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標題: | 甲氧西林敏感金黃色葡萄球菌之抗紅黴素基因結構分析 Genetic structure of erythromycin resistance genes in methicillin-susceptible Staphylococcus aureus |
作者: | Tsai-Wen Wan 萬采玟 |
指導教授: | 鄧麗珍(Lee-Jene Teng) |
關鍵字: | MSSA,MRSA,erythromycin,iMLSB,cMLSB,ermB,ermT, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 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 %。 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). |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57260 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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