禽源大肠杆菌质粒介导氟喹诺酮类耐药基因的检测及中药消除耐药性研究

发布时间：2019-01-07 20:44

【摘要】：目前已报道的喹诺酮类耐药机制有多种,其中由质粒介导的耐药机制合理地解释了细菌耐药性的高突变率及多种抗生素的耐药相关性。质粒上携带的耐药基因只介导细菌对喹诺酮类药物的低水平耐药,但是能够诱导靶位基因突变,引起高水平耐药。因此,国内外研究者一直在积极寻找能够消除细菌耐药性的方法。中药因其天然环保、副作用小、应用安全等优点,受到了研究者的青睐。本研究采用PCR扩增技术对83株禽源大肠杆菌进行质粒介导的喹诺酮类耐药(PMQR)基因的检测,同时微量肉汤稀释法测定恩诺沙星(ENR)的最小抑菌浓度。对其中6株大肠杆菌进行10味中药的抑菌试验,选取五味子、黄连、黄芩3味中药对6株耐药菌进行消除培养,筛选消除菌并通过抑菌试验及PCR方法确定耐药性及耐药基因的消除情况,同时RT-qPCR分析消除前后耐药基因表达量的变化。研究结果如下：83株禽源大肠杆菌PMQR基因检出率为86.7%(qnr、aac (6')-Ib-cr、oqxAB分别为49.4%、83.1%、9.6%,未检出qnrC和qepA基因)。PMQR基因在禽源大肠杆菌中具有很高的流行率。抑菌试验显示有50株大肠杆菌对ENR耐药,占全部菌株的比例为60%。说明本研究中禽源大肠杆菌对喹诺酮类药物的耐药情况严重。10种中药中山楂、五倍子、金银花、黄柏、鱼腥草的抑菌效果不明显,艾叶和连翘抑菌效果相对较好,五味子、黄连、黄芩的抑菌效果最好。高、中、低剂量的五味子、黄连、黄芩提取液对6株大肠杆菌的耐药性消除率在0-100%之间,高剂量组的消除效果最好。说明中药具有消除耐药性的作用而且存在完全消除的可能。进一步研究发现,中药也具有消除耐药基因的作用,黄连对耐药基因的消除率最高可达100%。不过不同中药和不同菌株间的消除效果有差异,而且并不都是高剂量组消除效果最好。阳性对照SDS对某一基因的消除率因菌株的不同有差异。RT-qPCR结果显示3味中药消除培养后,耐药基因mRNA表达量均下降,表明耐药基因mRNA表达水平与细菌耐药的强度有关。
[Abstract]:There are many mechanisms of quinolone resistance reported at present, among which the plasmide-mediated drug resistance mechanism reasonably explains the high mutation rate of bacterial resistance and the correlation of resistance to various antibiotics. The drug resistance gene carried on the plasmid only mediates the low level resistance of bacteria to quinolones, but it can induce the mutation of target gene and induce high level of drug resistance. Therefore, domestic and foreign researchers have been actively looking for ways to eliminate bacterial resistance. Traditional Chinese medicine is favored by researchers because of its advantages of natural environmental protection, small side effects and safe application. In this study, 83 strains of avian Escherichia coli were detected for quinolone-resistant (PMQR) gene mediated by plasmid, and minimal inhibitory concentration of enrofloxacin (ENR) was determined by broth dilution method. Six strains of Escherichia coli were tested by 10 kinds of traditional Chinese medicine, and 6 strains of drug-resistant bacteria were eliminated and cultured with Schisandra chinensis, Rhizoma Coptidis and Radix Scutellariae. The elimination of drug resistance and resistance genes were determined by bacteriostasis test and PCR method, and the changes of expression of drug resistance genes before and after elimination were analyzed by RT-qPCR. The results were as follows: the detection rate of PMQR gene in 83 strains of avian Escherichia coli was 86.7% (qnr,aac (6') -Ib-cr,oqxAB was 49.4% and 83.1%, respectively. No qnrC and qepA). PMQR genes were found to be highly prevalent in avian Escherichia coli. Bacteriostasis test showed that 50 strains of Escherichia coli were resistant to ENR, accounting for 60% of the total strains. The results showed that the resistance of avian Escherichia coli to quinolones was serious. The bacteriostatic effects of Hawthorn, Galla chinensis, Flos Lonicerae, Cortex Phellodendri and Houttuynia cordata were not obvious in 10 kinds of traditional Chinese medicine. The antibacterial effect of Artemisia argyi and Forsythia suspensa was relatively good, and Schisandra chinensis. The bacteriostatic effect of Coptis chinensis and Scutellaria baicalensis is the best. High, medium and low doses of Schisandra chinensis, Coptis chinensis, and Scutellaria baicalensis extract had a drug resistance elimination rate of 0-100% to 6 strains of Escherichia coli. It shows that Chinese medicine has the function of eliminating drug resistance and it is possible to eliminate the drug resistance completely. Further study showed that Chinese herbal medicine also had the effect of eliminating drug resistance gene, and the highest elimination rate of resistance gene of Coptis chinensis could reach 100%. However, the elimination effect of different Chinese medicine and different strains is different, and not all of the high dose groups have the best elimination effect. The results of RT-qPCR showed that the mRNA expression level of drug resistance gene decreased after three Chinese herbs were eliminated, indicating that the mRNA expression level of drug resistance gene was related to the strength of bacterial drug resistance.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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