本文選題：銅綠假單胞菌　切入點(diǎn)：臨床菌株　出處：《西北大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來,隨著多重耐藥菌株(Multiple Drug-resistant Strains,MDR)的頻頻出現(xiàn),越來越多的現(xiàn)有抗生素逐漸失去效用,細(xì)菌性疾病再次嚴(yán)重威脅人類健康。銅綠假單胞菌(Pseudomonas aeruginosa,PA)是人類機(jī)會(huì)病原菌,可以引起各種嚴(yán)重感染且常常對(duì)病人的生命造成威脅。MDR銅綠假單胞菌引起的感染在臨床上非常難以治療,這使其成為對(duì)人類健康威脅最大的病原菌之一。研究PA抗藥性機(jī)理不僅具有重要的理論價(jià)值也具有緊迫的臨床意義。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),PA四型菌毛(Type Ⅳ Pili,T4P)基因pilY1的轉(zhuǎn)座突變體(PA4554M)對(duì)羧芐青霉素(Carbenicillin,Car)抗性比野生型增加了 128倍;而另一個(gè)T4P基因pilE的轉(zhuǎn)座突變體對(duì)羧芐青霉素的抗性也提高了16倍。這暗示銅綠假單胞菌T4P與羧芐青霉素抗性有著某種聯(lián)系。本論文在此基礎(chǔ)上對(duì)T4P與羧芐青霉素抗性的關(guān)系進(jìn)行了初步研究。首先,對(duì)實(shí)驗(yàn)室已有的PA突變體菌株進(jìn)行篩選,得到蹭行(Twitching)降低的敲除突變體菌株5株,轉(zhuǎn)座突變體7株,對(duì)這12株突變體的羧芐青霉素抗性檢測發(fā)現(xiàn),其中5株突變體的羧芐青霉素抗性比野生型有增加,7株突變體的羧芐青霉素抗性比野生型降低。之后,我們挑選了 16種不同類抗生素對(duì)這些突變體的抗生素抗性做了濾紙片實(shí)驗(yàn),發(fā)現(xiàn)只有羧芐青霉素抗性在12株菌中改變一致,這說明在PA中,T4P特異性影響Car的抗生素抗性,與其它抗生素的抗性沒有相關(guān)性聯(lián)系。其次,本研究利用臨床菌株,對(duì)PA運(yùn)動(dòng)性與抗生素抗性的關(guān)系進(jìn)行了分析。我們從西安地區(qū)的醫(yī)院收集了 89株P(guān)A臨床菌株。對(duì)它們的蹭行運(yùn)動(dòng)和抗生素抗性表型進(jìn)行了調(diào)查。根據(jù)統(tǒng)計(jì)有70.78%臨床菌株twitching相對(duì)于實(shí)驗(yàn)室菌株P(guān)AO1有所降低;29.22%臨床菌株twitching不變;測定Car抗性發(fā)現(xiàn),與PAO1相比,敏感菌株2株,占2.24%,抗性不變的菌株3株,占3.37%,其余84株均表現(xiàn)為抗性,占93.38%,個(gè)別臨床菌株Car的MIC甚至達(dá)到了 8 mg/ml。這些結(jié)果再次表明,在臨床菌株中,銅綠假單胞菌T4P與羧芐青霉素抗性似乎存在著某種聯(lián)系,但這種聯(lián)系的機(jī)理需要進(jìn)一步的研究。最后,為了 了解這種聯(lián)系的機(jī)理,我們對(duì)T4P重要的功能、結(jié)構(gòu)基因進(jìn)行缺失敲除,得到突變體:△pilZ、△pilB、△pilJ、△pilS、△pilE、△fimX、△pilD和△pilY1,所有突變體的twitching運(yùn)動(dòng)表型明顯喪失,但是,羧芐青霉素抗性卻沒有變化,我們推測,也許只有特定的pilY1突變體才導(dǎo)致對(duì)羧芐青霉素抗性。pilYp的轉(zhuǎn)座突變體對(duì)羧芐青霉素的抗性可能取決于轉(zhuǎn)座子插入的特殊位點(diǎn),造成PilY1特殊的變化。因此T4P與羧芐青霉素抗性之間潛在聯(lián)系的具體機(jī)理還需要后期進(jìn)一步的研究。本論文的這些結(jié)果為進(jìn)一步研究PA羧芐青霉素抗性的產(chǎn)生機(jī)理奠定了初步基礎(chǔ)。
[Abstract]:In recent years, with the frequent emergence of multiple Drug-resistant strains, more and more existing antibiotics have gradually lost their efficacy, and bacterial diseases are once again a serious threat to human health. Pseudomonas aeruginosa PAA is a human opportunistic pathogen. Can cause a variety of serious infections and often threaten the lives of patients. Infection caused by Pseudomonas aeruginosa is very difficult to treat clinically. This makes it one of the most dangerous pathogens to human health. Studying the mechanism of PA resistance is not only of important theoretical value but also of urgent clinical significance. The resistance to Carbenicillinus carbenicillinus (Carbenicillin), a transgenic mutant of pilY1, was 128-fold higher than that of wild type. The resistance of another transposed mutant of T4P gene pilE to carbenicillin was also increased by 16 times. This suggests that Pseudomonas aeruginosa T4P has some relationship with resistance to carbenicillin. The relationship of element resistance was studied. First of all, Five knockout mutants and 7 transposed mutants were obtained by screening the PA mutants in the laboratory, and the resistance to carboxylpenicillin of the 12 mutants was detected. The resistance to Carbenicillin of 5 mutants was higher than that of wild type, and the resistance of 7 mutants was lower than that of wild type. We selected 16 different kinds of antibiotics for the antibiotic resistance of these mutants. We found that only the resistance to carbenicillin changed consistently in 12 strains, which indicated that T4P had specific effect on the antibiotic resistance of Car in PA. There is no correlation between the resistance of other antibiotics and the resistance of other antibiotics. Secondly, the clinical strains were used in this study. The relationship between exercise resistance and antibiotic resistance of PA was analyzed. 89 clinical strains of PA were collected from hospitals in Xi'an area. Compared with the laboratory strain PAO1, the clinical strain twitching decreased by 29.22% and the clinical strain twitching did not change. Compared with PAO1, 2 strains (2.24%) were sensitive to Car resistance, 3 strains (3.37%) were resistant, the other 84 strains showed resistance, accounting for 93.38%. The MIC of individual clinical strains Car even reached 8 mg / ml. In clinical strains, Pseudomonas aeruginosa T4P and Carbenicillin resistance seem to have some relationship, but the mechanism of this connection needs further study. Finally, in order to understand the mechanism of this connection, we are important to the function of T4P. The mutant: pilZ, pilB, pilJ, pilS, pilE, FimX, pilD and pilY1, all the mutants have obviously lost their twitching motional surface, but the resistance to Carbenicillin has not changed, we speculate. It may be that only a specific pilY1 mutant causes resistance to Carbenicillin. PilYp's transposable mutants may depend on the particular site of transposon insertion. Therefore, the specific mechanism of the potential relationship between T4P and resistance to carbenicillin needs to be further studied. These results in this paper are useful for further study of the mechanism of resistance to PA Carbenicillin. A preliminary foundation was laid.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378
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本文編號(hào)：1594831