一株分离自黄河污泥的金黄色葡萄球菌菌株LZ-01的抗生素和重金属共抗性研究

发布时间：2018-04-30 13:04

  本文选题：金黄色葡萄球菌 + 共抗性　； 参考：《兰州大学》2016年博士论文

【摘要】：抗生素作为人类20世纪最伟大的发明之一,在医疗发展史上具有里程碑式的意义。它的出现挽救了无数人的生命,让许多患者免除病痛的折磨。截至目前使用在医疗卫生活动中的抗生素已有千余种,其中的许多种还被大量用于农业、畜牧和水产养殖过程中以提高产量。在上世纪60年代以MRSA(Methicillin-resistant Staphylococcus aureus)菌株的出现为标志,一系列广谱耐药的超级细菌引起了人们的恐慌,如NDM-1,VRE(Vancomycin-resistant Enterococci)和CA-MASA等。滥用抗生素曾经被认为是造成这些超级耐药细菌的罪魁祸首,但随后限制抗生素的使用以后并没有减少或延缓广谱抗药病菌的出现速度。同时研究发现抗生素抗性与重金属或者其它抗菌剂抗性之间存在关联。环境中的重金属可以迫使微生物保持其金属抗性,而抗生素抗性也会同时保存下来。由于在自然环境中金属的存在时间远远长于抗生素,由金属诱导和保持的抗生素抗性在环境中会逐渐富集,而如果这些抗性菌引起疾病就会面临无药可用的局面,并演变成为一场灾难。因此,研究微生物尤其是病原微生物对于重金属和抗生素的共抗性机制是十分重要的,有助于寻找减缓抗生素抗性扩散的途径。在本实验中从黄河受工业废水污染的淤泥中分离出了一株具有较高的重金属抗性的菌株,该菌株对金属铬(Cr)、铅(Pb)和锰(Mn)的MIC分别达到了6 m M,1 m M和6 m M。该菌株同时还有较高的抗生素抗性,对氨苄青霉素(Amp)和氯霉素(Chlo)的MIC分别达到了0.51 mg/m L和0.25 mg/m L。同时还发现该菌株的重金属抗性和抗生素抗性之间可以相互诱导,当使用0.50 m M的铬(Cr)或者锰(Mn)诱导之后,菌株对氨苄的抗性水平分别达到了2.5 mg/m L和1.6 mg/m L。当时用0.15 mg/m L的Amp或者0.05 mg/m L Chlo诱导之后,该菌株对铬(Cr)的抗性水平分别达到了10 m M和8 m M。另外,当重金属和抗生素同时出现时,菌株对重金属和抗生素的抗性都会提高。在培养基中铬(Cr)的浓度达到6 m M时,菌株同时对氨苄青霉素(Amp)的抗性可以达到0.75 mg/m L。通过对该菌株核糖体16S序列的分析,表明该菌株为金黄色葡萄球菌的一个亚种,我们将其命名为Staphylococcus aureus LZ-01。为了寻找S aureus LZ-01菌株对重金属和抗生素共抗性的机制,我们首先通过转录组测序的方法分析了可以响应金属铬压力的基因。结果表明在可预测和注释的2370个基因中有512个基因的表达明显上调。通过这些上调基因的功能分析表明S.aureus LZ-01菌株对铬的抗性途径可能有三条,包括胞外还原、胞内还原和外排作用,其中还原途径对抗性的贡献较弱,外排作用为主要抗性机制。然后在这些上调基因中筛选出了43个与抗生素抗性相关的基因,并对这些基因使用q RT-PCR的方法进行验证。在添加氨苄青霉素压力的情况下,检测的43个基因中仅有少数几个上调表达,结合生物信息学方法,最后确定操纵子emr AB为S.aureus LZ-01菌株对重金属和抗生素共抗性的关键基因。利用基因敲除和回补的方法,在实验中验证了emr A/B基因对S.aureus LZ-01菌株金属和抗生素共抗性的贡献。基因emr A敲除后菌株对铬(Cr)和氨苄青霉素(Amp)的抗性平均下降了80%,而emr B基因敲除后菌株对铬(Cr)的抗性下降了30%,对氨苄青霉素(Amp)的抗性下降了60%。emr A/B基因的敲除会使菌株丧失金属和抗生素之间的诱导抗性和交叉抗性。实验结果表明emr A/B基因对于S.aureus LZ-01菌株金属和抗生素共抗性十分重要,这种共抗性主要基于三种途径:一是外排泵作用,二是生物膜作用,三是调控作用。随后我们对emr A/B基因的调控以及Emr A/B蛋白的结构做了初步分析,研究表明存在一个二元调控系统,可以感受铬(Cr)和氨苄青霉素(Amp)的压力,并在emr A/B基因的启动子区对基因转录进行调控。Emr A/B蛋白结构的初步分析表明这两个蛋白可以形成复合体结构,构成一个外排泵。本实验使用具有临床价值的金黄色葡萄球菌作为研究对象,深入研究了该菌株产生金属和抗生素共抗性的机制。并且首次发掘了emr A/B基因在革兰氏阳性菌中的功能,提出了产生共抗性的具体途径。本实验的结果对于控制抗生素抗性基因的扩散、设计新型抗菌药物都有一定的借鉴意义,同时为消除环境污染、评估污染生态危害提供科学支撑和理论依据。
[Abstract]:As one of the greatest inventions of mankind in the twentieth Century, antibiotics have a milestone in the history of medical development. The emergence of antibiotics has saved the lives of countless people and exempted many patients from the pain. As of now, there are more than a thousand antibiotics used in medical and health activities, many of which are also used in agriculture and animal husbandry. And in the process of aquaculture to increase production. A series of broad-spectrum resistant superbacteria caused panic, such as NDM-1, VRE (Vancomycin-resistant Enterococci) and CA-MASA, as marked by the emergence of MRSA (Methicillin-resistant Staphylococcus aureus) strain in the 60s of last century. This is the culprit of these super resistant bacteria, but subsequent restrictions on the use of antibiotics did not reduce or postpone the emergence of broad-spectrum antifungal pathogens. As the existence of metals in the natural environment is much longer than that of antibiotics, the resistance of metals induced and maintained is gradually enriched in the environment, and if these resistant bacteria cause disease, it will face the situation of non drug availability and become a disaster. Therefore, the study of microorganisms is especially important. It is important that the co resistance mechanism of pathogenic microbes to heavy metals and antibiotics is very important and helps to find a way to slow the diffusion of antibiotic resistance. In this experiment, a strain with high heavy metal resistance was isolated from the sludge contaminated by industrial wastewater in the Yellow River. This strain has a M of metal chromium (Cr), lead (Pb) and manganese (Mn). IC reached 6 m M, 1 m M and 6 m M., which had high antibiotic resistance. The MIC of ampicillin (Amp) and chloramphenicol (Chlo) reached 0.51 mg/m L and 0.25 respectively. After the strain, the resistance level of the strain to ampicin reached 2.5 mg/m L and 1.6 mg/m L., respectively, when the 0.15 mg/m L Amp or 0.05 mg/m L Chlo were induced. The resistance level of the strain to chromium (Cr) reached 10 m and 8 respectively. When the concentration of chromium (Cr) reached 6 m M in the medium, the resistance of the strain to ampicillin (Amp) could reach 0.75 mg/m L. through the analysis of the ribosome 16S sequence of the strain, indicating that the strain was a subspecies of Staphylococcus aureus, and we named it Staphylococcus aureus LZ-01. in order to find S aureus. The mechanism of strain resistance to heavy metals and antibiotics, we first analyzed the genes that could respond to metal chromium pressure by a transcriptional sequencing method. The results showed that 512 of the 2370 genes in the predicable and annotated genes were obviously up-regulated. Through the power analysis of these up - regulated genes, the S.aureus LZ-01 strain was shown to chromium. There are three pathways in the resistance pathway, including extracellular reduction, intracellular reduction and exocytosis, in which the contribution of the reductive pathway is weak and the efflux is the main resistance mechanism. Then, 43 genes related to antibiotic resistance are screened out in these up-regulated genes, and the Q RT-PCR method is used to verify these genes. In the case of benzylpenicillin pressure, only a few of the 43 genes were up-regulated, combined with bioinformatics methods, the operon EMR AB was finally identified as the key gene for the co resistance of S.aureus LZ-01 to heavy metals and antibiotics. The EMR A/B gene was verified by the method of gene knockout and supplementation. The EMR A/B gene was verified to be S.aureus LZ-0 in the experiment. The contribution of 1 strain metal and antibiotic co resistance. After gene EMR A knockout, the resistance to chromium (Cr) and ampicillin (Amp) decreased by an average of 80%, while the resistance to chromium (Cr) decreased by 30% after the EMR B knockout, and the decrease of the resistance to ampicillin (Amp) by 60%.emr A/B gene knockout would cause the strain to lose metal and antibiotics The experimental results show that the EMR A/B gene is very important for the resistance of the S.aureus LZ-01 strain metal and antibiotic resistance. This co resistance is mainly based on three ways: one is the efflux pump, the two is the biofilm action, and the three is the regulatory effect. Then, we regulate the EMR A/B gene and the structure of Emr A/B protein. A preliminary analysis shows that there is a two element control system that can feel the pressure of chromium (Cr) and ampicillin (Amp), and a preliminary analysis of the structure of.Emr A/B protein in the promoter region of the EMR A/B gene indicates that the two proteins can form a complex structure and form an outer row pump. The clinical value of Staphylococcus aureus was studied, and the mechanism of producing resistance to antibiotics was studied. The function of EMR A/B gene in Gram-positive bacteria was discovered for the first time, and the specific way to produce CO resistance was proposed. The results of this experiment were set up to control the spread of antibiotic resistance genes. It is of certain reference significance for new antibacterial agents to be calculated, and it also provides scientific support and theoretical basis for eliminating environmental pollution and assessing the ecological hazards of pollution.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：X703;X172
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本文编号：1824669