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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄧麗珍(Lee-Jene Teng) | |
dc.contributor.author | Tsai-Wen Wan | en |
dc.contributor.author | 萬采玟 | zh_TW |
dc.date.accessioned | 2021-06-16T06:39:33Z | - |
dc.date.available | 2024-12-31 | |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-30 | |
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J Clin Microbiol 44(6): 2268–2270. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57260 | - |
dc.description.abstract | 台灣金黃色葡萄球菌的抗藥性問題嚴重,以紅黴素抗藥狀況為例,在2000年時台灣21間醫院共161株methicillin-suseptible Staphylococcus aureus (MSSA) 中有35 %、239株Methicillin-resistant Staphylococcus aureus (MRSA) 有98 %具紅黴素抗藥性。目前大多針對MRSA的多重抗藥機討論,而MSSA的抗藥問題較被忽略。由Hsueh等人在2011年的統計指出台大醫院MSSA的分離率在1996-2006 年間維持30 % 左右,自2007年起MSSA分離率有上升趨勢,因此本研究選擇探討MSSA的抗紅黴素基因結構與其分子流行病學上的探討。
本研究挑選臺大醫院自血液檢體分離之具紅黴素抗性的MSSA,菌株收集時間為2000-2012年。所有臨床菌株先以nuc、mecA PCR確認確實為MSSA後才進行後續實驗,總共有274株菌株。並以double disk diffusion D test區分菌株抗藥表現型為inducible MLSB (iMLSB)、constitutive MLSB (cMLSB) 或M型,再以agar dilution確認菌株的紅黴素MIC值。分析紅黴素之抗藥基因,結果顯示274株菌株中有97株帶有ermB基因為最多的基因型,且這97株的抗藥表現型皆為cMLSB。分析其spa type、sequence type (MLST,ST) 與pulsotype,發現大多為spa type t437 (71 %)、ST59而pulsotype共28種 ( pulsotype類型較無一致性)。其中發現8株ermB攜帶方式不同於Hung等人發表的結構,以southern blot與conjugation確認這8株ermB攜帶方式結果顯示ermB位於質體DNA上且為單一copy數目,進一步解序發現此質體與Macrococcus caseolyticus的質體pMCCL2 (GenBank accession number AP009486) 相似度達99 %,且這8株為ST5、7、59,進一步篩選388株MRSA發現有6株ST188攜帶ermB基因的結構類似本研究於MSSA發現的結構。此外本研究發現2株帶ermT基因的MSSA,以southern blot確認ermT攜帶方式為插入染色體DNA且為單一copy數目,且將ermT基因插入染色體DNA範圍解序完成約17 kb,與MSSA ST398- t571編號71193 (GenBank accession number CP003045) 的ermT攜帶方式相似度達99 %。 | zh_TW |
dc.description.abstract | The problem of drug resistance in Staphylococcus aureus is serious. In Taiwan, the erythromycin resistance rates were 35 % in 161 methicillin-suseptible Staphylococcus aureus (MSSA) isolates and 98% in 239 Methicillin-resistant Staphylococcus aureus (MRSA) isolates from 21 hospitals in 2000. Most studies discussed about the mechanism of multi-drug resistance of MRSA, but less in MSSA. A previous study by Hsueh et al. indicated that the rate of MSSA isolates in National Taiwan University Hospital (NTUH) was about 30 % during 1996 to 2006, but increased since 2007. In this study, we analyzed the genetic structure of erythromycin resistance genes and molecular epidemiology focusing in MSSA.
A total of 274 erythromycin-resistant MSSA isolates were collected from blood cultures during the period 2000 to 2012 at the NTUH . MSSA was confirmed by the presence of nuc and lack of mecA genes. Determination of the inducible MLSB (iMLSB)、constitutive MLSB (cMLSB) and M phenotype was performed by the double disk diffusion D test. We also determined the MIC of erythromycin by agar dilution. Among erythromycin resistance genes, ermB was the predominant (97 of 274 isolates) determinant, and most were cMLSB phenotype. For ermB-carrying isolates, the majority were spa type t437 (71 %)、ST59 and distributed in 28 pulsotypes. The 8 of 97 isolates ermB-carrying structure were different from that in Hung’s report. By use of southern blot, the plasmid DNA location for the ermB gene was determined, and there was one ermB gene copy number at plasmid DNA. By comparing with the sequences in database, the best matches for the ermB-carrying regions (about 12 kb) were the Macrococcus caseolyticus plasmid pMCCL2 DNA (GenBank accession number AP009486) (99 % identity). The 8 of 97 isolates ST were ST5、7、59. Furthermore, we screened 388 MRSA isolates. The 6 of 388 MRSA isolates ermB-carrying structure were similar to MSSA ermB-carrying structure that founded in this study. Furthermore, we found 2 ermT-carrying MSSA isolates. The chromosomal DNA location for the ermT gene was determined by southern blotting, and there was one ermT gene copy number. The ermT and flanking sequence in one ermT-carrying isolate was determined. By comparing with the sequences in database, the best matches for the ermT-carrying regions (about 17 kb) were the MSSA ST398- t571 number 71193 (GenBank accession number CP003045) (99 % identity). | en |
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dc.description.tableofcontents | 總 目 次
致謝 I 中文摘要 II 英文摘要 IV 前言 1 1.1 金黃色葡萄球菌菌株特性簡介 1 1.2 抗生素藥物與抗藥性菌株產生的狀況概述 2 1.3 Macrolide - Lincosamide - Streptogramins抗藥機制 2 1.4 金黃色葡萄球菌ermB基因攜帶方式 4 1.5 金黃色葡萄球菌ermT基因攜帶方式 4 第二章 材料與方法 6 2.1文獻實驗用菌株 6 2.2寡核苷引子列表 6 2.3萃取金黃色葡萄球菌染色體核酸 (chromosomal DNA) 10 2.4以PCR確認菌株是否為金黃色葡萄球菌 11 2.5以PCR偵測抗紅黴素菌株所攜帶的抗紅黴素基因 12 2.6萃取金黃色葡萄球菌質體核酸 (plasmid DNA) 13 2.7南方墨點法 (southern blot) 14 2.8 LA PCR (Long and Accurate PCR) 18 2.9 InversePCR 21 2.10抗生素敏感性測試 (antibiotic susceptibility test)─最小抑菌濃度(Minimum Inhibitory Concentration,MIC) 23 2.11利用double disk diffusion 鑑別iMLSB 表現型、cMLSB 表現型與M型 24 2.12分析菌株spa type 25 2.13利用MLST (MultiLocus Sequence Typing) 分析菌株ST type (Sequence Type) 27 2.14大片瓊脂膠電泳條件 (質體分析使用大片膠體進行電泳) 28 2.15利用PFGE (Pulsed-Field Gel Electrophoresis) 分析菌株親緣關係 28 2.16 Elimination of plasmid 30 2.17 Conjugation 31 三、結果 32 3.1 篩選實驗菌株 (具紅黴素抗性的甲氧西林敏感性金黃色葡萄球菌 (MSSA)) 32 3.2 抗紅黴素基因的分佈 32 3.3 double disk diffusion 33 3.4 以agar dilution確認菌株紅黴素MIC值 34 3.5 spa type分析 34 3.6 以PCR分析帶ermB 基因菌株的基因體結構 35 3.7 以agar dilution確認菌株對其他藥物的MIC值 36 3.8 以MLST (Multilocus Sequence Typing) 分析特殊ermB攜帶菌株的sequence type (ST type) 37 3.9 PFGE分析 38 3.10 以MLST分析ermB攜帶菌株的ST type 38 3.11 以southern blot確認菌株ermB的攜帶 38 3.12 以LA (longaccurate) PCR與inverse PCR進行攜ermB的質體DNA解序 40 3.13以 PCR mapping分析以質體攜帶ermB基因的結構 40 3.14篩選MRSA菌株調查是否有以質體攜帶ermB基因的案例 41 3.15分析MRSA以質體攜帶ermB基因菌株的spa type、SCCmec type、ST 42 3.16 以southern blot確認MRSA菌株ermB的攜帶 42 3.17以conjugation確認ermB基因位於質體且提供紅黴素之抗性 43 3.18 Elimination of plasmid 44 3.19 篩選具ermT基因的菌株 45 3.20以southern blot確認菌株ermT基因的攜帶 45 3.21 以LA PCR與inverse PCR解序ermT基因插入染色體DNA位點 46 3.22 以PCR分析攜帶ermT基因的結構 46 四、討論 47 4.1 攜帶ermB的MSSA菌株在台灣北部流行 47 4.2 cMLSB表現型菌株以攜帶ermB基因的菌株為主要流行菌株 47 4.3 iMLSB表現型菌株盛行的隱憂 48 4.4 菌株攜帶相同作用之抗藥基因 49 4.5 MSSA基因型與抗藥表現型的關聯性 49 4.6 分子分型分析細菌演化關係之比較 50 4.7 攜帶ermB基因菌株之質體基因結構 51 4.8 攜帶ermB基因之質體的transfer frequency 51 4.9攜帶質體ermB基因之MRSA與MSSA的ST比較 51 4.10 攜帶質體ermB基因之MSSA與MRSA的抗藥樣式 52 4.11 攜帶ermT基因菌株之染色體基因結構 52 第五章 附圖 54 第六章 附表 68 第七章 參考文獻 76 圖 目 次 【圖一】MSSA攜帶之紅黴素抗性基因在2000-2012年的百分比 54 【圖二】以double disk diffusion結果判別紅黴素抗藥菌株表現型 55 【圖三】以agar dilution確認菌株紅黴素MIC 值 56 【圖四】以sasK基因完整性確認ermB是否插入染色體DNA 57 【圖五】具ermB基因的97株菌株PFGE結果 58 【圖六】以southern blot確認MSSA菌株ermB的攜帶 59 【圖七】解序列策略示意圖 60 【圖八】ermB攜帶菌株NTUH-3874解序列結果 61 【圖九】以PCR mapping 分析其他ermB攜帶菌株之基因結構 62 【圖十】以southern blot確認MRSA菌株ermB的攜帶、比較MRSA與MSSA攜帶ermB基因結構的異同 63 【圖十一】確認transconjugants以質體攜帶ermB 64 【圖十二】以southern blot確認NTUH-8300菌株ermT、cadD的攜帶 65 【圖十三】ermT攜帶菌株NTUH-3874解序列結果 66 【圖十四】以PCR mapping 分析其他ermT攜帶菌株之基因結構 67 表 目 次 【表一-a】臨床血液檢體分離MSSA菌株列表 68 【表一-b】臨床非血液檢體分離MSSA菌株列表 70 【表一-c】臨床血液檢體分離MRSA菌株列表 70 【表二】以PCR確認抗紅黴素菌株攜帶之抗藥基因 71 【表三】紅黴素抗藥菌株表現型與基因型分佈 72 【表四-a】攜帶ermB基因MSSA菌株的spa type分析 73 【表四-b】攜帶ermB基因MRSA菌株的spa type分析 73 【表五】MLST分析結果 74 【表六】conjugation的transfer frequency 75 | |
dc.language.iso | zh-TW | |
dc.title | 甲氧西林敏感金黃色葡萄球菌之抗紅黴素基因結構分析 | zh_TW |
dc.title | Genetic structure of erythromycin resistance genes in methicillin-susceptible Staphylococcus aureus | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 廖淑貞,邱浩傑,曾嵩斌 | |
dc.subject.keyword | MSSA,MRSA,erythromycin,iMLSB,cMLSB,ermB,ermT, | zh_TW |
dc.relation.page | 82 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-07-30 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-103-1.pdf 目前未授權公開取用 | 5.39 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。