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研究生:黃崇偉
研究生(外文):Chung-Wei Huang
論文名稱:包氏不動桿菌臨床菌株的分子特性分析
論文名稱(外文):Molecular Characterization of Clinical Acinetobacter baumannii isolates
指導教授:黃介辰簡麗鳳簡麗鳳引用關係
指導教授(外文):Chieh-Chen HuangLee-Feng Chien
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:98
中文關鍵詞:包氏不動桿菌水平傳播抗藥性轉位子
外文關鍵詞:Acinetobacter baumanniihorizontal transferantibiotic resistanceintegroncassettetransposon
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近年來台灣頻頻出現有最後一道防線號稱的imipenem抗生素具抗藥性的包氏不動桿菌(Acinetobacter baumannii),而且對其他抗生素抗藥性的普遍性也日益嚴重,因而喚起對包氏不動桿菌抗藥性之問題的注意與關切。包氏不動桿菌為常見的環境微生物,是人類皮膚與黏膜上的正常菌叢之一,但卻常使免疫功能缺損患者罹患許多嚴重感染疾病,如肺炎、尿道炎、心內膜炎、傷口感染、腦膜炎與敗血病等等。近年來在對於包氏不動桿菌所引發的嚴重感染疾病之治療上,常會引發多重抗藥性而使抗生素的治療失敗。
本研究以彰化基督教醫院中包氏不動桿菌臨床菌株的流行病學資料為基礎,分析其表現型與基因型的相關性,並探討其抗藥性基因是否經水平傳播的方式散佈於各菌株間之可能性,及其相關機制之解析。使用的臨床菌株以民國九十年四月一日到民國九十一年三月三十一日止,在彰化基督教醫院分離出的三十二株包氏不動桿菌為材料,以E-test與VITEK 2 Advance Expert System等抗生素藥物敏感性測試測定這32株菌株的抗生素抗藥性表現型。在這32株包氏不動桿菌中,較特別的是分別由Disk diffusion experiment與VITEK 2 AES所分別檢測出具有廣泛性抗藥性的1-43與2-45兩隻菌株。
隨即以聚合連鎖反應(PCR)方法進行抗藥性基因blaTEM-1與水平傳播單位integron的研究,發現在1-43與2-45兩菌株中,均帶有blaTEM-1基因(AY560328),經過NCBI BLAST的比對發現,也都與Escherichia coli中的blaTEM-1 基因(AF427133)達100%的相似性,就連非coding區也完全一致,這個結果顯示出菌種間水平傳播的可能性。以此為基礎,本研究再利用Mating Experiment的方法,檢討抗藥性基因水平傳播之移動性。此外,發現這兩株菌所帶有的水平移動單位integron等具cassette (AY557339),且排列方式也完全相同,經由南方墨點法與inverse PCR的結果,也發現這個integron所存在的位置具一致性,顯示抗藥性基因可經由一個比integron更大的水平移動單位進行傳播。
During the last decade, Acinetobacter baumannii strains were noticed as involving in increasingly reported of multiple-drug and imipenem resistance. Acinetobacter spp. are common environmental opportunistic pathogens, they can be found on the skin and mucosa of healthy people. But they cause lots of serious infectious disease such as pneumonia, urinary tract infection, endocarditis infection, wound infection, meningitis infection and septicemia, especially in immunocompromised patients. Antimicrobial treatment of these clinical infections, particularly those caused by A. baumannii strains, may be failed due to compromise by the multiple-resistance strains.
This research was based on the epidemiological study at Changhua Christian Hospital, trying to analysis the relationship between phenotype and genotype of A. baumannii clinic isolates. And the possibility and mechanism of horizontal transfer of those involved antibiotic resistance genes.
A total of 32 A. baumannii strains was isolated during April 1, 2001 through March 31, 2002 from the Microbiology Laboratory of Changhua Christian Hospital. The antibiotic resistance profile was determined by the disk diffusion experiment and VITEK 2 Advance Expert System. Two particular strains, 1-43 and 2-45, among the 32 A. baumannii isolates were identified as pan-drug resistance during the antibiotic sensitivity test and were further studied.
We used the PCR method for detecting the antibiotic resistance gene blaTEM-1 and the mobile genetic unit, integron. We found that both A. baumannii strains 1-43 and 2-45 own an identical Beta-lactamase gene blaTEM-1 (AY560328). Since not only the coding region but also the non-coding region are completely the same as the blaTEM-1 gene identified in Escherichia coli (AF427133), the results strongly indicate the possibility of horizontal transfer. The result from mating experiment also suggested the occurrence of horizontal transfer. The integrons PCR experiments showed that the mobile unit, integrons, found in these two strains are identical(AY557339), and the arrangement of the included cassettes are parallel to each other. By the southern blotting experiment, we also found the two integron may locate in the same genetic structure, and suggests the involvement of transposon. Concluded all the results, we suggested that the A. baumannii may acquire the antimicrobial resistance genes through a complex horizontal gene transfer mechanisms.
摘要 5
Abstract 7
第一章 緒論 9
1-1 現今細菌抗生素抗藥性的嚴重性 9
1-2 葛蘭氏陰性菌對抗抗生素的抗藥性機制 10
1-3 包氏不動桿菌在抗藥性問題上的角色 12
1-4 TEM族群b-lactamase之特性 14
1-5 基因水平傳播單位,Gene cassettes 16
1-6 研究動機與研究目的 21
第二章 材料與方法 22
2-1 菌種與質體 22
2-2 培養基 23
2-3 細菌的培養與保存 23
2-4 菌體總量DNA的萃取 24
2-5 聚合鏈反應(Polymerase chain reaction, PCR) 25
2-6 長距離聚合鏈反應(Long and Accurate PCR, LA PCR) 26
2-7 核酸膠體電泳分析與紀錄 27
2-8 DNA片段回收及純化 28
2-10 核酸純化的鑑定與定量分析 30
2-11 核酸的黏合反應 30
2-12 大腸菌的質體轉形作用(transformation) 31
2-13 大腸菌以及包氏不動桿菌質體萃取 32
2-14 Vitek 2 System藥物敏感性測試方法 33
2-15 以mating conjugation測試抗藥性的水平傳播 34
2-16 南方墨點法(Southern Blotting) 35
2-17 Integron兩端序列的解析 38
第三章 結果 40
3-1抗生素抗藥性表現型結果 40
3-1-1 Disk diffusion experiment 40
3-1-2 VITEK 2 Advance Expert System 41
3-2 b-lactamase TEM-1之探討 42
3-3 基因水平傳播平台Integron分析 43
3-4 藉由轉位子移動的Integron之探討 44
3-5以Mating測試抗藥性的水平傳播 45
3-6 水平移動平台Integron的兩端序列分析 46
3-6-1南方墨點法分析 46
3-6-2 Inverse PCR分析結果 47
第四章 討論 49
4-1 AB1-43與AB2-45中b-lactamase TEM-1特性分析 49
4-2 AB1-43與AB2-45中Integron的序列分析 50
4-3 AB1-43與AB2-45中Integron的存在位置與複合性水平傳播單位的可能性 50
第五章 參考文獻 52
第六章 圖與表與附錄 57
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