臺灣博碩士論文加值系統

鮑曼尼氏不動桿菌(Acinetobacter baumannii)已成為院內感染最常見的致病菌種，尤其是在加護病房內常引起與呼吸器有關的肺炎及菌血症。由於此菌的抗生素抗藥性的快速增加，已產生了多重抗藥性及全抗藥性的菌株，導致臨床上無藥可用的窘境。本研究的目的主要是探討在沒有抗生素的壓力之下，抗藥性鮑氏不動桿菌是否能改變其抗藥性。
由3家醫學中心蒐集而來的10株抗藥性鮑氏不動桿菌，進行在沒有抗生素壓力的環境下每天連續繼代培養為期一年，並利用抗生素紙錠擴散試驗和最低抑制濃度測試等2種方法來觀察其抗藥性的變化。結果發現10株在繼代培養90天後有2株(編號AB#1及AB#10)原本對amikacin呈抗藥性的菌株已轉變成感受性(從MIC>64μg/ml轉變為MIC 8 μg/ml)。同時利用2種基因分型的方法，ribotyping(用EcoR1切割)及PFGE (用Apa1切割)鑑定，確認此2株對amikacin具感受性的菌株與原本對amikacin具抗藥性的菌株為相同的基因型。
我們針對amikacin的已知的抗藥性基因(armA、rmtA、rmtB、rmtC、rmtD、aac(6')-Ib、ant(4’)-Ia、aph(3’)-IIIa)作PCR分析，而PCR的結果，確定此2株對amikacin具抗藥性的菌株(AB#1及AB#10)皆喪失了armA基因，並且其中1株(AB#10) 連同aac(6')-Ib 基因一併喪失。在過去的研究報告皆認為armA基因是存在於質體上，但我們也利用限制酵素I-ceu1切在23S rRNA的特性來證明armA存在染色體上的可能。質體的雜交和接合試驗無法排除位於質體的可能性，而染色體雜交試驗得到的結果，也無法確定armA是位在染色體上。經過DNA序列分析後，發現喪失的armA基因前後有跳躍子的存在。
至於這些抗藥性鮑氏不動桿菌所喪失的其他抗藥機制則需再作進一步的研究。

Acinetobacter baumannii is becoming an important pathogen causing nosocomial infections including ventilator-associated pneumonia and bacteremia especially in intensive care units. Because the rapid emergence of mutlidrug resistant- (MDR) and pandrug resistant-A. baumannii (PDRAB) in recent years, it is difficult to choose the effective antibiotics resulting a serious treatment problem. This study is designed to investigate the possibility of changing antimicrobial susceptibility of PDRAB after a subsequent daily subculture without antibiotic selection.
A total of 10 strains of DRAB were collected from 3 medical centers in Taiwan and serial daily sub-cultured without antibiotic selection for 360 days. Antimicrobial susceptibility test including disk diffusion test and minimum inhibitory concentration (MIC) were used to check the changes of resistance. There were 2 strains (AB#1 and AB#10) lost their resistance to amikacin (MIC > 64 μg/ml to MIC 8 μg/ml) after 90 days. Ribotyping (digested by Eco R1) and pulsed-field gel electrophoresis (digested by Apa 1) were used to identify these 2 susceptible strains showed that they were identical with their parent strains (resistant to amikacin).
We used PCR method to check the amikacin resistant genes (armA, rmtA, rmtB, rmtC, rmtD, aac(6')-Ib、ant(4’)-Ia、aph(3’)-IIIa) and found that both 2 resistant strains (AB#1 and AB#10) lost armA gene and 1 of them (AB#10) even lost aac(6')-Ib gene concomittantly. Although previous studies suggested that armA gene is plasmid-mediated, we did not find the armA gene in the plasmid after hybridization and conjugation of plasmid. The determination of chromosomal location was also did not show a positive result. However, the lost armA gene was found flanked by 2 transposons after DNA sequence analysis.
As for other lost resistance of these 10 strains of PDRAB and MDRAB, it is required for further study.

封面內頁………………………………………………...………………….i
中文摘要……………………………...………………………...………….ii
英文摘要…………………………………..…….………………...………iii
目錄…………………………………….……….…………………...……..v
圖目錄………………………………………….…………………...…..…ix
表目錄…………………………………..…………………………...……..x
附錄………………………………………………………………………..xi

第一章 緒論……………………………………………………….……..1
1.1 Acinetobacter之簡介……………….………..…….………...……...…1
1.2 Acinetobacter臨床上之分類及鑑定……………………………......…3
1.3 細菌抗藥性偵測……………………………………………...…...…..3
1.4 Aminoglycoside的作用機制………………….…………………..…...4
1.5 Aminoglycoside的抗藥機制…………………...…………………...…6
1.5.1 藉由幫浦排出藥物至菌體外………………………………...…6
1.5.2 產生酵素改變會被抗生素作用的標的位置…………...………6
1.5.2.1 armA gene………………………………………...…...…7
1.5.2.2 rmtA，B，C，D gene……………………………………7
1.5.3 產生酵素修飾aminoglycoside………………….……..…...……8
1.5.3.1 aminoglycoside acetyltransferases（AACs）……….…..8
1.5.3.2 aminoglycoside nucleotidyltransferases（ANTs）….….9
1.5.3.3 aminoglycoside phosphotransferases（APHs）…..…….10
1.6 Pulsed-field gel electrophoresis（PFGE）………………………….…10
1.7 Ribotyping………………………………………………………...…..11
1.8 抗藥基因之傳播……………………………………………....……..12
1.9 利用Solexa科技分析DNA序列………………………………...….13
1.10 微生物族群的抗藥性變化…………………………………...…….13
1.11 研究動機及目的………………………………………………...….14

第二章 材料與方法…………………………………….……………....15
2.1 實驗設計…………………………………………………..……...….15
2.2 菌株來源………………………………………………………...…...16
2.3 每天連續繼代培養方法……………………………..……...……….16
2.4 抗藥性試驗…………………………………………………......……17
2.4.1 紙錠擴散感受性試驗…………………………………...……17
2.4.2 抗生素最低抑制濃度……………………………………...…17
2.5 脈衝式膠體電泳PFGE…………………………………………..…..18
2.5.1 菌體培養…………………………………………………...…18
2.5.2 菌體包埋及菌體溶解…………………………...……………18
2.5.3 膠體清洗……………………………………………...………18
2.5.4 限制酵素切割………………………………………...………19
2.5.5 脈衝式電場電泳……………………………………...………19
2.6 Ribotyping……………………………………………………...……..20
2.7 利用聚合酶連鎖反應偵測amikacin抗藥基因…………….....……..20
2.8 PCR產物之純化……………………………………………......…….22
2.9 序列資料分析……………………………………………………..…22
2.10 armA基因在菌體位置之分析……………….……………………..23
2.10.1 armA基因位於染色體上之分析………………..……...……23
2.10.2 armA基因位於質體上之分析……………………..…...……24
2.10.2.1 質體DNA純化……………………………………24
2.10.2.2 質體用armA做Southern hybridization……......….24
2.10.2.3 質體接合轉移試驗Conjugation………………..…24
2.10.2.4 質體電穿孔轉移試驗Electroporation……..…...…25
2.11 南方雜交法Southern blot………………………………...…..……26
2.11.1 Southern blot……………….……………..……..…...….….26
2.11.2 Hybridization (DIG DNA Labeling and Detection Kit)...…..27
2.11.3 薄膜轉印……………………………………………...……28
2.11.4 探針製作………………………………………………...…28
2.11.5 雜交與呈色…………………………………………...……29
2.12 編號AB#1菌株之DNA序列分析……….………………………..30
2.13 微生物族群的抗藥性變化…………………………………......…..31

第三章 結果………………………………………………….…...…….32
3.1 抗生素感受性試驗測試結果…………………………………...…...32
3.1.1 Disk diffusion zone……………………………………...……..32
3.1.2 MIC……………………………………………………..……..32
3.2 基因分型結果………………………………………………......……33
3.2.1 Ribotyping的分子分型結果…………………………...……..33
3.2.2 PFGE的分子分型結果………………………………...……...33
3.3 Amikacin抗藥基因的PCR結果………………………..……...........33
3.3.1 armA抗藥基因的PCR結果………………..…....…………….33
3.3.2 rmtA、B、C、D抗藥基因的PCR結果…………………………33
3.3.3 AME抗藥基因的PCR結果……………………….……...……34
3.4 抗藥基因序列資料分析結果…………………………………......…34
3.5 抗藥基因armA位置之分析……………………………………...….34
3.5.1 抗藥基因armA位於染色體之分析結果………………….…34
3.5.2 抗藥基因armA位於質體之分析結果………….………...….34
3.5.2.1 Southern hybridization…………………………...…..34
3.5.2.2 質體接合轉移Conjugation試驗結果……………….35
3.5.2.3 質體電穿孔轉移Electroporation試驗結果…...….…35
3.6 編號AB#1之DNA序列分析結果……………………………….…35
3.7 微生物族群的抗藥性變化結果……………………………...…...…36

第四章 討論……………………………………………………...……..37

第五章 結論……………………………………………..………...……41

參考資料………………………………………..……………...…………42











圖目錄
圖1. Ribotyping的分子分型結果圖(EcoR1)…………………….…….47
圖2. PFGE的分子分型結果圖(Apa1)…………………..………….…..48
圖3. PCR電泳圖…………………………………………...……...……49
圖4. Southern blot of chromosome結果圖………………………..……52
圖 5. Southern blot of plasmid結果圖……………………………...…..53
圖 6. 編號AB#1 DNA sequence結果圖…………..…………....….….54


















表目錄
表1. 10株鮑氏不動桿菌菌株在未作繼代培養前之MIC判讀結果….55
表2.利用紙錠擴散測試法測試已從抗藥性改變為感受性之菌株結果.56
表3.對amikacin呈現感受性的2株菌株之最低抑制濃度結果…..…..57
表4. PCR引子表…………………………………………………....……58
表5.利用PCR之方法偵測aminoglycosid抗藥性基因之結果….……..59
表6.細菌族群抗藥基因分布結果………………………...…..……....…60


















附錄
附錄1. Acinetobacter baumanni佔院內感染菌百分比統計表…..……..61
附錄2. Acinetobacter baumanni菌落形態及鏡檢形狀…………………62
附錄3. 抗生素紙錠擴散檢測示意圖………………………………..….63
附錄4. aminoglycoside的分類…………………….…………………….64
附錄5. aminoglycoside化學結構圖…………………………….………..65
附錄6. aminoglycoside的作用機轉圖……………………………….…..66
附錄7. aminoglycoside抗藥機轉圖…………………………………..….67
附錄8. aminoglycoside modifying enzyme抗藥機轉圖……………..….68
附錄9. 南方雜交法流程圖………………………………………….…..69

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