华中地区空肠弯曲菌gyrA、CmeR-Box多态性与喹诺酮类药物耐药的相关性研究

发布时间：2018-04-01 04:20

本文选题：空肠弯曲菌　切入点：喹诺酮耐药　出处：《长江大学》2017年硕士论文

【摘要】：空肠弯曲菌(Campylobacter jejuni)是一种在世界范围内广泛流行的重要食源性致病菌,可引起人类肠胃炎,格林巴利综合症等疾病,是偏布环境链的重要共生菌。禽类是空肠弯曲菌的主要携带者之一,污染空肠弯曲菌禽类制品是人类感染空肠弯曲菌的重要途径;同时禽源可通过食品链、空气及排泄物对环境产生污染,进而威胁人类健康。在人类生活和畜牧养殖中抗生素的过度使用导致空肠弯曲菌的耐药率越来越高,近年来在欧美发达国家直至发展中国家,人类治疗常用的喹诺酮类药物经常失效,严重威胁公共卫生安全。实验室前期开展了华中地区家禽空肠弯曲菌流行病学调查,对分离株的药敏试验结果显示,分离株对环丙沙星等喹诺酮类抗生素耐药率达到了99.1%。因此,本研究基于空弯菌病的预防以及其耐药性对人类健康可能存在的威胁,深入开展了华中地区空肠弯曲菌喹诺酮类药物耐药机制研究。本研究从介导空肠弯曲菌喹诺酮耐药的促旋酶基因和耐药外排泵基因出发,对79株喹诺酮耐药分离株相关基因进行测序分析。发现了分离株中促旋酶基因gyrA上的78、81、86、110、119、120、149、157、161等多个突变位点,其中86位突变(Thr-86-Ile)存在于所有的喹诺酮类药物耐药菌株,与相关报道一致;gyrB基因在分离株中存在多个无义突变位点;parC基因在所有耐药分离株中均未检测到。对外排泵基因cmeABC启动子区测序比对发现,CmeR调控蛋白结合区域有10种突变的Cme R-Box类型,其中反向重复区域之间的点插入与点缺失均为首次发现。MIC试验显示,Cme R-Box发生突变的菌株的MIC值高于未突变菌株,其中点插入或者缺失的菌株最高(P0.05),荧光定量结果显示发生CmeR-Box突变的菌株外排泵基因cmeA表达量显著高于未突变菌株(P0.05),凝胶阻滞试验的结果显示rCme R蛋白与各CmeR-Box突变类型DNA结合都有所下降,其中点插入与点缺失下降最为明显。可见CmeR-Box突变导致其与负调控蛋白CmeR的结合能力下降,使得外排泵基因cmeABC表达上调,引起空肠弯曲菌喹诺酮类药物耐药性显著提高。本研究的结果显示喹诺酮类药物耐药的空肠弯曲菌均具有已报道的促旋酶基因gyrA 86位突变,同时本研究新发现了一系列CmeABC外排泵启动子区突变,其中CmeR-Box的点插入和缺失可引起了空肠弯曲菌喹诺酮类药物的高水平耐药。本研究初步解析了华中地区空肠弯曲菌喹诺酮类药物耐药的分子机制,由此,临床将有针对性地预防类似耐药机制产生,将真正为人类健康保驾护航。
[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.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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