本文選題：肺炎克雷伯菌 + 多重耐藥��； 參考：《上海交通大學(xué)》2015年博士論文

【摘要】：微生物能夠通過基因水平轉(zhuǎn)移的方式獲得致病和耐藥等相關(guān)基因。質(zhì)粒、插入序列(insertion sequence,IS)、轉(zhuǎn)座子、整合子、整合性接合元件(integrative and conjugative element,ICE)和其它基因組島等可移動(dòng)遺傳元件是推動(dòng)這一過程的關(guān)鍵遺傳介質(zhì),這些可移動(dòng)遺傳元件組成了可移動(dòng)基因組(mobile genome,mobilome)。肺炎克雷伯菌(Klebsiella pneumoniae)為條件致病菌,目前因引發(fā)嚴(yán)重耐藥問題引起廣泛的重視。肺炎克雷伯菌HS11286是2011年從上海某醫(yī)院臨床樣本中分離得到的耐碳青霉烯類抗生素的菌株,屬于ST11型,全基因組已完整測(cè)定。本文對(duì)其可移動(dòng)基因組進(jìn)行了識(shí)別和研究,包括6個(gè)質(zhì)粒、49個(gè)插入序列、9個(gè)轉(zhuǎn)座子、1個(gè)Class I整合子、2個(gè)ICE和7個(gè)前噬菌體。IS、轉(zhuǎn)座子和整合子多位于質(zhì)粒上。而HS11286攜帶超過20個(gè)預(yù)測(cè)的耐藥基因,其中17個(gè)也位于質(zhì)粒上,包括碳青霉烯類抗生素抗性基因blaKPC-2。遺傳背景分析表明轉(zhuǎn)座子Tn3、Tn1721、Tn5393,整合子ΔIn2和插入序列ISCR3、ISCR2對(duì)耐藥基因的獲得有重要貢獻(xiàn)。其它插入元件對(duì)耐藥遺傳背景進(jìn)化有重要作用,其中IS26是一個(gè)高度活躍的、推動(dòng)該進(jìn)化進(jìn)程的插入序列。進(jìn)而,利用λred重組和Flp-FRT重組突變方法對(duì)blaKPC-2和一段26 kb含有13個(gè)耐藥基因的區(qū)域進(jìn)行敲除。抗菌藥物敏感性試驗(yàn)表明,缺失突變株對(duì)臨床常用抗菌藥物的敏感性大幅提高,與突變后基因型相符,至此也成功構(gòu)建了可用于安全遺傳操作的模式菌株。此外,對(duì)HS11286的ICE進(jìn)行了研究。其中,ICEKpnHS11286-1帶有一個(gè)已知的毒力島;而ICEKpnHS11286-2功能未知。本研究開發(fā)了一種利用sacB反選擇的ICE敲除策略,通過篩選自然丟失該元件的方法獲得目標(biāo)突變株。應(yīng)用該方法成功獲得ICEKpnHS11286-1缺失的突變株。但發(fā)現(xiàn)該突變株在染色體上發(fā)生了非預(yù)期突變。結(jié)合序列分析和實(shí)驗(yàn)方法對(duì)突變進(jìn)行了準(zhǔn)確定位,確定這種非預(yù)期突變是一種染色體重組現(xiàn)象,與ICEKpnHS11286-1重組位點(diǎn)的序列相同,可能是由該ICE整合酶介導(dǎo)的,但相關(guān)機(jī)制有待于研究。ICE是一種廣泛存在于細(xì)菌中的可自主移動(dòng)元件,自身編碼重組和接合轉(zhuǎn)移所需的全部功能,攜帶多樣化附屬功能基因,是耐藥和致病基因播散的一種有效介質(zhì),目前尚缺乏系統(tǒng)研究。我們通過文獻(xiàn)挖掘方式和生物信息學(xué)方法收集了428個(gè)ICE,并進(jìn)行分類。ICE在DNA水平呈現(xiàn)多樣性,但在ICE家族內(nèi)進(jìn)行蛋白序列比較發(fā)現(xiàn)同一家族ICE擁有保守骨架和可變區(qū)域。通過基于隱馬爾科夫模型特征譜(profile hidden Markov model,profile HMM)的方法對(duì)保守蛋白進(jìn)一步比較,發(fā)現(xiàn)不同家族之間的ICE的重組和接合轉(zhuǎn)移模塊也具有保守特征。進(jìn)而,針對(duì)ICE整合模塊和接合轉(zhuǎn)移模塊收集和建立profile HMM數(shù)據(jù)集,尤其對(duì)與接合轉(zhuǎn)移相關(guān)的IV型分泌系統(tǒng)(T4SS)進(jìn)行重點(diǎn)挖掘,在531株已測(cè)序細(xì)菌基因組中識(shí)別出的811個(gè)T4SS,并進(jìn)行了功能分析和系統(tǒng)分類。以此為基礎(chǔ),提出一個(gè)基于生物學(xué)特征的ICE數(shù)學(xué)描述模型和識(shí)別策略。本研究將有助于我們深入理解和研究細(xì)菌遺傳多樣性、耐藥和致病機(jī)理,以期能夠?yàn)榘l(fā)展新一代預(yù)防性和治療性方法提供思路。
[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.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R378
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本文編號(hào)：1756001