大肠杆菌O157:H7对不同土壤矿物响应的分子生物学机制
发布时间：2018-09-11 12:40
【摘要】：全球养殖业每年产生100~1000亿吨的畜禽粪便,大部分畜禽粪便未经有效处理直接作为肥料施入农田,致使粪便中大量病原菌进入土壤环境,对人类健康和食品安全构成极大威胁。病原菌进入土壤后,会吸附在土壤固相组分上,形成单个微菌落或生物被膜,从而在土壤中持久存在。因此,阐明病原菌对土壤矿物响应的分子生物学机制,有助于深刻认识土壤中病原菌的存活行为,同时对于控制及修复病原菌污染土壤具有重要理论和实际意义。本文选取了致病菌株大肠杆菌O157:H7为实验菌株,研究了土壤常见矿物铁氧化物(针铁矿、赤铁矿)、铝氧化物(勃姆石)、层状硅酸盐矿物(高岭石、蒙脱石、伊利石)对大肠杆菌O157:H7有关存活及毒性等基因表达的影响。主要结果有:1.不同土壤矿物对大肠杆菌O157:H7存活的影响平板计数结果显示,与对照相比,在10 mmol/L KCl溶液中(p H=6.8),不同土壤矿物对大肠杆菌O157:H7的可培养数量没有显著影响,而结合荧光显微镜和LIVE/DEAD染色技术,发现不同土壤矿物与大肠杆菌O157:H7结合后均可破坏大肠杆菌O157:H7细胞膜的完整性,从而导致细胞失活。胞内活性氧(ROS)结果显示,土壤矿物能诱导细菌胞内ROS的产生,其中铁铝氧化物(针铁矿、赤铁矿、勃姆石)体系中,细菌胞内ROS的含量是对照组的3.7~4.2倍;而层状硅酸盐(高岭石、蒙脱石、伊利石)体系中,细菌胞内ROS的含量仅为对照组的1.8~2.1倍,表明铁铝氧化物对胞内活性氧的诱导作用强于层状硅酸盐矿物。2.不同土壤矿物对大肠杆菌O157:H7毒性、运动等基因表达的影响大肠杆菌O157:H7的关键致病因子志贺毒素(Stx)有两种主要形式:Stx1(stx1基因编码)和Stx2(stx2基因编码)。定量PCR结果显示,土壤矿物可抑制大肠杆菌O157:H7的stx1、stx2基因表达,而对细菌鞭毛调控基因flh D及编码鞭毛蛋白的fli C基因的表达没有显著影响。土壤矿物可引起自由基清除酶基因sod B及与DNA修复相关的rec A基因的大量表达,表明土壤矿物诱导的胞内ROS对细菌DNA造成了氧化损伤。
[Abstract]:100 ~ 100 billion tons of livestock and poultry manure are produced every year in the global breeding industry. Most of the animal manure is directly applied as fertilizer to farmland without effective treatment, which results in a large number of pathogenic bacteria in feces entering the soil environment, which poses a great threat to human health and food safety. When the pathogen enters the soil, it will adsorb on the solid component of the soil and form a single microbacterium colony or biofilm, which will exist in the soil for a long time. Therefore, to elucidate the molecular biological mechanism of pathogen response to soil minerals is helpful to understand the survival behavior of pathogens in soil, and has important theoretical and practical significance in controlling and remediation of soil contaminated by pathogens. In this paper, the common mineral iron oxides (goethite, hematite), aluminum oxide (Bohmite), layered silicate minerals (kaolinite, montmorillonite) in soil were studied by using Escherichia coli O157:H7 as the experimental strain. The effect of Illite on the survival and toxicity of Escherichia coli O157:H7. The main result was 1: 1. The effects of different soil minerals on the survival of Escherichia coli O157:H7 showed that, compared with the control, in 10 mmol/L KCl solution (pH6. 8), different soil minerals had no significant effect on the culturable quantity of Escherichia coli O157:H7. Combined with fluorescence microscope and LIVE/DEAD staining, it was found that different soil minerals combined with Escherichia coli O157:H7 could destroy the integrity of Escherichia coli O157:H7 cell membrane, resulting in cell inactivation. The results of intracellular reactive oxygen species (ROS) showed that soil minerals could induce the production of intracellular ROS of bacteria, and the content of ROS in ferric aluminum oxide (goethite, hematite, Boehmite) system was 3.740. 2 times of that in control group. In the layered silicate (kaolinite, montmorillonite, Illite) system, the intracellular ROS content of bacteria was only 1.8 ~ 2. 1 times of that of the control group, indicating that the inductive effect of iron and aluminum oxide on intracellular reactive oxygen species was stronger than that of layered silicate mineral. 2. Effects of different soil Minerals on the expression of O157:H7 and exercise genes in Escherichia coli (Stx), the key pathogenic factor of E. coli O157:H7, (Stx) has two main forms: stx1 gene encoding (stx1 gene coding) and Stx2 (stx2 gene coding). The results of quantitative PCR showed that soil minerals could inhibit the expression of O157:H7 stx1,stx2 gene in E. coli, but had no significant effect on the expression of flh D gene and fli C gene encoding flagellin. Soil minerals could induce the expression of free radical scavenging enzyme gene sod B and rec A gene associated with DNA repair, indicating that intracellular ROS induced by soil minerals caused oxidative damage to bacterial DNA.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
本文编号：2236716
[Abstract]:100 ~ 100 billion tons of livestock and poultry manure are produced every year in the global breeding industry. Most of the animal manure is directly applied as fertilizer to farmland without effective treatment, which results in a large number of pathogenic bacteria in feces entering the soil environment, which poses a great threat to human health and food safety. When the pathogen enters the soil, it will adsorb on the solid component of the soil and form a single microbacterium colony or biofilm, which will exist in the soil for a long time. Therefore, to elucidate the molecular biological mechanism of pathogen response to soil minerals is helpful to understand the survival behavior of pathogens in soil, and has important theoretical and practical significance in controlling and remediation of soil contaminated by pathogens. In this paper, the common mineral iron oxides (goethite, hematite), aluminum oxide (Bohmite), layered silicate minerals (kaolinite, montmorillonite) in soil were studied by using Escherichia coli O157:H7 as the experimental strain. The effect of Illite on the survival and toxicity of Escherichia coli O157:H7. The main result was 1: 1. The effects of different soil minerals on the survival of Escherichia coli O157:H7 showed that, compared with the control, in 10 mmol/L KCl solution (pH6. 8), different soil minerals had no significant effect on the culturable quantity of Escherichia coli O157:H7. Combined with fluorescence microscope and LIVE/DEAD staining, it was found that different soil minerals combined with Escherichia coli O157:H7 could destroy the integrity of Escherichia coli O157:H7 cell membrane, resulting in cell inactivation. The results of intracellular reactive oxygen species (ROS) showed that soil minerals could induce the production of intracellular ROS of bacteria, and the content of ROS in ferric aluminum oxide (goethite, hematite, Boehmite) system was 3.740. 2 times of that in control group. In the layered silicate (kaolinite, montmorillonite, Illite) system, the intracellular ROS content of bacteria was only 1.8 ~ 2. 1 times of that of the control group, indicating that the inductive effect of iron and aluminum oxide on intracellular reactive oxygen species was stronger than that of layered silicate mineral. 2. Effects of different soil Minerals on the expression of O157:H7 and exercise genes in Escherichia coli (Stx), the key pathogenic factor of E. coli O157:H7, (Stx) has two main forms: stx1 gene encoding (stx1 gene coding) and Stx2 (stx2 gene coding). The results of quantitative PCR showed that soil minerals could inhibit the expression of O157:H7 stx1,stx2 gene in E. coli, but had no significant effect on the expression of flh D gene and fli C gene encoding flagellin. Soil minerals could induce the expression of free radical scavenging enzyme gene sod B and rec A gene associated with DNA repair, indicating that intracellular ROS induced by soil minerals caused oxidative damage to bacterial DNA.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61
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