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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
| dc.contributor.author | Yun-Ting Tsai | en |
| dc.contributor.author | 蔡昀庭 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:49:08Z | - |
| dc.date.available | 2020-09-25 | |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-07 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53177 | - |
| dc.description.abstract | 近年來克雷伯氏肺炎桿菌(Klebsiella pneumoniae)造成的院內感染以及社區型肝膿瘍的案例不斷上升，抗藥性的報導也持續增加，從帶有超廣效β-內醯胺酶(Extended spectrum beta-lactamase; ESBL)基因的克雷伯氏肺炎桿菌，到近年來對最後一線藥物碳青黴烯類(carbapenem)藥物也出現抗藥性，產生了carbapenem- resistant K. pneumoniae(CRKP)。老虎黴素(tigecycline)是新一代的藥物，可以用來治療CRKP的感染，在全球陸續有抗藥性的報導。目前文獻指出在克雷伯氏肺炎桿菌中，造成老虎黴素抗藥性的原因主要為AcrAB-TolC外排幫浦(efflux pump)及OqxAB外排幫浦及一個新的外排幫浦KpgA大量表現，也有報導指出30S核醣體次單元S10 蛋白質的突變也會降低對老虎黴素的感受性。本研究收集分離自台北榮總31株抗老虎黴素菌株並研究其抗藥機轉，發現有14株有AcrAB-TolC外排幫浦過量表現的情形及12株有OqxAB外排幫浦過量表現，此外也有5株KpgA外排幫浦表現量上升，另外有1株帶有30S核醣體次單元S10 蛋白質突變，而仍有12株未帶有目前所報導的抗藥機轉，其中有一株臨床菌株229其最小抑菌濃度(minimal inhibitory concentration; MIC )高達64 μg/ml，為了瞭解其抗藥機轉，我們建構表現基因庫(Expression library)以尋找抗藥機制。篩選結果發現帶有tetracycline resistance protein(TetA)，會使大腸桿菌對於老虎黴素抗藥性顯著上升，而後tetA補至老虎黴素感受性克雷伯氏肺炎桿菌菌株NTUH-K2044染色體上，可以使其對於老虎黴素MIC上升2倍，隨後發現tetA基因位於菌株229中大小約為10kb的質體上，將此質體送入NTUH-K2044中也可使其MIC上升8倍，而我們在菌株229中將tetA剔除，發現其MIC下降4倍至16 μg/ml。因此在本研究中，我們證實在克雷伯氏肺炎桿菌中tetA能夠造成老虎黴素的抗藥性。此外也發現菌株229中ramR基因被transposase插入，將野生型的ramR補回至229中可使MIC下降8倍，進一步補回229 tetA基因剔除株中可使MIC下降至1 μg/ml回到敏感型，因此確認菌株229高抗藥性的情形藉由tetA過度表現和ramR失去功能這兩個機轉參與。 | zh_TW |
| dc.description.abstract | Recently, nosocomial infection and community-acquired disease caused byKlebsiella pneumoniae were increasing. A large proportion of them developed drug resistance including ESBL-producing K. pneumoniae or even carbapenem-resistant K. pneumoniae(CRKP) which confer resistance to last-line drugs like carbapenem and monobactam. However, tigecycline is first glycylcycline drugs derived from tetracycline which were used to treat CRKP infected patients. But bacteria also acquired tigecycline resistance recently. Previous studies indicated that overexpression of efflux pump such as AcrA pump, OqxA pump or KpgA pump contributed in tigecycline resistance of K. pneumonia. Mutation of S10 protein which is a subunit of 30S ribosome was also reported to involve in tigecycline resistance. In this study, 31 tigecycline-resistant K. pneumoniae strains from Taipei Veterans General Hospital(VGH) were collected. Among 31 tigecycline-resistant K. pneumonia strains, 14 isolates possessed overexpression of AcrA pump, 12 strains possessed overexpression of OqxA pump, 5 strains acquired overexpression of KpgA pump and 1 isolate had S10 protein mutation. Therefore, no reported mechanisms were detected in 12 isolates including one isolate (229) with high minimal inhibitory concentration(MIC) up to 64 μg/ml. In order to elucidate its resistant mechanism, we constructed an expression library. After selection, we found that harboring tetA gene in E.coli can increase tigecycline MIC significantly. Complementation of tetA gene in the sensitive K. pneumoniae strain NTUH-K2044 also increased tigecycline MIC by two fold. Furthermore, deletion of tetA gene in strain 229 decreased its tigecycline MIC by four fold. We found that tetA gene was located in a plasmid with a size of 10kb in strain 229. Transfering this plasmid to NTUH-K2044 resulted in increased MIC to tigecycline from 1 μg/ml to 8 μg/ml. Therefore, in this study we demonstrated that tetA gene involved in tigecycline resistance. On the other hands, we found that ramR gene was inserted by a transposase in strain 229. Complementation of an intact ramR gene in 229ΔtetA strain decreased tigecycline MIC to 1 μg/ml. In conclusion, strain 229 aquired high-resistance to tigecycline by two mechanisms including tetA overexpression and ramR mutation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T16:49:08Z (GMT). No. of bitstreams: 1 ntu-104-R02445113-1.pdf: 1936260 bytes, checksum: 09c78c1d1a7ad3db3272bb3900be5900 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 口試委員審定書 I 致謝 II 中文摘要 III Abstract V 目錄 VII 表目錄 X 圖目錄 XI 第一章、緒論 1 1.1 克雷伯氏肺炎桿菌簡介 1 1.2 帶有超廣效β-內醯胺酶(Extended spectrum beta-lactamase; ESBL)基因的克雷伯氏肺炎桿菌(klebsiella pneumoniae) 1 1.3抗carbapenem類的克雷伯氏肺炎桿菌(carbapenem-resistant klebsiella pneumoniae; CRKP) 2 1.4多重抗藥性克雷伯氏肺炎桿菌的臨床治療 3 1.5 老虎黴素(Tigecycline, TGC)，新一代的glycylcline的藥物 4 1.6 研究動機 5 第二章、材料與方法 6 2.1材料 6 2.1.1菌株與載體 6 2.1.2培養基(Media) 6 2.1.3抗生素(Antibiotics) 6 2.1.4引子(Primers) 6 2.2方法 7 2.2.1抗生素感受性測試(Antimicrobial susceptibility test) 7 2.2.2萃取克雷伯氏肺炎桿菌的全基因表現體(total RNA) 8 2.2.3反轉錄定量聚合酶連鎖反應(Quantitative real-time reverse- 8 2.2.4建構表現型基因庫 10 2.2.5基因補回細菌基因體上(chromosomal complementation) 12 2.2.6克雷伯氏肺炎桿菌突變株庫 13 2.2.7突變株插入之序列分析 14 2.2.8篩選抗藥性下降突變株之方法 15 2.2.9克雷伯氏肺炎桿菌基因剔除株之建構 16 2.2.10基因剔除 16 第三章、結果 18 3.1、抗生素感受性測試 (Susceptibility Testing) 18 3.2、外排幫浦表現量(Expression Level of Efflux Pump) 18 3.3、探討外排幫浦上游調控基因 19 3.4核醣體S10蛋白質定序 19 3.5、建構表現型基因庫(Construction of Expression Library) 20 3.6再次轉型作用(Retransformation) 22 3.7將tetA和pecM利用染色體互補至NTUH-K2044 22 3.8將菌株229中帶有tetA的質體送至NTUH-K2044 22 3.9建構229菌株的tetA基因剔除突變株 23 3.10 tetA基因在老虎黴素抗藥菌株的盛行率(prevalence) 23 3.11、建構突變型基因庫(Construction of Mutant Library) 23 3.12建構菌株229的無抗生素標記基因剔除突變株 24 3.13 建構菌株229基因剔除株 25 3.14 突變株41-4B及229ΔromA的外排幫浦RNA表現量 25 3.15將ramR補回至菌株229ΔtetA 25 3.16總結(conclusion) 26 第四章、討論 27 4.1外排幫浦RNA表現量分析 27 4.2探討幫浦上游調控基因 28 4.3表現型基因庫的涵蓋度與篩選 28 4.4突變基因庫篩選結果探討 30 4.5臨床菌株229對老虎黴素高抗藥性的原因 32 參考文獻 62 | |
| dc.language.iso | zh-TW | |
| dc.title | 克雷伯氏肺炎桿菌對老虎黴素之抗藥機制研究 | zh_TW |
| dc.title | Tigecycline Resistant Mechanism(s) in Klebsiella pneumoniae strains | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡丰喬(Feng-Chiao Tsai),董馨蓮(Shin-Lian Doong),林雅容 | |
| dc.subject.keyword | 克雷伯氏肺炎桿菌,抗藥性,老虎黴素,外排幫浦,四環黴素抗藥基因, | zh_TW |
| dc.subject.keyword | Klebsiella pneumonia,tigecycline,efflux pump,AcrA,ramR,OqxA,tetracycline-resistant gene(tetA), | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-07 | |
| 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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