Taipei Medical University Institutional Repository:Item 987654321/61023
English  |  正體中文  |  简体中文  |  全文筆數/總筆數 : 45069/58245 (77%)
造訪人次 : 2372528      線上人數 : 175
RC Version 7.0 © Powered By DSPACE, MIT. Enhanced by NTU Library IR team.
搜尋範圍 查詢小技巧:
  • 您可在西文檢索詞彙前後加上"雙引號",以獲取較精準的檢索結果
  • 若欲以作者姓名搜尋,建議至進階搜尋限定作者欄位,可獲得較完整資料
  • 進階搜尋
    請使用永久網址來引用或連結此文件: http://libir.tmu.edu.tw/handle/987654321/61023


    題名: 次世代定序偵測沙門氏桿菌之抗生素抗性
    Using Next Generation Sequencing to Detect Antimicrobial Resistance of Salmonella
    作者: 周宛萱
    Chou, Wan-Hsuan
    貢獻者: 臨床藥物基因體學暨蛋白質體學碩士學位學程
    關鍵詞: 沙門氏菌;抗生素抗性;次世代定序
    Salmonella;antimicrobial resistance;next generation sequencing
    日期: 2015
    上傳時間: 2021-11-11 15:36:08 (UTC+8)
    摘要: 非傷寒性沙門氏菌(NTS)是最常見的細菌性腸道病原體之一。他在人類及其他動物都有顯著的發病率和死亡率。然而,其抗生素抗性對於此種細菌感染的臨床治療帶來莫大的挑戰。此外,傳統的藥敏試驗曠日費時且需要許多具有專業訓練的技術人員執行,因而拖慢臨床決策效率。由於細菌能夠透過自身基因突變或是從外界獲取抗藥性基因而對藥物產生抗性,本研究利用次世代定序(NGS)來識別潛在的基因標記以檢測細菌的抗藥性。我們的目標是提供一個執行策略,用以開發高效率、低成本的診斷工具以檢測細菌抗藥性。
    首先,我們從雙和醫院小兒胃腸科病房取得19位的病人同意後,蒐集他們的糞便檢體並分離出其感染的沙門氏菌作為發現集,其中包含8隻對ampicillin 有抗藥性、2隻有ceftriaxone抗性,以及1隻對ciprofloxacin有抗性。我們一方面將對分離出的細菌株進行藥敏試驗,一方面萃取他們的DNA進行次世代定序。經過數據處理後,我們將發現集的DNA序列與線上抗藥性基因資料庫ResFinder比對。我們發現了存在有blaTEM176, blaCMY2, blaCARB2, blaTEM1B, ampC 或 blaPSE1 任一基因,與對ampicillin的抗藥性具有關聯性;而blaCMY2 and ampC 則只存在於具有ceftriaxone抗性的細菌株。接下來我們針對這些基因設計引子並蒐集了另外30位病人的檢體做為驗證集,利用聚合酶連鎖反應(PCR)檢測這些基因是否存在於驗證集細菌株的DNA中。最後,我們針對結果,計算以這些基因的存在作為預測抗生素抗性的敏感性及特異性。利用存在有blaTEM176, blaCMY2, blaCARB2, blaTEM1B, ampC 或 blaPSE1 任一基因作為ampicillin抗性的預測,其敏感性高達90%,特異性則有80%。
    此外,我們透過文獻搜尋,找到過去報導與抗生素抗性相關的基因突變,並將發現集菌株的DNA序列利用BLAST與之比對。在具有ciprofloxacin抗性的菌株中,我們發現了兩個未曾被發現過的突變點:一是parC g.1307delA ,此讀框轉移突變會造成轉譯出的胺基酸序列與原本完全不同;另外則是parE g.1031C>T,會造成轉譯出的胺基酸從丙胺酸變成纈胺酸。同時,兩個在先前文獻報導過的突變點在本次研究也被再次觀察到 (gyrA g.248C>T 和 parC g.170C>G)。此外,另兩個先前報導過與ciprofloxacin抗性相關的突變在本次研究中同時在對ciprofloxacin具抗性及不具抗性的菌株都有觀察到 (parC g.170C>G 和gyrB g.2044del),暗示其可能不是造成細菌ciprofloxacin抗性的主要變異。
    總結而言,本研究提供一個執行策略用以發展可靠、有效且較低成本的方法,以檢測抗生素抗性。若未來能應用到全球各地,或許能減緩抗生素抗性的問題。另外,本研究新發現與ciprofloxacin抗性相關的突變,需進一步製造基因重組細菌,以確認真正造成ciprofloxacin抗性的突變為何。
    Non-typhoid Salmonella (NTS) is one of the most common bacterial enteropathogens worldwide and leads to significant morbidity and mortality in humans and animals. However, antibiotic resistance in NTS is a serious problem worldwide. Besides, traditional antimicrobial susceptibility tests are labor-demanding and time-consuming, making clinical decisions inefficient. Since bacteria are able to acquire resistance through gene exchange or mutation on drug target genes, we conducted this study using the next-generation sequencing (NGS) to identify potential markers to detect antimicrobial resistance and novel genetic mutations in NTS. We aim to provide a workflow to develop efficient and affordable diagnosis of antimicrobial resistance.
    First, a total of 19 Salmonella clinical isolates were collected in TMU-SHH, including 8 ampicillin-resistant, 2 ceftriaxone-resistant and 1 ciprofloxacin-resistant strains. The bacterial DNAs were extracted from these strains and NGS was performed using the Illumina® MiSeq sequencer. The big genomic data were analyzed to examine the presence of plasmid-mediated antimicrobial resistance genes reported in ResFinder, which are potential markers to detect antimicrobial resistance. In this discovery set, our data demonstrated that existence of blaTEM176, blaCMY2, blaCARB2, blaTEM1B, ampC or blaPSE1 is associated with ampicillin resistance. Furthermore, presence of blaCMY2 and ampC were found mutually exclusively in the 2 ceftriaxone-resistant strains. Next, we designed primers of the genes detected and validated it on the validation set of another 30 isolates with a double-blinded study design. Using the existence of blaTEM176, blaCMY2, blaCARB2, blaTEM1B, ampC or blaPSE1 is shown to have the high sensitivity (90%) and specificity (80%) to predict ampicillin resistance.
    Furthermore, genes of which there were mutations reported in previous studies correlating with antimicrobial resistance were also analyzed using BLAST. In the ciprofloxacin-resistant strain, we discovered one novel frameshift mutation in parC g.1307delA that leads to totally different translation in amino acid sequences and one novel point mutation in parE g.1031C>T that leads to amino acid change from Alanine to Valine. Besides, 2 previously reported point mutations (gyrA g.248C>T and parC g.170C>G) were also observed in this ciprofloxacin-resistant strain. Furthermore, the point mutation in parC g.170C>G and another previously reported point mutation in gyrB g.2044del were also found in one of the 18 ciprofloxacin-susceptible strains, suggesting that parC g.170C>G and gyrB g.2044del are not major genetic mutations contributing to ciprofloxacin resistance.
    In conclusion, we provide a workflow to develop reliable, efficient and affordable way to detect antimicrobial resistance. If applied worldwide, it may be a promising strategy to lessen the worsening situation of antimicrobial resistance. Furthermore, a novel genetic frameshift mutation in parC g.1307delA and point mutation in parE g.1031C>T are identified as potentially decisive genetic loci to ciprofloxacin resistance in NTS, which warrants further confirmation in their phenotype by generating a recombinant mutated NTS strain.
    描述: Non-typhoid Salmonella (NTS) is one of the most common bacterial enteropathogens worldwide and leads to significant morbidity and mortality in humans and animals. However, antibiotic resistance in NTS is a serious problem worldwide. Besides, traditional antimicrobial susceptibility tests are labor-demanding and time-consuming, making clinical decisions inefficient. Since bacteria are able to acquire resistance through gene exchange or mutation on drug target genes, we conducted this study using the next-generation sequencing (NGS) to identify potential markers to detect antimicrobial resistance and novel genetic mutations in NTS. We aim to provide a workflow to develop efficient and affordable diagnosis of antimicrobial resistance.
    First, a total of 19 Salmonella clinical isolates were collected in TMU-SHH, including 8 ampicillin-resistant, 2 ceftriaxone-resistant and 1 ciprofloxacin-resistant strains. The bacterial DNAs were extracted from these strains and NGS was performed using the Illumina® MiSeq sequencer. The big genomic data were analyzed to examine the presence of plasmid-mediated antimicrobial resistance genes reported in ResFinder, which are potential markers to detect antimicrobial resistance. In this discovery set, our data demonstrated that existence of blaTEM176, blaCMY2, blaCARB2, blaTEM1B, ampC or blaPSE1 is associated with ampicillin resistance. Furthermore, presence of blaCMY2 and ampC were found mutually exclusively in the 2 ceftriaxone-resistant strains. Next, we designed primers of the genes detected and validated it on the validation set of another 30 isolates with a double-blinded study design. Using the existence of blaTEM176, blaCMY2, blaCARB2, blaTEM1B, ampC or blaPSE1 is shown to have the high sensitivity (90%) and specificity (80%) to predict ampicillin resistance.
    Furthermore, genes of which there were mutations reported in previous studies correlating with antimicrobial resistance were also analyzed using BLAST. In the ciprofloxacin-resistant strain, we discovered one novel frameshift mutation in parC g.1307delA that leads to totally different translation in amino acid sequences and one novel point mutation in parE g.1031C>T that leads to amino acid change from Alanine to Valine. Besides, 2 previously reported point mutations (gyrA g.248C>T and parC g.170C>G) were also observed in this ciprofloxacin-resistant strain. Furthermore, the point mutation in parC g.170C>G and another previously reported point mutation in gyrB g.2044del were also found in one of the 18 ciprofloxacin-susceptible strains, suggesting that parC g.170C>G and gyrB g.2044del are not major genetic mutations contributing to ciprofloxacin resistance.
    In conclusion, we provide a workflow to develop reliable, efficient and affordable way to detect antimicrobial resistance. If applied worldwide, it may be a promising strategy to lessen the worsening situation of antimicrobial resistance. Furthermore, a novel genetic frameshift mutation in parC g.1307delA and point mutation in parE g.1031C>T are identified as potentially decisive genetic loci to ciprofloxacin resistance in NTS, which warrants further confirmation in their phenotype by generating a recombinant mutated NTS strain.
    碩士
    指導教授:張偉嶠
    資料類型: thesis
    顯示於類別:[臨床藥物基因體學暨蛋白質體學碩士學位學程] 博碩士論文

    文件中的檔案:

    檔案 描述 大小格式瀏覽次數
    index.html0KbHTML1049檢視/開啟


    在TMUIR中所有的資料項目都受到原著作權保護.

    TAIR相關文章

    著作權聲明 Copyright Notice
    • 本平台之數位內容為臺北醫學大學所收錄之機構典藏,包含體系內各式學術著作及學術產出。秉持開放取用的精神,提供使用者進行資料檢索、下載與取用,惟仍請適度、合理地於合法範圍內使用本平台之內容,以尊重著作權人之權益。商業上之利用,請先取得著作權人之授權。

      The digital content on this platform is part of the Taipei Medical University Institutional Repository, featuring various academic works and outputs from the institution. It offers free access to academic research and public education for non-commercial use. Please use the content appropriately and within legal boundaries to respect copyright owners' rights. For commercial use, please obtain prior authorization from the copyright owner.

    • 瀏覽或使用本平台,視同使用者已完全接受並瞭解聲明中所有規範、中華民國相關法規、一切國際網路規定及使用慣例,並不得為任何不法目的使用TMUIR。

      By utilising the platform, users are deemed to have fully accepted and understood all the regulations set out in the statement, relevant laws of the Republic of China, all international internet regulations, and usage conventions. Furthermore, users must not use TMUIR for any illegal purposes.

    • 本平台盡力防止侵害著作權人之權益。若發現本平台之數位內容有侵害著作權人權益情事者,煩請權利人通知本平台維護人員([email protected]),將立即採取移除該數位著作等補救措施。

      TMUIR is made to protect the interests of copyright owners. If you believe that any material on the website infringes copyright, please contact our staff([email protected]). We will remove the work from the repository.

    Back to Top
    DSpace Software Copyright © 2002-2004  MIT &  Hewlett-Packard  /   Enhanced by   NTU Library IR team Copyright ©   - 回饋