suggesting that this gene may be induced by the presence of antibiotics in Pseudomonas aeruginosa resistance. At the same time, a comparative study of pathogenicity found PA2800 and Pseudomonas aeruginosa with pathogenic, wild type PAO and PA2800 mutant Drosophila mutants of Drosophila melanogaster infection, a higher mortality rate. The virulence factor in the mutant and the wild type in table As indicated that the expression of rhlR and znuA in PA2800 mutant, which may lead to mutant compared to the wild type lethal factors of high rate. At the same time, we also found that the expression of oprH in mutant PA2800 enhanced.OprH is an outer membrane protein, its overexpression can enhance the uptake of Pseudomonas aeruginosa to quinolones and chloramphenicol, so oprH expression is enhanced in the mutant may be one of the reason of bad ciprofloxacin increased mutant and chloramphenicol sensitivity. Experiments prove that PA2800 mutant and wild type were not significantly different between film movement and biological, PA2800 gene and motility and biofilm formation is not directly related.

【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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本文編號：1428411