医院获得性及社区获得性肺炎克雷伯菌的比较基因组学研究

发布时间：2018-06-23 15:52

本文选题：肺炎克雷伯菌 + 医院获得性　；参考：《中国科学院北京基因组研究所》2016年博士论文

【摘要】：肺炎克雷伯菌是医院内三大条件致病菌之一,属于革兰氏阴性菌。肺炎克雷伯菌因其自身超强的基因组可塑性及携带的多重耐药质粒,常造成多种抗生素失效,导致多重耐药、泛耐药的发生,引起非常严重的感染和呼吸道疾病,严重者会危及生命。由于具有广泛的传播性和致病性的特点,肺炎克雷伯菌也被称为“超级细菌”。除长期存在于院内的肺炎克雷伯菌,近年来一些非院内的肺炎克雷伯菌即社区获得性肺炎克雷伯菌不断出现,也会引起人体呼吸道疾病,同样值得关注。本课题综合利用第一代、第二代以及第三代测序技术,针对医院获得性以及社区获得性肺炎克雷伯菌进行了较系统的研究。我们从7个地区收集了338株医院获得性肺炎克雷伯菌,利用第一代测序技术对每一株进行MLST (Multilocus Sequence Typing)序列测定及相应的最小抑菌浓度MIC (Minimum Inhibitory Concentration)检测,利用软件将338株MLST中的SNPs (Single Nucleotide Polymorphism)与MIC值进行关联分析,共找到两个基因中各自含有一个SNP位点与14种抗生素的耐药程度相关,另外两个基因中各自含有一个SNP位点与13种抗生素的耐药程度相关。我们利用第二代测序技术(Next Generation Sequencing)系统比较了医院获得性以及社区获得性肺炎克雷伯菌在基因类型以及拷贝数上的不同,发现了一些医院获得性和社区获得性各自特异的基因,并针对外排泵进行了较深入的分析。利用核心基因集对医院获得性以及社区获得性肺炎克雷伯菌构建系统发育树,发现它们在进化树中呈“混合”分布,说明医院获得性以及社区获得性肺炎克雷伯菌是在不同环境压力下生存的同一物种。同时,我们利用泛基因组学的方法分别构建了医院获得性和社区获得性的核心基因集和非必需基因集,通过比较发现社区获得性核心基因集比医院获得性在基因家族数量上多(社区获得性为2198,医院获得性为1752),这可能是由于环境不同导致的基因集增加或减少。在非必需基因集中,医院获得性和社区获得性也有各自独特的基因集,医院获得性主要与代谢相关,例如半乳糖转移酶、水解酶、以及DNA引物酶等,而社区获得性则主要与环境适应相关,如整合酶、外膜蛋白、以及葡萄糖基转移酶等。我们利用SMRT测序(Single Molecule Real Time Sequencing)对从上述NGS测序(Next Genome Sequencing)的株菌中挑选出两株医院获得性肺炎克雷伯菌进行三代精细图构建。经过基因组装、注释后分别得到一株耐药肺炎克雷伯菌H11的质粒基因组和肺炎克雷伯菌H39菌株基因组精细图。序列分析结果表明,肺炎克雷伯菌H11质粒除携带多种耐药基因,如blaTEM-1,blaSHV-12, sull, qacE delta 1, ere A, arr2, aac3等外,还具有3个耐重金属(碲、铅以及汞元素)的基因簇和两种独立的毒素抗毒素系统HipBA和RelBE。H39菌株则表现出更为独特的特征,虽然其分离自医院病人体内,但却与植物性肺炎克雷伯菌(即寄生于植物体内的肺炎克雷伯菌)342进化地位很相近；在单基因ParC系统发育树中,H39也被聚类到通常与环境相关的肺炎克雷伯菌Ⅲ类中。全基因组序列分析表明,H39共包含46个基因组岛及48个耐药基因。通过比较基因组学发现,H39具有完整的植物性肺炎克雷伯菌特有的固氮基因簇,说明‘H39很有可能是一株既可以感染动物同时也可以感染植物的特殊的肺炎克雷伯菌。
[Abstract]:Klebsiella pneumoniae, one of the three major pathogenic bacteria in the hospital, belongs to the Gram-negative bacteria. Klebsiella pneumoniae, because of its super strong genome plasticity and carrying multidrug resistant plasmids, often causes a variety of antibiotics failure, resulting in multiple drug resistance, widespread drug resistance, causing very serious infection and respiratory diseases. Life - threatening. Klebsiella pneumoniae is also known as "superbacteria" because of its widespread transmission and pathogenicity. In addition to chronic Klebsiella pneumoniae, a number of non hospital Klebsiella pneumoniae, community-acquired Klebsiella pneumoniae, can also cause respiratory disease in recent years. The first generation, second generation, and third generation sequencing technologies were used to systematically study the hospital acquired and community-acquired Klebsiella pneumoniae. We collected 338 Klebsiella pneumoniae from 7 regions and used the first generation sequencing technology to carry out MLST (Multilocus Seq) for each strain. Uence Typing) sequence determination and the corresponding minimum inhibitory concentration MIC (Minimum Inhibitory Concentration) detection, using software to associate SNPs (Single Nucleotide Polymorphism) in 338 strains of MLST with MIC values. A total of two genes were found to contain one locus and 14 antibiotics, the other two were related to the degree of resistance. Each gene contained one SNP locus in relation to the degree of resistance to 13 antibiotics. We compared hospital acquired and community-acquired Klebsiella pneumoniae in gene types and copies using the second generation sequencing technology (Next Generation Sequencing) system, and found some hospital acquired and community acquisition. An in-depth analysis of the specific genes and the external drainage pump was carried out. Using the core gene set to construct the phylogenetic tree of hospital acquired and community-acquired Klebsiella pneumoniae, it was found that they were "mixed" distribution in the evolutionary tree, indicating that the hospital acquired and community-acquired Klebsiella pneumoniae were under different environmental pressures. At the same time, we constructed the core gene set and non essential gene set of hospital acquired and community acquired by means of Pan genomics. Through comparison, we found that the core gene set of community acquired genes is more than the hospital acquired in the number of gene families (community acquired 2198 and hospital acquired 1752). This may be due to the increase or decrease of the gene set caused by different environment. In the non essential gene concentration, the hospital acquired and community acquired also have their own unique gene sets. The hospital acquired is mainly related to metabolism, such as galactose transferase, hydrolase, and DNA primers, while community acquisition is mainly related to environmental adaptation, such as Integrase, epicoprotein, and glucosaminotransferase. We used SMRT sequencing (Single Molecule Real Time Sequencing) to select two strains of Klebsiella pneumoniae from the above NGS sequencing (Next Genome Sequencing) strains for three generation of Klebsiella pneumoniae. After gene assembly, a drug resistance was obtained. The plasmid genome of Klebsiella pneumoniae H11 and the fine genome of Klebsiella pneumoniae strain H39 showed that Klebsiella pneumoniae H11 plasmid has 3 gene clusters, except for many resistant genes, such as blaTEM-1, blaSHV-12, sull, qacE delta 1, ere A, arr2, AAC3, etc. The two independent toxin antitoxin systems HipBA and RelBE.H39 showed more unique characteristics. Although they were isolated from hospital patients, they were closely related to the phylogenetic status of Klebsiella pneumoniae (that is, kleo Lei Bojun parasitic in plants); in the single gene ParC phylogeny tree, H39 was also clustered to the normal. The whole genome sequence analysis showed that H39 contains 46 genome islands and 48 resistant genes. Through comparative genomics, H39 has a complete nitrogen fixing gene cluster of Klebsiella pneumoniae, indicating that 'H39 can be an animal that can infect animals at the same time as well. A special Klebsiella pneumoniae that infects plants.
【学位授予单位】：中国科学院北京基因组研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R378;Q78

【相似文献】