本文選題：空腸彎曲菌　切入點：喹諾酮耐藥　出處：《長江大學》2017年碩士論文

【摘要】：空腸彎曲菌(Campylobacter jejuni)是一種在世界范圍內廣泛流行的重要食源性致病菌,可引起人類腸胃炎,格林巴利綜合癥等疾病,是偏布環(huán)境鏈的重要共生菌。禽類是空腸彎曲菌的主要攜帶者之一,污染空腸彎曲菌禽類制品是人類感染空腸彎曲菌的重要途徑;同時禽源可通過食品鏈、空氣及排泄物對環(huán)境產生污染,進而威脅人類健康。在人類生活和畜牧養(yǎng)殖中抗生素的過度使用導致空腸彎曲菌的耐藥率越來越高,近年來在歐美發(fā)達國家直至發(fā)展中國家,人類治療常用的喹諾酮類藥物經常失效,嚴重威脅公共衛(wèi)生安全。實驗室前期開展了華中地區(qū)家禽空腸彎曲菌流行病學調查,對分離株的藥敏試驗結果顯示,分離株對環(huán)丙沙星等喹諾酮類抗生素耐藥率達到了99.1%。因此,本研究基于空彎菌病的預防以及其耐藥性對人類健康可能存在的威脅,深入開展了華中地區(qū)空腸彎曲菌喹諾酮類藥物耐藥機制研究。本研究從介導空腸彎曲菌喹諾酮耐藥的促旋酶基因和耐藥外排泵基因出發(fā),對79株喹諾酮耐藥分離株相關基因進行測序分析。發(fā)現了分離株中促旋酶基因gyrA上的78、81、86、110、119、120、149、157、161等多個突變位點,其中86位突變(Thr-86-Ile)存在于所有的喹諾酮類藥物耐藥菌株,與相關報道一致;gyrB基因在分離株中存在多個無義突變位點;parC基因在所有耐藥分離株中均未檢測到。對外排泵基因cmeABC啟動子區(qū)測序比對發(fā)現,CmeR調控蛋白結合區(qū)域有10種突變的Cme R-Box類型,其中反向重復區(qū)域之間的點插入與點缺失均為首次發(fā)現。MIC試驗顯示,Cme R-Box發(fā)生突變的菌株的MIC值高于未突變菌株,其中點插入或者缺失的菌株最高(P0.05),熒光定量結果顯示發(fā)生CmeR-Box突變的菌株外排泵基因cmeA表達量顯著高于未突變菌株(P0.05),凝膠阻滯試驗的結果顯示rCme R蛋白與各CmeR-Box突變類型DNA結合都有所下降,其中點插入與點缺失下降最為明顯。可見CmeR-Box突變導致其與負調控蛋白CmeR的結合能力下降,使得外排泵基因cmeABC表達上調,引起空腸彎曲菌喹諾酮類藥物耐藥性顯著提高。本研究的結果顯示喹諾酮類藥物耐藥的空腸彎曲菌均具有已報道的促旋酶基因gyrA 86位突變,同時本研究新發(fā)現了一系列CmeABC外排泵啟動子區(qū)突變,其中CmeR-Box的點插入和缺失可引起了空腸彎曲菌喹諾酮類藥物的高水平耐藥。本研究初步解析了華中地區(qū)空腸彎曲菌喹諾酮類藥物耐藥的分子機制,由此,臨床將有針對性地預防類似耐藥機制產生,將真正為人類健康保駕護航。
[Abstract]:Campylobacter jejunii (Campylobacter jejunius) is an important foodborne pathogen that is widely prevalent in the world. It can cause gastroenteritis, Guillain-Barre syndrome and other diseases. Poultry is one of the major carriers of Campylobacter jejuni, and the contamination of Campylobacter jejuni poultry products is an important way for human to infect Campylobacter jejuni. Air and excreta pollute the environment and threaten human health. The overuse of antibiotics in human life and animal husbandry leads to a higher rate of drug resistance of Campylobacter jejuni, and in recent years, in developed countries of Europe and America, even in developing countries, the drug resistance of Campylobacter jejuni has been increasing. Quinolones, commonly used in human treatment, often fail and threaten public health and safety. An epidemiological survey of Campylobacter jejuni in poultry in central China was carried out in the early stage of the laboratory. The resistance rate of the isolates to quinolones such as ciprofloxacin has reached 99.1.Therefore, this study is based on the prevention of vibriosis and the possible threat of drug resistance to human health. The mechanism of quinolone resistance of Campylobacter jejuni in central China was studied. The sequence analysis of 79 quinolone-resistant isolates was carried out. It was found that there were many mutation sites on the gyrA gene, such as 78r-8110119120149157161, in which 86 loci of Thr-86-Ile) existed in all quinolone-resistant strains. In accordance with the relevant reports, there were many nonsense mutation sites in the isolated strain. The sequence analysis of the cmeABC promoter region of the efflux pump gene revealed that the binding region of the CmeR regulatory protein was found to be in all drug-resistant isolates. The sequence analysis of the cmeABC promoter region of the efflux pump gene revealed that there was a CmeR regulatory protein binding region in the isolated strain. Ten mutant Cme R-Box types, Among them, the point insertion and deletion between the reverse repeat regions were the first time to find. MIC test showed that the MIC value of the mutant strain Cme R-Box was higher than that of the unmutated strain. The results of fluorescence quantitative analysis showed that the cmeA expression of efflux pump gene of the strain with CmeR-Box mutation was significantly higher than that of the non-mutant strain P0.05. the result of gel block test showed that rCme R protein and all CmeR-Box mutations were significantly higher than those of the non-mutant strain P0.05. the results of gel block test showed that the expression of rCme R protein was higher than that of the non-mutant strain. The type of DNA combination has declined, The decrease of point insertion and point deletion is the most obvious. It can be seen that CmeR-Box mutation leads to a decrease in its binding ability to negative regulatory protein CmeR, resulting in up-regulation of cmeABC expression of efflux pump gene. The results of this study showed that all of the quinolones resistant to quinolones had the reported gyrA 86 mutation. At the same time, a series of mutations in the promoter region of CmeABC efflux pump were discovered. In this study, the molecular mechanism of quinolone resistance of Campylobacter jejuni quinolones in central China was analyzed. Clinical prevention of similar drug-resistance mechanisms will truly protect human health.
【學位授予單位】：長江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R378

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