臺灣博碩士論文加值系統

包氏不動桿菌(Acinetobacter baumannii，簡稱AB菌)是一種新興的伺機性革蘭氏陰性致病菌，近年來逐漸對許多抗生素產生抗藥性，在國內已陸續出現對carbapenem 類抗生素具抗藥性的AB菌。為了瞭解多重抗藥性包氏不動桿菌(multidrug-resistant resistant Acinetobacter baumannii；簡稱MDRAB )對carbapenem 類藥物產生的抗藥性基因差異及院內感染及非院內感染菌株的流行分布，因此收集童綜合醫院自2010年1月至12月間分離的A. baumannii共計91株進行研究；其中包含院內感染菌株36株，非院內感染41株，及14株加護病房環境採檢菌株。其中55株為carbapenem resistant A. baumannii (CRAB），包括36株醫院相關感染個案、13株環境採檢及6株與非醫院感染相關。這55株CRAB均呈現多重抗藥性，且55株CRAB中有53株具有OXA-23(53/55；96.4%)，9株具有OXA-24(9/55；16.4%)。本研究中所有菌株均未帶有IMP、VIM、OXA-58 等其他抗carbapenem基因。分析91株 A. baumannii PFGE分型共有45種基因型，加護病房之AB主要來自4個主要cluster：cluster Z 12株；cluster J 8株；cluster V 7株；及cluster T 5株。MLST 分析顯示本研究91株菌株中有68株（75%）屬於clonal complex 2。比較PFGE及MLST兩種分子生物學方法，PFGE具有較高鑑別力，依親緣性能區分更多型別。此外院內相關及非院內相關菌株在多重抗生素抗藥性、oxacillinase 基因組成皆有顯著差異。本研究結果可應用於協助制訂防止CRAB進一步擴散及流行之策略。

Acinetobacter baumannii, an important emerging opportunistic pathogen, recently showed a significantly increasing incidence of resistance to multiple antibiotics including carbapenem. The study was conducted to explore the difference between healthcare associated and non-healthcare associated multidrug-resistant resistant A. baumannii (MDRAB) in antibiotic resistance mechanisms and epidemiology. A total of 91 isolates of A. baumannii, including 36 isolates recovered from patient with healthcare-associated infection, 41 isolates from non-healthcare associated infection, and 14 isolates from environment surveillance, were collected from Tungs’ Taichung MetroHarbor Hospital during the period: Jan 2010 and Dec 2010. Fifty-six out of 91 isolates were carbapenem-resistant, including 36, 6, and 13 isolates from patient with healthcare-associated infection, patient with non-healthcare associated infection, and environment surveillance of intensive care units, respectively. All carbapenem-resistant A. baumannii (CRAB) were multidrug-resistant. Fifty-three out of 56 (94.6%) CRAB strains harbored OXA-23, and 9 strains (16.1%) harbored OXA-24. IMP- or VIM-type metallo-β-lactamases were not detected. The analysis of PFGE patterns showed 45 clusters (< 80% similarity). Most isolates of A. baumannii of intensive care units belonged to 4 clusters: cluster Z, 12 isolates; cluster J, 8 isolates; cluster V, 7 isolates, and cluster T, 5 isolates. MLST analysis revealed 68 out of 91 isolates (75%) belonged to clonal complex 2. Above results indicated that PFGE has a higher discriminatory power than MLST in A. baumannii typing. There were significant difference between clonal complex 2 isolates and others in multiple antibiotics resistance and oxacillinase types。The study results can help to build a strategy to prevent CRAB spreading.

摘要（中文）…………………………………………………………………………...i
摘要（英文）……………………………………………………………………….…iii
目次..……………………………………………………………………………………v
圖目次…………………………………………………………………………………vii
表目次…………………………………………………………………………….…..viii
附錄………………………………………………………………………………...…..ix
一、 前言……………………………………………………………………………..1
（一） 研究背景及動機…………………………………………………………1
1. 全球細菌抗藥性發展趨勢………………………………………………1
2. 國內細菌抗藥性現況……………………………………………………2
3. 研究目的…………………………………………………………………4
（二） 包氏不動桿菌……………………………………………………………7
1. 命名及特性………………………………………………………………7
2. 微生物學特徵……………………………………………………………7
3. 培養特性…………………………………………………………………8
4. 生化特性…………………………………………………………………9
5. 致病性及危險因子………………………………………………………9
（三） 抗生素的總類與功能……………………………………….…………..11
（四） 乙內醯胺類抗生素介紹………………………………………………...12
（五） 碳青黴烯類抗生素介紹……………………………………………...…13
（六） 碳青黴烯酶簡介…………………………………………………...……14
（七） 多重抗藥包氏不動桿菌抗藥機制……………………………………...16
（八） 乙內醯胺酶的分類系統……………………………………………...…18
（九） 多位基因序列介紹……………………………………………………...19
（十） 包氏不動桿菌在院內感染所扮演的角色……………………………...20
二、 材料與方法…………………………………………………………………….21
（一） 研究材料…………………………………………………………….…..21
（二） 菌株收集………………………………………………………….……..22
1. 定義……………………………………………………………………...22
2. 菌株來源………………………………………………………………...23
3. 菌株保存………………………………………………………………...23
（三） 菌株鑑定與藥物敏感性試驗方法……………………………………...25
1. 鑑定流程…………………………...……………………………………25

2. 藥物敏感性試…………………………………………………….……..25
3. 自動化儀器分析…………………………………………………….…..25
4. E test……………………………………………………………………..26
（四） 菌體總量DNA的萃取方法……………………………………………28
（五） 聚核酶連鎖反應…………………………………………….…………..29
（六） 核酸膠體脈衝電泳分析法……………………………………………...30
1. 菌體溶解及包埋步驟…………………………………………………...30
2. 膠塊清洗………………………………………………………………...30
3. 限制酵素消化切割……………………………………………………...31
4. 電泳操作流程及設定條件……………………………………………...31
5. 染色及照膠……………………………………………………………...32
6. 圖譜分析………………………………………………………………...33
（七） 多位基因座序列分析法………………………………………………...34
1. 核苷酸序列……………………………………………………………...34
2. 操作流程………………………………………………………………...34
（八） 統計分析方法…………………………………………………………...36
SPSS statistic 17.0…………………………………………………………...36
三、 結果…………………………………………………………………………….37
（一） 菌株分離來源分析……………………………………………………...37
（二） 藥物感受性試驗及最低抑菌濃度測量………………………………...39
（三） 具有碳青黴烯酶之包氏不動桿菌分類………………………………...41
（四） 核酸膠體脈衝電泳菌株比對結果……………………………………...42
（五） 多位基因座序列比對結果……………………………………………...43
四、 討論…………………………………………………………………………….44
（一） carbapenem具抗藥性的包氏不動桿菌(carbapenem resistant Acinetobacter baumannii; CRAB)的比率大幅上升……………..….44
（二） 包氏不動桿菌在本院造成院內感染的流行病學現況…………….…..46
（三） PFGE分型與院內感染的關係…………………………………………48
（四） Complex clonal ST2 與Carbapenem resistant A. baumannii…….51
（五） 比較多位基因座序列分析法和脈衝式電泳分析法……………….…..53
五、 結論…………………………………………………………………………….54
六、 參考文獻……………………………………………………………………….56

中文
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