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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 潘子明(Tzu-Ming Pan) | |
| dc.contributor.author | Jia-Chi Chiou | en |
| dc.contributor.author | 邱家琪 | zh_TW |
| dc.date.accessioned | 2021-06-13T17:29:15Z | - |
| dc.date.available | 2004-07-27 | |
| dc.date.copyright | 2004-07-27 | |
| dc.date.issued | 2004 | |
| dc.date.submitted | 2004-07-22 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39468 | - |
| dc.description.abstract | 由志賀氏痢疾桿菌 (Shigella spp.) 所引起的細菌性痢疾為全球最常發生之腸胃疾病之一。台灣地區桿菌性痢疾的主要感染源為志賀氏副痢疾菌 (S. flexneri) 及志賀氏宋內菌 (S. sonnei)，臨床上以抗生素治療可縮短病程並達治癒之目的，但若未完成一完整療程，則會導致細菌在抗生素壓力下產生抗藥性。
頭孢菌素 (cephalosporin) 為全球最常使用的抗生素，而頭孢曲松 (ceftriaxone) 為頭孢菌素中血清半衰期最長的一種抗生素，其殺菌機制在於抑制細菌細胞壁之合成。細菌可能藉由排藥幫浦之表現或產生乙型單環醯胺? (beta-lactamase) 而對頭孢菌素產生抗藥性，其中又以後者所造成的抗藥性表現較為明顯。將臨床分離所得之志賀氏宋內菌培養於添加頭孢曲松的培養基進行誘導培養，以提高其抗藥性表現，觀察其最低抑制濃度，並分別以聚合?鏈反應及二維式電泳檢測其乙型單環醯胺?基因及其他蛋白質在抗藥性產生過程中的表現情形。 結果顯示 2 株臨床為敏感性之菌株在培基中之 ceftriaxone 添加濃度增加至 16 μg/mL 時，其抗生素感受性形式直接由敏感性變成抗藥性，推測在這個過程中，菌株可能啟動了某種抗藥性機制而使得對藥物的耐受性急速增加。而以聚合?鏈反應對抗藥性基因進行檢測，發現在所有臨床及誘導菌株之質體 DNA 上均帶有與 Salmonella enterica subsp. enterica serovar Choleraesuis plasmid ampC 具有 99% 相似度的基因存在。在蛋白質表現層面上，也發現高度抗藥性菌株增強表現 AmpC β-lactamase，而在敏感性菌株上則無該蛋白質點表現，證明經 ceftriaxone 誘導抗藥性產生之 S. sonnei 藉由 AmpC β- lactamase 之產生的確對該抗生素產生高度抗藥性，同時本研究中以基因及蛋白質層面探討抗藥性之表現所得之結果相符合。 | zh_TW |
| dc.description.abstract | Shigellosis caused by Shigella spp. is one of the most frequently happened gastrointestinal disease worldwide. The shigellosis outbreaks in Taiwan were mainly caused by S. flexneri and S. sonnei. Antibiotics are used to treat or to shorten the carrier phase, however, Shigella spp. may acquire antibiotic-resistance on account of incomplete course of treatment.
Cephalosporin is the most commonly used antibiotics in the whole world. Ceftriaxone has the longest serum half-life among the cephalosporins and it impedes the synthesis of bacteria cell wall. Bacteria might acquire drug-resistance by efflux pump overexpression or beta-lactamase producing in order to fight the antibiotics pressure, and the effect of the latter one is more helpful. The clinical isolated S. sonnei was incubated in media with ceftriaxone in order to enhance the ceftriaxone-resistance. Minimal inhibition concentration (MIC) value of experimental strains is determined. Polymerase chain reaction (PCR) and two-dimensional electrophoresis are used to detect the beta-lactamase gene and protein expression. Two clinical ceftriaxone-susceptible strains became resistant ones when the ceftriaxone concentrations in the media increased to 16 μg/mL. We supposed that some mechanisms may be triggered in the process among the susceptible strains. The PCR results revealed that the clinical and ceftriaxone-resistant strains carry the plasmid-mediated ampC, having 99% homology with Salmonella enterica subsp. enterica serovar Choleraesuis plasmid ampC. The expression of AmpC beta-lactamase on ceftriaxone-resistant strains, which did not observed on susceptible strains, increased on the two-dimensional electrophoresis gel. The results of studying the drug-resistance based on both the gene and protein level are coincident in this study. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T17:29:15Z (GMT). No. of bitstreams: 1 ntu-93-R91623519-1.pdf: 2558532 bytes, checksum: 5029b6f16462c7c04dbaaafa503cc26e (MD5) Previous issue date: 2004 | en |
| dc.description.tableofcontents | 第一章 前言-------------------------------------------- 1
1.1 痢疾與志賀氏宋內菌------------------------------- 1 1.2 抗生素與細菌之抗藥性----------------------------- 1第二章 文獻回顧---------------------------------------- 2 2.1 志賀氏桿菌--------------------------------------- 2 2.1.1 志賀氏桿菌之分類----------------------------- 2 2.1.2 一般生化特性--------------------------------- 4 2.2 世界各國桿菌性痢疾感染事件概述------------------- 4 2.2.1 美洲地區桿菌性痢疾感染事件------------------- 6 2.2.2 亞洲地區桿菌性痢疾感染事件------------------- 6 2.2.3 台灣區桿菌性痢疾感染事件---------------------10 2.3 抗生素總論---------------------------------------12 2.3.1 一般抗生素種類及作用機制---------------------12 2.3.1.1 Tetracycline 及 aminoglycoside 之作用機制-12 2.3.1.2 Quinolone之作用機制----------------------14 2.3.1.3 Sulfonamides之作用機制-------------------14 2.3.2 頭孢菌素之分類及作用機制---------------------14 2.3.3 Ceftriaxone----------------------------------15 2.4 細菌抗藥性與抗生素之關係-------------------------17 2.4.1 細菌抗藥性產生之機制種類---------------------17 2.4.1.1 對 tetracycline 及aminoglycoside 抗藥性產生 之機制--------------------------------------------------20 2.4.1.2 對 quinolone抗藥性產生之機制-------------21 2.4.1.3 對 sulfonamides抗藥性產生之機制----------21 2.4.2 細菌對頭孢菌素抗藥性之產生-----------------------21 2.4.3 β-lactamase 之發現及種類-------------------------21 2.5 一般常用於分析細菌抗藥性之方法-------------------23 2.5.1 抗生素敏感性試驗-----------------------------25 2.5.1.1 瓊脂紙錠擴散試驗-------------------------25 2.5.1.2 稀釋試驗---------------------------------27 2.5.1.3 E-test-----------------------------------27 2.5.2 聚合?鏈反應-------------------------------------27 2.5.3 二維式電泳---------------------------------------28 2.6 研究目的-----------------------------------------30 第三章 材料與方法--------------------------------------31 3.1 材料---------------------------------------------31 3.1.1 菌株與質體-----------------------------------31 3.1.2 培養基---------------------------------------31 3.1.3 血清凝集測試---------------------------------31 3.1.4 抗生素---------------------------------------31 3.1.5 藥品試劑-------------------------------------33 3.1.6 實驗套組-------------------------------------33 3.1.7 儀器設備-------------------------------------34 3.2 方法---------------------------------------------34 3.2.1 抗生素敏感性實驗-----------------------------34 3.2.1.1 紙錠擴散法 (agar disk d-iffusion)--------34 3.2.1.2 Minimal inhibitory concentration (MIC)---34 3.2.1.3 E-test-----------------------------------------34 3.2.1.4 Vitek test-------------------------------------35 3.2.2 試管誘導志賀氏宋內菌對 CRO 產生抗藥性--------35 3.2.3 血清凝集測試---------------------------------35 3.2.4 質體 DNA 輪廓分析(Plasmid Profile Analysis, PPA)-36 3.2.5 Genomic DNA 抽取--------------------------------36 3.2.6 聚合?鏈反應------------------------------------36 3.2.7 β-Lactamase 粗萃取------------------------------37- 3.2.7.1 細胞質間蛋白質抽取------------------------37 3.2.7.2 蛋白質沉澱------------------------------41 3.2.7.3 蛋白質定量------------------------------41 3.2.7.4 β-lactamase 活性測試--------------------41 3.2.8 二維式電泳----------------------------------41 3.2.8.1 第一維：等電點焦集電泳------------------41 3.2.8.2 第二維：變性聚丙醯胺膠體電泳------------42 3.2.8.3 Coomassie Brilliant R-250 (CBR) 染色----42 3.2.8.4 銀染色法--------------------------------43 3.2.9 蛋白質質譜分析------------------------------43 3.2.9.1 膠體內水解------------------------------43 3.2.9.2 基質輔助雷射脫附離子化質譜法------------45 3.2.10 DNA 選殖 (cloning)-------------------------46 3.2.10.1 聚合?鏈反應產物純化-------------------46 3.2.10.2 質體接合作用 (ligation)----------------48 3.2.10.3 轉形作用–勝任細胞 (compentent cell) 製備---------------------------------------------------------48 3.2.10.4 轉形作用–熱休克轉形-------------------48 3.2.10.5 轉形株篩選-----------------------------48 3.2.10.6 DNA 定序作用---------------------------49 第四章 結果與討論-------------------------------------50 4.1 實驗菌株之紙錠擴散試驗及 E-test 資料數據分析------50 4.1.1 臨床菌株----------------------------------------50 4.1.2 試管誘導志賀氏宋內菌對 CRO 產生抗藥性-----------53 4.1.3 誘導菌株----------------------------------------53 4.2 質體輪廓分析--------------------------------------57 4.3 聚合?鏈反應--------------------------------------57 4.4 二維式電泳與蛋白質質譜分析------------------------65 4.4.1 建立細胞質間蛋白質二維式電泳參考圖------------67 4.4.2 細胞質間蛋白質於抗藥性產生過程中之變化情形----69 第五章 結論-------------------------------------------79 第六章 參考文獻---------------------------------------81--------- | |
| dc.language.iso | zh-TW | |
| dc.subject | 抗藥性 | zh_TW |
| dc.subject | 二維式電泳 | zh_TW |
| dc.subject | 志賀氏宋內菌 | zh_TW |
| dc.subject | 頭孢曲松 | zh_TW |
| dc.subject | two-dimensional electrophoresis | en |
| dc.subject | ceftriaxone | en |
| dc.subject | drug-resistance | en |
| dc.subject | Shigella sonnei | en |
| dc.title | 志賀氏宋內菌對頭孢曲松抗藥性機制之研究 | zh_TW |
| dc.title | Study on the mechanisms of ceftriaxone-resistance of Shigella sonnei | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 91-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 蘇遠志(Yung-Chi Su),陳陸宏(Lu-Hung Cheng),施養志,李智隆 | |
| dc.contributor.oralexamcommittee | 蘇遠志(chinyuan@ms.cc.ntu.edu.tw) | |
| dc.subject.keyword | 抗藥性,志賀氏宋內菌,頭孢曲松,二維式電泳, | zh_TW |
| dc.subject.keyword | Shigella sonnei,drug-resistance,ceftriaxone,two-dimensional electrophoresis, | en |
| dc.relation.page | 91 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2004-07-22 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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