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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
| dc.contributor.author | Li-Chen Ma | en |
| dc.contributor.author | 馬立珍 | zh_TW |
| dc.date.accessioned | 2021-06-13T16:52:43Z | - |
| dc.date.available | 2005-07-05 | |
| dc.date.copyright | 2005-07-05 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-06-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/38924 | - |
| dc.description.abstract | 由侵襲性克雷伯氏肺炎桿菌所造成的原發性肝膿瘍合併敗血症，目前已成為全球性的新興感染症。我們利用基因微陣列的方式，在侵襲性菌株中找尋到一段長約20 kb的染色體片段，並將其命名為kfu/PTS片段。此片段共涵蓋15個開放讀架，其中包括一組與鐵攝取有關的基因（kfuABC）、一組phosphoenolpyruvate sugar phosphotransferase system（PTS）和6個功能未知的開放讀架。這個片段多分佈於侵襲性菌株中(35/46)，而在非侵襲性菌株則分佈較少（19/98），統計分析兩者間有顯著的差異（p＜0.0001, Chi-Square test）。為了證實此長片段基因與細菌本身致病力的關係，我們設計了3組基因剔除菌株（ΔkfuABC, ΔORF7-9, ΔPTS），其中只有ΔkfuABC菌株與原生菌株比較，喪失對鼷鼠的致病力。進一步的實驗證明kfuABC基因組可幫助細菌在缺鐵的生長條件下與外界競爭鐵的來源。此外，我們發現kfu/PTS片段與另兩段與組織侵襲性有關的染色體片段（allS和magA）有極高的相關性（85％）；且臨床上能同時造成原發性肝膿瘍合併眼內炎或腦膜炎的菌株都共同包含此3個片段。因此，我們推測侵襲性克雷伯氏肺炎桿菌菌株間有顯著的遺傳差異，同時帶有此3個片段的基因型菌株和肝膿瘍與其他轉移性的併發症發生有強烈的相關性。這3個片段可以應用於臨床上作為快速診斷侵襲性感染菌株的方便指標。 | zh_TW |
| dc.description.abstract | Primary pyogenic liver abscess (PLA) with septic complication by Klebsiella pneumoniae is an emerging infectious disease. Using DNA microarray hybridization, we identified a 20-kb chromosomal region that contained 15 open-reading frames (ORFs), including an iron-uptake system (kfu), a phosphoenolpyruvate sugar phosphotransferase system (PTS), and 6 unknown ORFs. The region was more prevalent among tissue-invasive strains (35/46) than among noninvasive strains (19/98) (p＜0.0001, Chi-Square test). To test the role played by this region in pathogenesis, 3 different deletion mutants (NTUH-K2044 [Δkfu], K2044 [ΔORF7–9], and K2044 [ΔPTS]) were constructed. Only the ΔkfuABC mutants showed decreased virulence in mice, compared with the wild-type strain. An in vitro assay confirmed the involvement of kfu in iron acquisition. There was a high correlation rate (85%) between the kfu/PTS region and 2 tissue invasion–associated chromosomal regions (allS and magA). Moreover, all 3 regions were present in strains that caused PLA plus endophthalmitis or meningitis. Our results suggest that chromosomal heterogeneity is present in tissue-invasive K. pneumoniae strains. A genotype containing all 3 regions is strongly associated with PLA and metastatic infection. These regions may serve as convenient markers for the rapid diagnosis of emergent tissue-invasive strains. | en |
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| dc.description.tableofcontents | 目 錄
中文摘要………………………………………………………….……. 1 英文摘要……………………………………………………………….. 3 第一章 緒論………………………………………………………….. 5 第二章 材料及方法………………………………………………….. 12 2.1 材料： 2.1.1 克雷伯氏肺炎桿菌…………………………...………………. 12 2.1.2 大腸桿菌………………………………..…………………….. 13 2.1.3 質體……………………………………………………….…… 15 2.1.4 培養基………………………………………………………… 16 2.1.5 抗生素………………………………………………………… 16 2.1.6 引子…………………………………………………………… 16 2.2 研究方法： 2.2.1 脈衝電泳……………………………………………………… 17 2.2.2 克雷伯氏肺炎桿菌基因體DNA的抽取…………………… 19 2.2.3 克雷伯氏肺炎桿菌基因微陣列的介紹.............................. 20 2.2.4 基因微陣列探針的合成……………………………………… 21 2.2.5 基因微陣列的雜交…………………………………………… 22 2.2.6 基因微陣列呈色反應及影像分析…………………………… 23 2.2.7 殖株（clone 3~10）上下游基因定序……………………… 25 2.2.8 基因剔除缺陷株的建構……………………………………… 25 2.2.9 異位補回kfuABC基因，並證明其功能…………………… 27 2.2.10 克雷伯氏肺炎桿菌total RNA的抽取……………………… 28 2.2.11 反轉錄PCR………………………………………………….. 30 2.2.12 動物實驗……………………………………………………… 31 2.2.13 狹縫墨點法…………………………………………………… 33 第三章 結果…………………………………………………………. 34 3.1 基因微陣列分析…………………………………………………. 34 3.2 9 kb NTUH-K2044獨有基因片段鄰近區域分析…………….. 35 3.3 kfu/PTS片段和大質體在臨床克雷伯氏肺炎桿菌分佈的關連性 37 3.4 20 kb染色體片段和細菌毒力的關係…………………………. 39 3.5 克雷伯氏肺炎桿菌kfu system的功能分析………………….. 40 3.6 kfu/PTS、magA和allS三大片段與臨床菌株間的關連性….. 42 第四章 討論…………………………………………………………. 44 第五章 參考文獻……………………………………………………. 74 表 目 錄 表一： 本研究中所使用的臨床克雷伯氏桿菌…………………….. 52 表二： 本研究中所使用的引子……………………………………… 53 表三： 12個雜交訊號在侵襲性菌株強於非侵襲性菌株的殖株 基因定序結果………………………………………………… 54 表四： NTUH-K2044獨有的20 kb片段其可能帶有的開放讀 架及功能……………………………………………………… 55 表五： kfuABC 基因與其它菌種的 ABC transport system比較 56 表六： 大質體與三大染色體片段在臨床克雷伯氏肺炎桿菌菌株 間的分布情形………………………………………………… 57 表七： 三大染色體於臨床菌株間分佈與造成疾病型態的關係表 58 圖 目 錄 圖一： 脈衝場膠電泳法分型基因微陣列所使用的臨床菌株......... 59 圖二： 侵襲性與非侵襲性菌株基因微陣列比較圖....................... 60 圖三： 20 kb染色體片段上的基因排列組成............................... 61 圖四： 偵測kfu/PTS片段所使用的引子位置圖.......................... 62 圖五： 三組克雷伯氏肺炎桿菌基因剔除缺陷菌株建構圖............ 63 圖六： 小鼠以腸胃道餵食原生株與三組基因剔除缺陷菌株的 存活比例與時間圖（104 CFU）...................................... 64 圖七： 小鼠以腸胃道餵食原生株與三組基因剔除缺陷菌株的 存活比例與時間圖（105 CFU）...................................... 65 圖八： 小鼠餵食克雷伯氏肺炎桿菌後肝組織切片圖.................. 66 圖九： 小鼠餵食克雷伯氏肺炎桿菌後腦組織切片圖.................. 67 圖十： 侵襲性菌株NTUH-K2044的kfuABC基因上游區域位 置圖............................................................................. 68 圖十一： RT-PCR 結果顯示圖................................................. 69 圖十二： 比較原生株、kfuABC基因剔除缺陷株及非侵襲性 菌株於含有鐵鉗合因子的培養基中生長情形.............. 70 圖十三： 於含有鐵鉗合因子的培養基中證明kfuABC基因功能 71 圖十四： 偵測allS及magA片段所使用的引子位置圖............. 72 圖十五： 以狹縫墨點法觀察kfu/PTS、magA和allS三大片段 與侵襲性菌株的關聯性.............................................. 73 | |
| dc.language.iso | zh-TW | |
| dc.subject | 侵襲性克雷伯氏肺炎桿菌 | zh_TW |
| dc.subject | kfu/PTS 片段 | zh_TW |
| dc.subject | tissue invasive K. pneumoniae | en |
| dc.subject | kfu/PTS region | en |
| dc.title | 造成化膿性肝膿瘍及其他轉移性感染的
克雷伯氏肺炎桿菌菌株之基因體差異性 | zh_TW |
| dc.title | Genomic Heterogeneity in Klebsiella pneumoniae Strains Is Associated with Primary Pyogenic Liver Abscess and Metastatic Infections | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 何漣漪(Lien-I Hor),賴信志(Hsin-Chih Lai),鄧述諄(Shu-Chun Teng),李芳仁(Fang-Jen Lee) | |
| dc.subject.keyword | kfu/PTS 片段,侵襲性克雷伯氏肺炎桿菌, | zh_TW |
| dc.subject.keyword | kfu/PTS region,tissue invasive K. pneumoniae, | en |
| dc.relation.page | 88 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-06-20 | |
| 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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