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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂 | |
| dc.contributor.author | Chun-Tang Chen | en |
| dc.contributor.author | 陳俊堂 | zh_TW |
| dc.date.accessioned | 2021-06-16T05:17:11Z | - |
| dc.date.available | 2019-10-09 | |
| dc.date.copyright | 2014-10-09 | |
| dc.date.issued | 2014 | |
| dc.date.submitted | 2014-08-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56155 | - |
| dc.description.abstract | 克雷伯氏肺炎桿菌具有由多醣體所組成的莢膜，目前已知至少有79種莢膜型，不同莢膜型的克雷伯氏肺炎桿菌的致病力和其造成的疾病也會有所不同，因此在臨床上鑑定其莢膜型具有一定的重要性。近年來克雷伯氏肺炎桿菌造成院內感染增加，且多為抗藥性菌株，甚至已有菌株對於用來治療多重抗藥性細菌最後防線的碳青黴烯類抗生素產生抗藥性，但目前對於其莢膜分型的相關研究鮮少有報告。本實驗室先前研究來自台大、長庚、成大、榮總四家醫院的85株碳青黴烯類抗生素抗藥性的克雷伯氏肺炎桿菌之菌株，發現其莢膜型以K64的比例最高32/85 (38%)，而後依序是K62為11/85 (13%)、K24為7/85 (8%)、KN2為6/85 (7%)和K28為5/85 (6%)，而其它莢膜型之菌株皆少於或等於3株，顯示特定莢膜型似乎和細菌獲得抗藥性有關聯。本研究由接合實驗結果，發現不論是碳青黴烯類抗生素抗藥性菌株或是藥物敏感性菌株，抗藥盛行莢膜型(K64、K62、K24、KN2和K28)菌株之平均接合效率，相較非抗藥盛行莢膜型之菌株皆來得高，且在統計上有顯著差異。利用聚合酶鏈鎖反應，發現抗藥盛行莢膜型之碳青黴烯類抗生素抗藥性菌株，其具有第1類整合子的比例，相較非抗藥盛行莢膜型之菌株有較高的趨勢，但在統計上並無顯著差異。另一方面，因為85株碳青黴烯類抗生素抗藥性菌株只有12株具有carbapenemase基因(~14%)，因此進一步探討其它可能的抗藥機制。以EtBr- agar cartwheel和RT-qPCR方法，發現碳青黴烯類抗生素抗藥性菌株，其外排幫浦活性和外排幫浦蛋白AcrA的RNA表現量，相較藥物敏感性菌株有較高的趨勢，但在統計上無顯著差異。除此之外，以質體補回完整的外膜蛋白OmpK35或OmpK36，發現有11/12 (91%)和3/12 (25%)碳青黴烯類抗生素抗藥性菌株的最小抑制濃度下降2-8倍。本研究結果顯示，造成碳青黴烯類抗生素抗藥性菌株莢膜型集中的現象，可能是因為抗藥盛行莢膜型之菌株較容易獲取抗藥基因；此外，外膜蛋白在克雷伯氏肺炎桿菌對碳青黴烯類抗生素之抗藥性也扮演重要的角色。 | zh_TW |
| dc.description.abstract | Klebsiella pneumoniae is enveloped by a polysaccharide capsule. At least 79 capsular types were defined in K. pneumoniae so far. Capsular types were associated with different clinical settings and severity of illness caused by K. pneumoniae. Therefore, to determine the capsular type of clinical K. pneumoniae isolates will be important. Recently, nosocomial infections caused by K. pneumoniae were increasing, and a great number of them developed drug resistance. K. pneumoniae has been reported to exhibit resistance to carbapenem which is one of last-line defense for multiple drug resistant strains, but the capsular type of multi-drug-resistant K. pneumoniae was poorly understood. Previously we determined the capsular types of 85 carbapenem-resistant K. pneumoniae (CRKP) strains from NTUH, CGMH, NCKUH and VGH, and found that K64 (38%) was the dominant type among these strains, followed by K62 (13%), K24 (8%), KN2 (7%) and K28 (6%), suggesting that specific capsular types may be associated with drug resistance. Conjugation experiments revealed that conjugation efficiency of prevalent capsular types (K64, K62, K24, KN2 and K28) strains was higher than non-prevalent capsular types strains in CRKP or drug-sensitive strains. The prevalence rate of class 1 integron among prevalent capsular types strains was slight higher than non-prevalent capsular types strains (48/61, 79% vs. 14/24, 58%) in CRKP by PCR. Because carbapenemase were only present in 12 of 85 strains (~14%), we further to explore other possible carbapenem resistance mechanisms. Detection of efflux pump activity by EtBr-agar cartwheel method and determination of RNA expression levels of efflux pump protein AcrA by RT-qPCR showed no significantly different among CRKP and drug-sensitive strains. Restoration of plasmid carrying intact OmpK35 or OmpK36 significantly decreased the MICs of imipenem in 11/12 (91%) and 3/12 (25%) CRKP strains, respectively. In summary, K. pneumoniae strains with prevalent capsular types are more easily to acquire drug resistance genes and result capsular types clustering among CRKP strains. Besides, outer membrane proteins, OmpK35 and OmpK36, play important roles in carbapenem resistance of CRKP. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T05:17:11Z (GMT). No. of bitstreams: 1 ntu-103-R01445103-1.pdf: 2763491 bytes, checksum: 216162d13b7dbf574285b5a7bc40f90a (MD5) Previous issue date: 2014 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書 I 誌謝 II 中文摘要 III Abstract V 第一章、緒論 1 1.1克雷伯氏肺炎桿菌之基本介紹 1 1.2 克雷伯氏肺炎桿菌之莢膜分型方法 2 1.3 克雷伯氏肺炎桿菌對抗生素的感受性 3 1.4 細菌獲得抗藥性基因的方式 4 1.5 碳青黴烯類抗生素抗藥性的克雷伯氏肺炎桿菌(Carbapenem-Resistant Klebsiella pneumoniae, CRKP) 6 1.6 研究動機 7 第二章、材料與方法 8 2.1 材料 8 2.1.1 克雷伯氏肺炎桿菌之臨床菌株 8 2.1.2 其它菌株以及質體 8 2.1.3培養基(Media) 9 2.1.4抗生素(Antibiotics) 9 2.1.5引子(Primers) 9 2.2方法 10 2.2.1 電穿孔(Electroporation) 10 2.2.2 自然轉形(Natural transform) 10 2.2.3 建構具有oriT序列的質體pMN402 11 2.2.4 接合實驗(Conjugation assay) 11 2.2.5 突變率測試(Determination of mutation rate) 12 2.2.6 聚合酶鏈鎖反應PCR(polymerase chain reaction) 13 2.2.7 抗生素感受性測試(Antimicrobial susceptibility test) 15 2.2.8 外排幫浦活性(efflux pump activity)測試 16 2.2.9 克雷伯氏肺炎桿菌之RNA萃取 17 2.2.10 RT-qPCR相對定量外排幫浦蛋白AcrA之RNA表現量 18 2.2.11建立具有完整的外膜蛋白OmpK35基因和OmpK36基因之質體 20 2.2.12以具有完整的外膜蛋白OmpK35基因和OmpK36基因之質體補回試驗 20 2.2.13外膜蛋白基因OmpK35和OmpK36定序分析 21 2.2.14 ESBLs和AmpC-β-lactamase基因檢測和定序 21 第三章、結果 22 3.1抗藥盛行莢膜型和非抗藥盛行莢膜型克雷伯氏肺炎桿菌之獲得基因能力…22 3.1.1電穿孔 (Electroporation) 22 3.1.2自然轉形 (Natural transformation) 22 3.1.3接合實驗 (Conjugation assay) 23 3.1.4克雷肺炎桿菌之臨床菌株具有第1類整合子及抗藥基因卡匣 23 3.2抗藥盛行莢膜型和非抗藥盛行莢膜型克雷伯氏肺炎桿菌之突變率 24 3.3 碳青黴烯類抗生素抗藥性菌株具有carbapenemase基因的比例 25 3.4碳青黴烯類抗生素抗藥性菌株之外排幫浦分析 25 3.4.1 克雷伯氏肺炎桿菌之外排幫浦活性(efflux pump activity) 25 3.4.2 以RT-qPCR相對定量外排幫浦蛋白AcrA之RNA表現量 26 3.5外膜蛋白OmpK35及OmpK36之分析 26 3.5.1 以質體補回完整的外膜蛋白OmpK35或OmpK36對碳青黴烯類抗生素抗藥性之影響 26 3.5.2外膜蛋白OmpK35或OmpK36基因序列之分析 27 3.6 CRKP之ESBLs和AmpC-β-lactamase基因分析 28 第四章、討論 29 參考文獻 57 圖目錄 圖一、第1類整合子以及基因匣之引子設計位置 47 圖二、以電穿孔實驗評估臨床菌株之轉形效率結果 48 圖三、以接合實驗評估臨床菌株之接合效率結果 49 圖四、以Rifampicin評估臨床菌株之突變率結果 50 圖五、碳青黴烯類抗生素抗藥性克雷伯氏肺炎桿菌之外排幫浦分析 51 圖六、定序及排列(aligment)外膜蛋白OmpK35之coding region胺基酸序列 52 圖七、定序及排列(aligment)外膜蛋白OmpK35之coding region胺基酸序列 53 圖八、定序及排列(aligment)外膜蛋白OmpK35之promoter DNA序列 54 圖九、定序及排列(aligment)外膜蛋白OmpK36之coding region胺基酸序列 55 圖十、定序及排列(aligment)外膜蛋白OmpK36之promoter DNA序列 56 表目錄 表一、碳青黴烯類抗生素抗藥性克雷伯氏肺炎桿菌之臨床菌株 34 表二、藥物敏感性克雷伯氏肺炎桿菌之臨床菌株 37 表三、本研究使用的引子 38 表四、以電穿孔實驗評估臨床菌株之轉形效率結果 40 表五、以接合實驗評估臨床菌株之獲取質體能力結果 41 表六、臨床菌株具有第1類整合子的比例統計分析圖表 43 表七、第1類整合子基因匣中之定序結果 44 表八、補回外膜蛋白OmpK35或OmpK36對carbapenem抗藥性菌株(CRKP)之MICs變化 45 表九、外膜蛋白OmpK35或OmpK36之基因分析序列總表 46 | |
| dc.language.iso | zh-TW | |
| dc.subject | 碳青黴烯類抗藥性克雷伯氏肺炎桿菌 | zh_TW |
| dc.subject | 莢膜 | zh_TW |
| dc.subject | 外排幫浦 | zh_TW |
| dc.subject | 外膜蛋白 | zh_TW |
| dc.subject | capsule | en |
| dc.subject | carbapenem-resistant Klebsiella pneumoniae | en |
| dc.subject | efflux pump | en |
| dc.subject | outer membrane protein | en |
| dc.title | 碳青黴烯類抗生素抗藥性克雷伯氏肺炎桿菌莢膜型分析和抗藥性機制之探討 | zh_TW |
| dc.title | Capsular type and carbapenem resistance of Klebsiella pneumoniae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 102-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 董馨蓮,楊宏志,蔡丰喬,林稚容 | |
| dc.subject.keyword | 莢膜,碳青黴烯類抗藥性克雷伯氏肺炎桿菌,外排幫浦,外膜蛋白, | zh_TW |
| dc.subject.keyword | capsule,carbapenem-resistant Klebsiella pneumoniae,efflux pump,outer membrane protein, | en |
| dc.relation.page | 65 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2014-08-18 | |
| 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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