低剂量抗生素刺激条件下耐药基因水平传播的机制研究

发布时间：2018-09-19 06:32

【摘要】：食品的安全性，不仅是民生大计，更是国家文明程度的重要象征。而食源性致病菌，作为食品安全最重要的风险因素之一已成为了全球性公共安全问题。而耐药基因在食物链中的传递在食品安全的研究中往往被忽略。由于抗生素在医疗领域和畜牧业中的大量使用，使一些残留抗生素随医疗垃圾和动物粪便流入到了环境当中并发生扩散，形成亚抑菌浓度的抗生素。然而，目前对于人们对于低剂量抗生素刺激下耐药基因水平传播的影响机制了解的尚不透彻，，这限制了人们对于食源微生物中的耐药基因向人体传递这一潜在风险的认知。本研究从食品安全角度出发，以食源性致病菌作为研究对象，围绕耐药基因水平传播的机理及抑制方法进行了系统和深入的研究，首先在四种不同抑菌机制的抗生素的分别刺激条件下，研究了对大肠杆菌耐药基因水平传播效率的影响，发现在低于50%MIC（最小抑菌浓度）的环丙沙星、链霉素、氨苄西林的刺激条件对于耐药基因水平传播具有促进作用。随后利用蛋白质双向电泳技术，研究了亚抑菌浓度抗生素刺激条件下发生表达差异的蛋白，在所有差异蛋白中有7种与细菌耐药基因水平传播相关，分别是gspE、tesB、lexA、ftsZ、ftsY、kdsD、sul1。经过实时荧光定量PCR对编码上述7种蛋白的基因在RNA转录水平验证，证实了其中2个基因gspE、tesB在四种不同抑菌机制抗生素的分别刺激下均发生了差异表达，且变化趋势一致。然后通过构建gspE和tesB基因表达载体，并在大肠杆菌体内进行诱导，发现tesB基因高效表达时能够有效抑制耐药基因的水平传播（p0.01）；而gspE基因的高效表达具有促进耐药基因水平传播的作用（p0.01）。研究结果说明，tesB基因和gspE基因是大肠杆菌耐药基因水平传播的过程的关键基因。这一发现对于科学地缓解耐药基因水平传播、抑制耐药基因在食物链中的传递提供可行的研究方向。
[Abstract]:Food safety is not only a livelihood plan, but also an important symbol of national civilization. Food-borne pathogens, as one of the most important risk factors for food safety, have become a global public safety problem. The transmission of drug-resistant genes in the food chain is often ignored in food safety studies. Because of the extensive use of antibiotics in the medical field and animal husbandry, some residual antibiotics flow into the environment with medical waste and animal feces and spread, forming antibiotics with sub-inhibitory concentration. However, the mechanism of drug resistance gene transmission induced by low dose antibiotics has not been fully understood, which limits the understanding of the potential risk of drug resistance gene transmission in food source microorganisms. In this study, from the point of view of food safety, the mechanism of drug resistance gene transmission and inhibition methods were studied systematically and deeply. In this paper, we first studied the effect of four different antibiotic stimuli on the transmission efficiency of Escherichia coli resistance gene. We found that ciprofloxacin and streptomycin were lower than 50%MIC (minimum inhibitory concentration). The stimulation conditions of ampicillin can promote the level transmission of drug resistance gene. Then protein two-dimensional electrophoresis technique was used to study the expression of differentially expressed proteins under the stimulation of antibiotic subinhibitory concentration. Seven of the differential proteins were related to the horizontal transmission of bacterial drug resistance genes (gspE,tesB,lexA,ftsZ,ftsY,kdsD,sul1.). By real-time fluorescence quantitative PCR, the genes encoding the seven proteins were verified at the RNA transcription level. It was confirmed that two of the genes were differentially expressed under the stimulation of four antimicrobial mechanisms, and the change trend was the same. Then the expression vector of gspE and tesB gene was constructed and induced in E. coli. It was found that the high expression of tesB gene could effectively inhibit the horizontal transmission of drug resistance gene (p0.01). The high expression of gspE gene can promote the level transmission of drug resistance gene (p0.01). The results showed that the genes of TSE B and gspE were the key genes in the horizontal transmission of drug resistance genes of Escherichia coli. This finding provides a feasible research direction for scientific mitigation of drug resistance gene transmission and inhibition of drug resistance gene transmission in the food chain.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：TS201.6;R155.5

【相似文献】