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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張紹光(Shao-Kuang Chang) | |
| dc.contributor.author | Yu-Chen Yeh | en |
| dc.contributor.author | 葉昱辰 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:52:20Z | - |
| dc.date.copyright | 2012-08-22 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15796 | - |
| dc.description.abstract | 鮑氏不動桿菌 (Acinetobacter baumannii) 是一種分布廣泛的伺機性革蘭氏陰性菌，為人體皮膚上的常在菌叢（normal flora），目前也已在多種動物及相關的產品發現其存在。近年來，多重抗藥性鮑氏不動桿菌 (multidrug- resistant A. baumannii，MDRAB) 隨著抗菌劑的廣泛使用而出現，目前已成為醫療照護相關感染（healthcare associated infections，HAIs）中，難以控制細菌之一。由於MDRAB的出現，以及在藥理學上的重新認識，colistin再次的被用於臨床醫療，但抗藥性菌株也漸漸的開始出現。本研究自2010年9月至2011年3月間，自台灣北、中、南三區的傳統市場和超級市場進行檢體的逢機採樣，共216件。並以改良式Leeds Acinetobacter Medium（MLAM）培養基及馬康奇培養基進行初步分離。再以聚合酶鏈鎖反應增幅16S-23S核糖核酸基因內轉錄區，並搭配序列分析進行比對鑑定。結果共分離28/216（13％）株鮑氏不動桿菌，而傳統市場及超級市場的分離率分別為27/108（25%）及1/108（1％），北、中、南三區的分離率分別為6/36（17％）、10/36（28％）及12/36（33％），以及自台北地區醫院收集72株非重複之鮑氏不動桿菌。本研究亦進行抗菌劑感受性試驗及利用脈衝式電泳（pulsed-field gel electrophoresis，PFGE）調查兩來源菌株的抗藥性及親緣性。由結果得知，市售雞肉來源的菌株對cefazolin外藥品都保持一定的感受性（0∼36％），而人類臨床菌株則有較高的抗藥性（72∼100％），僅對colistin（7％）與ampicillin/sulbactam（10％）保持較佳的感受性。親緣性分析的結果發現，市售雞肉與人類臨床來源的菌株並不共有相同的基因型，故兩來源間應鮮少發生菌株及抗藥性的交流。本研究另外以蛋白質體學方法比較鮑氏不動桿菌標準菌株ATCC 19003及人工誘導抗colistin菌株19003 CR間蛋白質體的差異，結果發現誘導colistin的抗藥性產生，並不會造成菌株基因型別上的變化。藉由二維蛋白質電泳與質譜儀鑑定差異性蛋白質的身分，並以即時定量聚合酶鏈鎖反應（real-time PCR，qPCR）測定這些蛋白質的表現量，結果發現外膜蛋白Omp38在標準菌株有較高的表現量；sensor protein QseC、kdsA蛋白和aacD蛋白則是在抗colistin菌株有較高的表現量，推測colistin抗藥性的產生與細胞膜的合成相關，並受到二元訊號轉導系統調控，但還需更進一步研究其發生的機制。 | zh_TW |
| dc.description.abstract | Acinetobacter baumannii are widely distributed opportunistic Gram-negative bacteria, and also a part of normal bacteria flora of human skin. It can be found in a variety of animals and related product. In recent years, multidrug-resistant A. baumannii ( MDRAB) were emerged, caused by extensive use of antimicrobial agents, and become a part of difficult to control bacterial in healthcare associated infections (HAIs). Due to the emergence of MDRAB, and advances in pharmacology of colistin, colistin is used for clinical again. But colistin-resistant strains began to appear when it is used. In this study, two hundred and sixteen fresh raw chicken meat samples from the traditional markets and supermarkets of northern, medium and southern areas of Taiwan were collected during Sep. 2010 to Mar. 2011. Modified Leeds Acinetobacter medium (MLAM) and MacConkey agar was used as the isolation and selective medium. The samples were identified by amplify the 16S-23S internal transcribed spacers (ITS) region by polymerase chain reaction and sequence method. The total ratio of isolation was 13% (28/216). The ratio of isolation for traditional markets and supermarkets were 25% (27/108) and 1% (1/108), respectively. The prevalence of the northern, middle and southern regions was 17% (6/36), 28% (10/36) and 33% (12/36), respectively. A total of 72 isolates were consecutively collected from regional hospital in Taipei. In this study, antimicrobial susceptibility testing and pulsed field gel-electrophoresis (PFGE) are used to analysis antimicrobial resistance and phylogenic between two origin strains. The results indicated that strains from fresh raw chicken meat samples are sensitive to most tested antimicrobials (0-36%) except cefazolin. Most human clinical origin strains are highly resistant to all tested antimicrobials (72-100%); only colistin (7%) and ampicillin/sulbactam (10%) have good susceptibility. Phylogenetic analysis didn’t show any genetic relativity between chicken isolates and human isolates. The results showed isolates from two different sources were genetic and antimicrobial resistance independent. In this study, also use proteomic tool to compare the proteome differences between reference strain ATCC 19003 and artificial induced colistin-resistant strain 19003 CR. The results showed there was no genetic changes caused by induce reference strain to colistin-resistant strain by using PFGE analysis. Two-dimensional electrophoresis and MALDI-TOF analysis were used to identify the differentially expression proteins and real-time PCR was used to confirm the expression levels of these proteins. The results showed that the expression levels of outer membrane protein 38 (Omp38) increased in reference strain; sensor protein QseC, kdsA protein and aacD protein increased in artificial induced colistin-resistant strain, alternatively. It is speculate that A. baumannii may obtain their resistance against colistin through membrane biosynthesis, and it may be regulated by two component regulatory systems, but still needs further studies to verify its mechanism. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:52:20Z (GMT). No. of bitstreams: 1 ntu-101-R98629033-1.pdf: 1608624 bytes, checksum: 354bfa146e7aa440d811ff7dd6c8f31c (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書 i 誌謝 ii 中文摘要 iii 英文摘要 v 目錄 vii 表次 xi 圖次 xii 第一章 緒言 1 第二章 文獻探討 3 第一節 鮑氏不動桿菌 3 1.1鮑氏不動桿菌之分類與命名 3 1.2 鮑氏不動桿菌之形態學及生化特性 5 1.3 鮑氏不動桿菌之培養、分離及基因型鑑定 6 1.3.1 鮑氏不動桿菌之選擇性培養基 6 1.3.2 不動桿菌基因型之鑑定與分型 7 1.3.2.1 表現型及血清學鑑定與分型 7 1.3.3.2 基因體及分子生物學鑑定與分型 7 1.4 鮑氏不動桿菌對公共衛生之影響 11 1.4.1 鮑氏不動桿菌之病原性 11 1.4.2多重抗藥性鮑氏不動桿菌之崛起 12 1.5 鮑氏不動桿菌之治療與控制 13 第二節 多黏桿菌素 16 2.1 多黏桿菌素之歷史 16 2.2 多黏桿菌素之作用機制及抗菌圖譜 16 2.3 多黏桿菌素之藥物動力學及毒理學 17 2.4 多黏桿菌素之抗藥性 18 第三節 蛋白質體學 20 第三章 材料與方法 23 第一節 實驗材料 23 1.1 培養基、藥品及試劑 23 1.1.1篩選、增菌及存菌用培養基與試劑 23 1.1.2 抗菌劑感受性試驗之培養基與藥品 23 1.1.3 染色體去氧核糖核酸萃取之試劑 24 1.1.4 聚合酶鏈鎖反應及瓊脂膠體電泳之試劑 24 1.1.5 脈衝式電泳之試劑 24 1.1.6 抗colistin株19003CR人工誘導之試劑 25 1.1.7 細菌蛋白質萃取及定量之試劑 25 1.1.8 二維蛋白質電泳之試劑 25 1.1.9 膠內蛋白質水解之試劑 26 1.1.10 核糖核酸萃取及互補型去氧核糖核酸製備之試劑 26 1.1.11 即時定量聚合酶鏈鎖反應之試劑 27 1.2 標準菌株 27 1.3 器具 27 1.4 儀器設備及分析軟體 28 第二節 實驗方法 29 2.1 實驗菌株之收集 29 2.1.1 人類臨床菌株 29 2.1.2 市售雞肉菌株 29 2.2 鮑氏不動桿菌之分離、純化與鑑定 30 2.2.1 生化試驗 30 2.2.2 聚合酶鏈鎖反應增幅核糖核酸基因內轉錄區鑑定鮑氏不動桿菌 31 2.2.2.1 染色體去氧核糖核酸之萃取 31 2.3 菌種保存 32 2.4 脈衝式電泳 32 2.4.1 菌體包埋、清洗 32 2.4.2 限制酶切割及脈衝式電泳分析 33 2.5 抗菌劑感受性試驗 34 2.5.1 抗微生物藥劑原液之製備 34 2.5.2 微量肉湯稀釋法之操作流程 34 2.6 抗多黏桿菌素株19003CR之人工誘導 35 2.7 細菌外膜蛋白萃取及定量 36 2.7.1細菌全蛋白之萃取 36 2.7.2 蛋白質定量 36 2.8 二維蛋白質電泳分析 37 2.8.1 第一維蛋白質電泳 37 2.8.2 第二維蛋白質電泳 37 2.8.3 膠內蛋白質水解 38 2.8.4基質輔助雷射解析電離飛行時間質譜分析及質譜資料分析 38 2.9 核糖核酸萃取及反轉錄 39 2.10 即時定量聚合酶鏈鎖反應 40 第三節 統計分析 42 3.1 脈衝式電泳圖譜 42 3.2 抗菌劑感受性試驗 42 3.3 即時定量聚合酶鏈鎖反應之分析 43 第四章 結果 44 第一節 菌株收集 44 第二節 人類臨床與市售雞肉來源菌株之親緣性分析 45 第三節 抗菌劑感受性試驗 47 第四節 抗colistin株19003CR人工誘導 49 第五節 二維蛋白質電泳及質譜儀資料分析 50 5.1 二維蛋白質電泳圖譜分析的結果 50 5.2 質譜資料分析之結果 50 第六節 即時定量聚合酶鏈鎖反應 52 第五章 討論 53 第一節 鮑氏不動桿菌分離結果之探討 53 第二節 市售雞肉與人類臨床菌株間親緣性分析結果的探討 55 第三節 抗菌劑感受性試驗結果的探討 57 第四節 抗colistin株19003CR的人工誘導 61 第五節 二維蛋白質電泳及質譜儀資料分析 62 5.1 細菌蛋白質的萃取與純化 62 5.2 二維蛋白質電泳 63 5.3 二維蛋白質圖譜及質譜儀分析 63 第六節 即時定量聚合酶鏈鎖反應 65 第六章 結論 66 參考文獻 84 APPENDIX 1. Delineation of Acinetobacter baumannii genomic species 99 APPENDIX 2. CLSI recommended ranges of minimum inhibitory concentration test of Acinetobacter baumannii and interpretive standard of antimicrobial agents tested in this study 100 表次 TABLE 1. The area, type, number of markets and sample number of retail fresh raw chicken meat 69 TABLE 2. Results of Acinetobacter baumannii isolated from retail chicken meat 70 TABLE 3. Phylogenetic relationship of Acinetobacter baumannii isolated from fresh raw chicken meat and human sources 71 TABLE 4. The minimum inhibitory concentration (mg/L) of Acinetobacter baumannii isolated from fresh raw chicken meat and human sources 72 TABLE 5. The minimum inhibitory concentration (mg/L) of Acinetobacter baumannii isolated from fresh raw chicken meat in three district areas 73 TABLE 6. Identity of 14 protein spots with MALDI-TOF-MS analysis 74 TABLE 7. List of primer sequences used in this study for quantitative real-time polymerase chain reaction. 75 TABLE 8. Mean cycle threshold (Ct) and expression levels of target gene measured by quantitative real-time PCR 76 圖次 FIGURE 1. Dendrogram of phylogenetic relationship of Acinetobacter baumannii isolates from human source 77 FIGURE 2. Dendrogram of phylogenetic relationship of Acinetobacter baumannii isolates from retail fresh raw chicken meat 78 FIGURE 3. Dendrogram of phylogenetic relationship of Acinetobacter baumannii isolates from human source and retail fresh raw chicken meat 79 FIGURE 4. Dendrogram of antibiotic resistances of Acinetobacter baumannii isolates from human source 80 FIGURE 5. Dendrogram of antibiotic resistances of Acinetobacter baumannii isolates from retail fresh raw chicken meat 81 FIGURE 6. Dendrogram of antibiotic resistances of Acinetobacter baumannii isolates from human source and retail fresh raw chicken meat 82 FIGURE 7. Two-dimensional master gel made by combine reference strain gel with artificial induced colistin-resistant strain gel 83 | |
| dc.language.iso | zh-TW | |
| dc.subject | 多黏桿菌素 | zh_TW |
| dc.subject | 鮑氏不動桿菌 | zh_TW |
| dc.subject | 蛋白質體學 | zh_TW |
| dc.subject | 抗藥性 | zh_TW |
| dc.subject | 親緣性分析 | zh_TW |
| dc.subject | phylogenetic analysis | en |
| dc.subject | proteomics | en |
| dc.subject | Acinetobacter baumannii | en |
| dc.subject | antimicrobial resistance | en |
| dc.subject | colistin | en |
| dc.title | 以蛋白質體學技術研究鮑氏不動桿菌之多黏桿菌素抗藥性 | zh_TW |
| dc.title | Proteomic Analysis of Polymyxin-Resistance Acinetobacter baumannii | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡向榮(Hsiang-Jung Tsai),周濟眾(Chi-Chung Chou),周崇熙(Chung-Hsi JiuJiu Chou),郭鴻志(Hung-Chih Kuo) | |
| dc.subject.keyword | 鮑氏不動桿菌,抗藥性,多黏桿菌素,親緣性分析,蛋白質體學, | zh_TW |
| dc.subject.keyword | Acinetobacter baumannii,antimicrobial resistance,colistin,phylogenetic analysis,proteomics, | en |
| dc.relation.page | 100 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2012-08-20 | |
| dc.contributor.author-college | 獸醫專業學院 | zh_TW |
| dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
| 顯示於系所單位： | 獸醫學系 | |
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