纳米材料促进质粒介导的细菌耐药基因接合转移及机制研究

发布时间：2018-05-10 04:50

本文选题：纳米材料 + 细菌耐药性　；参考：《中国人民解放军军事医学科学院》2012年博士论文

【摘要】：细菌耐药性的扩散是一个严重威胁全球公共卫生安全的重要问题,尤其是2010年,在南亚和我国发现新的“超级细菌”后,人们更加关注细菌耐药性问题。细菌获得抗生素抗性基因,一方面是由于抗生素过度使用和滥用产生的抗生素选择压力造成细菌产生抗生素抗性突变；另一方面是细菌通过水平转移的方式获得抗生素抗性基因。后者是细菌获得耐药性的主要方式。细菌抗生素抗性基因往往编码在可移动质粒上,通过质粒接合转移在细菌间进行传播。质粒介导的细菌耐药基因接合转移对细菌耐药性快速扩散起到了至关重要作用。许多环境因素都能够影响质粒的接合转移过程。纳米材料由于具有新颖的物理、化学和生物学特性,已经被应用于多个领域,尤其是作为高效吸附剂和氧化剂,正用于水处理行业。然而,纳米材料在环境中的残留对环境、生物以及人体的负面影响正在引起人们重视。纳米材料也正在成为一种新的环境污染物。大量研究表明,纳米材料可产生大量活性氧,破坏细菌细胞膜,影响蛋白质和基因,载带DNA或RNA分子进入动物和植物细胞。现在,水环境中存在着大量耐药菌和纳米材料,纳米材料是否促进耐药基因在细菌间传播还不清楚。本研究基于以上事实提出,水中纳米材料可作用于细菌细胞膜和/或调控基因表达,促进细菌耐药基因接合转移的假说,并建立质粒介导的接合转移模型,研究水中常见纳米材料对细菌耐药基因接合转移的影响及规律,分析纳米材料作用过程中各种因素的影响规律,利用正交实验的方法分析其中的主要因素,从生物化学、细胞生物学和分子生物学多个角度探讨了纳米材料促进细菌耐药基因接合转移机制。主要研究结果如下：利用RP4质粒建立了四个接合转移模型,包括耐药基因在大肠杆菌种属内、大肠杆菌到沙门氏菌、大肠杆菌到粪肠球菌、粪肠球菌到粪肠球菌的转移；利用RK2质粒建立了耐药基因在大肠杆菌内的转移模型；利用pCF10建立了耐药基因在粪肠球菌内的转移模型。建立的6个耐药基因接合转移模型,既包括了耐药基因在种属内的接合转移,又包括了耐药基因的跨种属转移。研究结果表明,建立的6个耐药基因接合转移模型比较稳定,可以用来评价纳米材料对耐药基因接合转移的影响。纳米TiO2、纳米SiO2、纳米Fe2O3和纳米Al2O3都能促进RP4质粒从大肠杆菌到沙门菌的转移。纳米材料浓度为5mmol/L时,与空白对照和大颗粒材料相比,可使RP4接合转移分别提高89倍、10倍、20倍和142倍,纳米Al2O3的促进作用最强。不同接合转移体系中,纳米材料都有促进作用。5mmol/L纳米Al2O3可使RP4从大肠杆菌到大肠杆菌、革兰阴性菌到革兰阳性菌、粪肠球菌到粪肠球菌的接合频率转移分别增加200倍、50倍和100倍。纳米Al_2O_3对RK2和pCF10接合转移的促进作用可分别达到170倍和120倍。这些数据说明,纳米材料对接合质粒介导的耐药基因转移的促进作用可能是一个普遍现象。而且相同成分的大颗粒材料对耐药基因的接合转移无明显的促进作用,也说明对接合转移的促进作用来自纳米材料的纳米结构。以纳米Al_2O_3作为代表材料研究了接合转移中的影响因素。结果表明,随着浓度升高,纳米Al_2O_3对RP4接合转移促进作用增强。纳米Al_2O_3浓度为5mmol/L时达到最高,可以提高RP4接合转移100倍以上。纳米Al_2O_3浓度高于5mmol/L时,促进作用减弱,但是与空白对照组和大颗粒组比较,仍能显著促进RP4接合转移。纳米Al_2O_3的作用效果随浓度变化有峰值出现,原因可能是纳米材料,尤其是高浓度纳米材料能使细胞产生严重的氧化应激反应,造成细菌大量死亡。利用数学模型排除细菌死亡影响,5mmol/L纳米Al_2O_3能提高接合转移常数5个数量级。纳米Al_2O_3作用下,随着接合菌密度升高和接合时间延长,接合子数量逐渐增加,与空白对照和大颗粒组比较,增加显著。纳米Al_2O_3作用后,接合温度低于20℃时,随温度升高生成的接合子数量增加；温度高于20℃时,接合子生成数量增加不明显。接合体系的pH值对接合子生成无明显影响。在富营养条件下(LB培养基),接合子生成的数量更多。利用正交实验分析各因素的作用情况发现,各因素对接合子生成影响的重要次序为：初始菌密度纳米Al_2O_3浓度接合时间接合温度初始菌密度与纳米浓度的交互作用。纳米Al_2O_3浓度已成为仅次于初始菌密度的影响因素。在最适宜的条件下能够提高RP4在PBS中跨种属转移效率200倍,提高RP4在LB中同种属内的转移效率250倍。纳米Al_2O_3促进RP4接合转移机制研究发现：纳米Al_2O_3能促使细菌细胞产生大量OH·,激发细菌的氧化抗氧化反应。透射电镜与原子力显微镜的结果也表明,纳米Al_2O_3对细菌细胞膜产生影响,细胞膜表面变得不光滑,甚至部分细胞膜发生溶解。浓度越高,纳米Al_2O_3对细菌细胞膜影响越严重。大颗粒Al_2O_3却无类似的结果,说明纳米Al_2O_3的纳米结构是产生细胞膜损伤的主要因素。纳米Al_2O_3对细菌细胞膜的影响为供体菌和受体菌的细胞膜融合和DNA跨膜转运提供了便利条件。纳米Al_2O_3通过抑制korA和korB的mRNA表达,激活了trbBp基因的mRNA表达,进而促使供体菌与受体菌间形成“接合桥”,促进了接合转移的第一个过程。电镜结果也证实纳米Al_2O_3使形成的“接合桥”数量增加。同时纳米Al_2O_3通过抑制korB和trbA的mRNA的表达,激活了trfAp基因的mRNA表达,促使RP4质粒的转移与复制,促进了接合转移的第二个过程。总的来说,纳米材料能通过影响细菌细胞膜状态,影响与接合转移过程相关的调控基因mRNA表达而促进了接合质粒的接合转移。本课题利用生命科学的新技术,研究材料领域的纳米粒子对环境安全领域的细菌耐药性接合转移的影响规律与机制,是典型的学科交叉研究,也是相关学科的前沿领域,将开辟纳米材料环境安全研究的新领域。本课题的研究结果将进一步增加人们对细菌耐药性转移与获得的认识程度,丰富相关的科学知识,同时也将提高人们对环境中残留纳米材料所引起的环境与生态风险的认识。本研究提示我们应更加认真的对纳米材料进行相关的安全性评价和谨慎地使用纳米材料,以便控制或减少由纳米材料导致的环境中耐药菌的形成与增加。
[Abstract]:The spread of bacterial drug resistance is an important issue that poses a serious threat to global public health safety , especially in 2010 , after the discovery of new " super - bacteria " in South Asia and China , people pay more attention to bacterial drug resistance .
Bacterial antibiotic resistance genes are often encoded on a movable plasmid and propagated through a plasmid joint transfer between bacteria . The plasmid - mediated drug resistance gene - binding transfer plays a crucial role in the rapid diffusion of bacterial drug resistance . Many environmental factors can influence the junction transfer process of the plasmid .

Nano - materials have been used in many fields due to their novel physical , chemical and biological characteristics , especially as high - efficiency adsorbents and oxidants , and are being used in water treatment industries . However , nano - materials are becoming a new environmental pollutant . Nano - materials are also becoming a new environmental pollutant . Many researches have shown that nano - materials can produce large amounts of active oxygen , destroy bacterial cell membranes , affect proteins and genes , carry DNA or RNA molecules into animals and plant cells . Nowadays , there are a large number of drug - resistant bacteria and nano - materials in the water environment .

Based on the above facts , it is suggested that nano - materials in water can act on the expression of bacterial cell membrane and / or regulate gene expression , promote the transfer of drug - resistant genes , and establish a plasmid - mediated junction transfer model .

Four conjugative transfer models were constructed by using RP4 plasmid , including the transfer of drug resistance genes in E . coli , E . coli to Salmonella , E . coli , E . coli , E . coli , E . coli , E . coli , E . coli , E . coli , E . coli and E . coli .
The transfer model of resistance gene in E . coli was established by using RK2 plasmid .
The transfer model of drug resistant gene was established by using pCF10 . Six drug resistant gene transfer models were established , which included both drug resistance gene and drug resistance gene transfer . The results showed that the 6 drug resistant gene transfer models were stable and could be used to evaluate the effect of nano - material on drug resistance gene transfer .

Nano - TiO2 , nano - SiO2 , Fe2O3 and Al2O3 can promote the transfer of RP4 plasmid from E . coli to Salmonella .

The effects of nano Al _ 2O _ 3 on the bonding transfer of RP4 were studied . The results showed that the effect of nano - Al _ 2O _ 3 was higher than that of the blank control group and the large - particle group . The effect of nano - Al _ 2O _ 3 was higher than that of the control group and the large - particle group .
Under the condition of rich nutrition ( LB medium ) , the number of zygotes was increased . In the condition of rich nutrition ( LB medium ) , the number of zygotes was much more . It was found that the influence factors of the initial bacterial density were the interaction of initial bacterial density with nano - concentration . Under optimum conditions , the efficiency of RP4 was 200 - fold higher than that in PBS and 250 - fold increase in RP4 ' s transfer efficiency in LB medium .

The effect of nano Al _ 2O _ 3 on the cell membrane of bacteria was investigated . The results of TEM and AFM showed that the nano - Al _ 2O _ 3 had an effect on cell membrane damage .

In general , the nano - material can promote the junction transfer of the bonding plasmid by influencing the state of the cell membrane of the bacteria , affecting the expression of the regulating gene mRNA related to the junction transfer process .

The research results of this project will further increase people ' s understanding of bacterial drug resistance transfer and acquisition , enrich relevant scientific knowledge , and raise people ' s awareness of environmental and ecological risks caused by residual nano materials in the environment .

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：TB383.1;R318.08

【参考文献】