多重耐药肺炎克雷伯菌可移动基因组的研究

发布时间：2018-04-15 22:08

  本文选题：肺炎克雷伯菌 + 多重耐药　； 参考：《上海交通大学》2015年博士论文

【摘要】：微生物能够通过基因水平转移的方式获得致病和耐药等相关基因。质粒、插入序列(insertion sequence,IS)、转座子、整合子、整合性接合元件(integrative and conjugative element,ICE)和其它基因组岛等可移动遗传元件是推动这一过程的关键遗传介质,这些可移动遗传元件组成了可移动基因组(mobile genome,mobilome)。肺炎克雷伯菌(Klebsiella pneumoniae)为条件致病菌,目前因引发严重耐药问题引起广泛的重视。肺炎克雷伯菌HS11286是2011年从上海某医院临床样本中分离得到的耐碳青霉烯类抗生素的菌株,属于ST11型,全基因组已完整测定。本文对其可移动基因组进行了识别和研究,包括6个质粒、49个插入序列、9个转座子、1个Class I整合子、2个ICE和7个前噬菌体。IS、转座子和整合子多位于质粒上。而HS11286携带超过20个预测的耐药基因,其中17个也位于质粒上,包括碳青霉烯类抗生素抗性基因blaKPC-2。遗传背景分析表明转座子Tn3、Tn1721、Tn5393,整合子ΔIn2和插入序列ISCR3、ISCR2对耐药基因的获得有重要贡献。其它插入元件对耐药遗传背景进化有重要作用,其中IS26是一个高度活跃的、推动该进化进程的插入序列。进而,利用λred重组和Flp-FRT重组突变方法对blaKPC-2和一段26 kb含有13个耐药基因的区域进行敲除。抗菌药物敏感性试验表明,缺失突变株对临床常用抗菌药物的敏感性大幅提高,与突变后基因型相符,至此也成功构建了可用于安全遗传操作的模式菌株。此外,对HS11286的ICE进行了研究。其中,ICEKpnHS11286-1带有一个已知的毒力岛;而ICEKpnHS11286-2功能未知。本研究开发了一种利用sacB反选择的ICE敲除策略,通过筛选自然丢失该元件的方法获得目标突变株。应用该方法成功获得ICEKpnHS11286-1缺失的突变株。但发现该突变株在染色体上发生了非预期突变。结合序列分析和实验方法对突变进行了准确定位,确定这种非预期突变是一种染色体重组现象,与ICEKpnHS11286-1重组位点的序列相同,可能是由该ICE整合酶介导的,但相关机制有待于研究。ICE是一种广泛存在于细菌中的可自主移动元件,自身编码重组和接合转移所需的全部功能,携带多样化附属功能基因,是耐药和致病基因播散的一种有效介质,目前尚缺乏系统研究。我们通过文献挖掘方式和生物信息学方法收集了428个ICE,并进行分类。ICE在DNA水平呈现多样性,但在ICE家族内进行蛋白序列比较发现同一家族ICE拥有保守骨架和可变区域。通过基于隐马尔科夫模型特征谱(profile hidden Markov model,profile HMM)的方法对保守蛋白进一步比较,发现不同家族之间的ICE的重组和接合转移模块也具有保守特征。进而,针对ICE整合模块和接合转移模块收集和建立profile HMM数据集,尤其对与接合转移相关的IV型分泌系统(T4SS)进行重点挖掘,在531株已测序细菌基因组中识别出的811个T4SS,并进行了功能分析和系统分类。以此为基础,提出一个基于生物学特征的ICE数学描述模型和识别策略。本研究将有助于我们深入理解和研究细菌遗传多样性、耐药和致病机理,以期能够为发展新一代预防性和治疗性方法提供思路。
[Abstract]:Microorganisms can through horizontal gene transfer obtained pathogenic and drug resistance related gene. The plasmid insertion sequence (insertion, sequence, IS), transposons and integrons, integrated joint element (integrative and conjugative element, ICE) and other genomic island mobile genetic elements is the key to promote genetic mediators of this process. These mobile genetic elements form mobile genome (mobile genome, mobilome). Klebsiella pneumoniae (Klebsiella pneumoniae) as opportunistic pathogens, at present due to cause serious problems of drug resistance of Klebsiella pneumoniae. HS11286 was isolated carbapenem resistant strains in Shanghai province the hospital clinical samples in 2011, belongs to ST11 type, the whole genome has been determined. The complete mobile genome were identified and studied, including 6 plasmid, 49 insertion sequence, 9 Transposon, 1 Class I integron, 2 ICE and 7.IS prophage, transposon and integron located in plasmid. HS11286 carrying resistance genes more than 20 predicted, 17 of which are located on plasmids, including analysis of carbapenem resistance gene showed that the genetic background of blaKPC-2. transposon Tn3, Tn1721, Tn5393, In2 and integron insertion sequence ISCR3, ISCR2 on resistance genes have important contributions. Other insertion element plays an important role in the genetic background of resistance evolution, where IS26 is a highly active, promote the evolution of the inserted sequence. Then, using a recombinant red and recombinant Flp-FRT the mutation method contains 13 resistance genes of blaKPC-2 and a 26 KB region by knockout. Antimicrobial susceptibility test showed that the mutant sensitivity to antibiotics increases, consistent with the type for base mutation, to This is also successfully constructed can be used to model strain genetic operation safety. In addition, the HS11286 ICE was studied. The ICEKpnHS11286-1 pathogenicity island with a known and unknown function; ICEKpnHS11286-2. This study developed a way to use sacB anti choice ICE knockout strategy, target mutant screening method by nature the loss of the elements. The method was successfully used to obtain ICEKpnHS11286-1 deletion mutant. The mutant but found in chromosomes had unintended mutations. Combined with sequence analysis and experimental methods for the accurate position of mutation, determine the expected mutation is a chromosome recombination, ICEKpnHS11286-1 sequence and recombination sites may be the same. By the ICE integrase mediated, but the mechanism remains to be studied and.ICE is a widely existed in bacteria in the autonomous mobile element, its encoding weight Joint group and all functions required for the transfer, with diversified subsidiary function gene, is a kind of effective medium resistant gene dissemination and pathogenesis, there is still a lack of system research. Through literature mining and bioinformatics methods to collect 428 ICE,.ICE classification and diversity at the DNA level, but the protein sequence in the ICE family that same family ICE has the conservative and variable regions. Through the framework of hidden Markov model based on characteristic spectrum (profile hidden Markov model, profile HMM) for further comparison of a conserved protein, found in different family between recombinant ICE and conjugative transfer module also has conservative characteristics. Then, aiming at the ICE integration module and conjugative transfer module to collect and establish the profile HMM data set, especially related to type IV and conjugation secretion system (T4SS) to focus on mining, in the 531 strains The 811 T4SS identified in sequenced bacterial genome, and analyses the function and classification system. On this basis, put forward a description model and identification methods of biological characteristics of ICE based on mathematics. This study will help us to understand and study the genetic diversity of bacteria, drug resistance and pathogenic mechanism, in order to to provide ideas for the development of a new generation of preventive and therapeutic methods.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R378
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本文编号：1756001