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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
| dc.contributor.author | Meng-Chuan Wu | en |
| dc.contributor.author | 吳孟娟 | zh_TW |
| dc.date.accessioned | 2021-06-17T00:44:45Z | - |
| dc.date.available | 2012-03-02 | |
| dc.date.copyright | 2012-03-02 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2012-01-09 | |
| dc.identifier.citation | 1. Murray PR, Rosenthal KS, Kobayashi GS and Pfaller MA. Enterobacteriaceae. Medical Microbiology. 3rd ed: Mosby, 1998:232-244
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66586 | - |
| dc.description.abstract | 克雷伯氏肺炎桿菌（Klebsiella pneumoniae）為革蘭氏陰性桿菌，屬於腸內菌科，常在免疫功能不全的病患造成泌尿道感染、呼吸道感染、及菌血症，為重要的伺機性感染病原菌。近二十多年來，克雷伯氏肺炎桿菌在許多地區造成社區感染症的病例逐年上升，且其症狀顯著不同，經常造成細菌性肝膿瘍合併菌血症及轉移性的眼內炎或腦膜炎。形成生物膜可使細菌對於抗生素及免疫反應有較強的扺抗能力膜，因此細菌形成生物膜的能力在致病過程中扮演了重要的角色。我們利用微量培養盤探討克雷伯氏肺炎桿菌造成的細菌性肝膿瘍與生物膜形成的相關性，發現肝膿瘍菌株生物膜形成的能力明顯較非組織侵襲性菌株的能力高。接著由肝膿瘍菌株的跳躍子突變株庫中篩選出23株生物膜生成下降與4株上升的突變菌株並分析被影響的基因。在生物膜生成改變的跳躍子突變菌株中，treC突變菌株的生物膜形成、黏性與莢膜多醣的產生量都減少；反之sugE的這些表型都上升。treC與sugE突變菌株生物膜形成能力改變的情況在玻片培養時亦與在微量培養盤中相似，而兩基因的基因剔除菌株也驗證了在跳躍子突變菌株的觀察。treC基因剔除菌株利用海藻醣的能力受到損害，在培養基中加入葡萄糖可使treC基因剔除菌株的生物膜形成能力與莢膜多醣表現量回復。體內競爭性分析的結果顯示treC基因剔除菌株在胃內感染老鼠時，拓殖的競爭力下降。另一方面，轉錄表現分析顯示sugE基因剔除菌株中莢膜多醣產生量的增加是由於莢膜生合成基因群（經由莢膜調控基因rmpA）與treC基因的RNA表現量上升所導致。treC基因在腸胃道感染時的重要性顯示生物膜形成應有助於克雷伯氏肺炎桿菌感染的建立與持續。 | zh_TW |
| dc.description.abstract | Background: Community-acquired pyogenic liver abscess (PLA) complicated with meningitis and endophthalmitis caused by Klebsiella pneumoniae is an emerging infectious disease. To investigate the mechanisms and effects of biofilm formation of K. pneumoniae causing PLA, microtiter plate assays were used to determine the levels of biofilm formed by K. pneumoniae clinical isolates and to screen for biofilm-altered mutants from a transposon mutant library of a K. pneumoniae PLA-associated strain.
Methodology/Principal Findings: The biofilm formation of K. pneumoniae was examined by microtiter plate assay. Higher levels of biofilm formation were demonstrated by K. pneumoniae strains associated with PLA. A total of 23 biofilm-decreased mutants and 4 biofilm-increased mutants were identified. Among these mutants, a biofilm-decreased treC mutant displayed less mucoviscosity and produced less capsular polysaccharide (CPS), whereas a biofilm-increased sugE mutant displayed higher mucoviscosity and produced more CPS. The biofilm phenotypes of treC and sugE mutants also were confirmed by glass slide culture. Deletion of treC, which encodes trehalose-6-phosphate hydrolase, impaired bacterial trehalose utilization. Addition of glucose to the culture medium restored the capsule production and biofilm formation in the treC mutant. Transcriptional profile analysis suggested that the increase of CPS production in ΔsugE may reflect elevated cps gene expression (upregulated through rmpA) in combination with increased treC expression. In vivo competition assays demonstrated that the treC mutant strain was attenuated in competitiveness during intragastric infection in mice. Conclusions/Significance: We have identified 25 genes important for biofilm formation in a K. pneumoniae PLA strain using microtiter plate assay and confirmed by slide cultures. Among these genes, treC and sugE affect biofilm formation by modulating CPS production. The importance of treC in gastrointestinal tract colonization suggests that biofilm formation contributes to the establishment and persistence of K. pneumoniae infection. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T00:44:45Z (GMT). No. of bitstreams: 1 ntu-100-D94445008-1.pdf: 3254192 bytes, checksum: 920ca1b9789a780d2ecbd5029baef2e3 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 摘 要 I
Abstract II 目 錄 IV 圖 目 錄 VII 表 目 錄 VIII 表 目 錄 VIII 第一章 緒論 1 1.1. 克雷伯氏肺炎桿菌（Klebsiella pneumoniae） 1 1.1.1. 克雷伯氏肺炎桿菌與疾病 1 1.1.2. 克雷伯氏肺炎桿菌的致病因子 2 1.2. 生物膜（Biofilms） 4 1.3. 生物膜與疾病 5 1.4. 克雷伯氏肺炎桿菌生物膜相關基因 6 1.5. 研究目的 6 第二章 材料與方法 8 2.1 細菌菌株、質體與細菌培養 8 2.2 微量培養盤試驗（microtiter plate assay） 8 2.3 克雷伯氏肺炎桿菌跳躍子突變菌株庫的建構 8 2.4 突變株基因序列分析 9 2.3.1. 半隨機聚合酶鏈鎖反應（semi-random polymerase chain traction, semi-random PCR） 9 2.3.2. 反轉聚合酶鏈鎖反應 （Inverse PCR） 9 2.5 克雷伯氏肺炎桿菌黏性檢驗（mucoviscosity assay） 9 2.6 克雷伯氏肺炎桿菌基因剔除突變株 (deletion mutant)之建構 10 2.7 克雷伯氏肺炎桿菌染色體異位補回菌株（chromosome complementation strain）之建構 10 2.8 以玻片培養（slide culture）觀察生物膜生成量 11 2.9 克雷伯氏肺炎桿菌胞外多醣之萃取與測定 11 2.9.1 以熱的酚-水（Hot phenol-water）方式萃取胞外多醣 11 2.9.2 酵素連結免疫吸附法（Eznyme-linked immunosorbent assay）測定莢膜多醣 11 2.9.3 總醣量含量測定(Phenol-sulfuric acid method) 12 2.10 不同碳源培養盤生長情形之檢驗 12 2.11 DNA微陣列分析法（Microarray analysis） 12 2.11.1 克雷伯氏肺炎桿菌RNA之萃取 12 2.11.2 互補DNA（complementary DNA,cDNA）探針製備 13 2.11.3 預雜交反應（Prehybridization） 13 2.11.4 雜交反應（Hybridization） 13 2.11.5 偵測雜交訊息 14 2.12 即時定量聚合酶連鎖反應（Real-time quantitative polymerase chain reaction, real-time qPCR） 14 2.13 treB與treC基因在生物膜生長與懸浮狀態表現之比較 15 2.14 動物感染 15 2.15 競爭性分析 15 2.15.1 體外競爭性分析（in vitro competition assay） 16 2.15.2 體內競爭性分析（in vivo competition assay） 16 2.16 最低抑制濃度（Minimum inhibitory concentration, MIC）測試 16 2.17 統計分析 17 第三章 結果 18 3.1 克雷伯氏肺炎桿菌臨床菌株的生物膜形成 18 3.2 克雷伯氏肺炎桿菌生物膜形成相關基因之篩選 18 3.3 跳躍子突變株黏性與莢膜多醣的表現 21 3.4 treC與sugE基因剔除菌株與異位補回菌株的建構與特性 22 3.5 treB對克雷伯氏肺炎桿菌生物膜形成的影響 23 3.6 treC和sugE突變株在玻璃表面上的生物膜形成 23 3.7 treC突變菌株的生物膜發展（biofilm development） 24 3.8 treC基因在克雷伯氏肺炎桿菌生物膜中的表現模式 24 3.9 利用克雷伯氏肺炎桿菌基因微陣列尋找treC基因所影響的基因 25 3.10 treC的生物功能 26 3.11 treC的功能在莢膜表現上所扮演的角色 26 3.12 動物感染 27 3.13 體外競爭力測試 27 3.14 體內競爭性分析 28 3.15 sugE蛋白質的生物功能之預測與最小抑制濃度之檢測 28 3.16 莢膜基因群在sugE基因剔除菌株中的表現量 29 第四章 討論 30 第五章 參考文獻 69 | |
| dc.language.iso | zh-TW | |
| dc.subject | 克雷伯氏肺炎桿菌 | zh_TW |
| dc.subject | 肝膿瘍 | zh_TW |
| dc.subject | 生物膜 | zh_TW |
| dc.subject | 海藻醣-六-水解酶 | zh_TW |
| dc.subject | 競爭性分析 | zh_TW |
| dc.subject | biofilm | en |
| dc.subject | treC | en |
| dc.subject | klebsiella pneumoniae | en |
| dc.subject | pyogenic liver abscess | en |
| dc.subject | sugE | en |
| dc.title | 由肝膿瘍之克雷伯氏肺炎桿菌菌株分離生物膜生成相關基因 | zh_TW |
| dc.title | Isolation of Genes Involved in Biofilm formation of a Klebsiella pneumoniae strain causing pyogenic liver abscess | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 張晃猷,董馨蓮,楊宏志,林稚容 | |
| dc.subject.keyword | 克雷伯氏肺炎桿菌,肝膿瘍,生物膜,海藻醣-六-水解酶,競爭性分析, | zh_TW |
| dc.subject.keyword | klebsiella pneumoniae,pyogenic liver abscess,biofilm,sugE,treC, | en |
| dc.relation.page | 78 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-01-10 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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