臺灣博碩士論文加值系統

抗藥感染性微生物劇增是日益嚴重的世界問題，針對最後一線抗生素「碳青黴烯類 (carbapenem)」藥物，在濫用下導致許多抗藥性細菌的出現。臺灣2010年的調查，發現有6%的臨床克雷伯氏肺炎菌被偵測出帶有碳青黴烯分解酶基因，但不含克雷伯氏肺炎菌碳青黴烯分解酶 (Klebsiella pneumonia carbapenemase, KPC)。但在2012年時，帶有碳青黴稀分解酶的臨床克雷伯氏肺炎菌上升至22.3%，其中有75%為KPC-2。KPC-2基因坐落於Tn3型轉因子 (Tn4401) 上，可轉移至革蘭氏陰性細菌的質體中，作為傳播來源。且由於克雷伯氏肺炎菌存在於腸道中，所以KPC-2基因會轉移至其他菌種，導致KPC-2基因的擴散。目前在臨床微生物實驗室中尚未有有效檢測KPCs基因的方法，為了有效提升檢測方式，本研究目標是以高效率抗體為平台，用來檢測臨床菌株是否表現KPC碳青黴烯分解酶，作為臨床投藥參考指標。
首先自臨床對碳青黴烯抗生素有抗藥性的克雷伯氏肺炎菌 (Carbapenem-resistant K. pneumonia , CRKP)，利用菌落PCR取得KPC-2基因片段，接合至pQE30表現載體，並轉形至E. coli M15中，以IPTG誘導大量KPC-2蛋白表現，經Ni-column來純化，並進一步利用SDS-PAGE將蛋白完全純化，再以免疫兔子產生抗血清。
取得的KPC-2抗血清利用西方墨點法及斑點墨點法偵測純化的KPC-2蛋白，顯示具高度敏感性。再用此抗血清檢測五十株臨床的CRKP，發現表現KPC蛋白的共有二十六株 (52%)。為確定西方墨點法檢測的正確性，利用菌落PCR檢測KPC基因，也發現西方墨點法為陽性的二十六株菌皆攜帶有KPC基因，而西方墨點法陰性的菌株則沒有PCR的產物。這些西方墨點法陰性菌株的抗碳青黴烯抗生素的機制正在調查中。本研究數據顯示,以抗體檢測CRKP是否帶有KPC是有效且快速的方法。

Antibiotic resistance among infectious microorganisms is a serious and growing crisis worldwide. The carbapenem antibiotics are drugs of last resort, however, their misuse has already led to carbapenem-resistant bacteria. In Taiwan carbapenemase genes were detected in only 6.0% of Klebsiella pneumoniae isolates but no Klebsiella pneumoniae carbapenemase (KPC)-producing isolates detected in 2010. However, in 2012 the carbapenemase genes were detected in 22.3% of K. pneumoniae isolates and 75% of them are KPC-2. The KPC carbapenemase gene has been identified within Tn3-type transposon, Tn4401, and it can insert into diverse plasmids of Gram-negative bacteria and functions as origin of blaKPC-like gene dissemination. Identification of isolates harboring KPCs has proved to be especially challenging in clinical microbiology laboratories. The aim of this study is to establish a quick antibody-based detecting platform to screen KPC-2 harboring clinical isolates. The outcome can serve as a reference for clinical antibiotic administration.
The KPC-2 gene from a carbapenem-resistant K. pneumonia (CRKP) isolate was obtained by colony PCR, subcloned into pQE30 expression vector and KPC-2 protein was overproduced as insoluble inclusion bodies upon IPTG induction in E. coli M15. The urea-solubilized KPC-2 proteins were purified with Ni-column and further resolved by SDS-PAGE to exclude trace contaminates and used to immunize rabbit to produce antiserum against KPC-2 protein by a commercial biotech company.
The KPC-2 antiserum showed high sensitivity toward purified KPC-2 protein by Western blot and Dot blot. Screening 50 clinical CRKP isolates showed that 26 (52%) of them expressing KPCs proteins by Western blot using this KPC-2 antiserum. Colony PCR also confirmed presence of KPCs genes in these 26 corresponding immunoblot-positive isolates, but not in other immunoblot-negative isolates. The mechanisms of carbapenem resistance of those KPC-negative isolates are being under investigation. Nevertheless, our data suggested that this antibody-based method can serve as a quick and efficient screening for KPC-harboring CRKP.

謝辭 i
中文摘要 ii
Abstract iii
目錄 iv
表目錄 viii
圖目錄 ix
第一章 簡介 1
1. 前言 1
2. 克雷伯氏肺炎菌 (Klebsiella pneumoniae) 2
3. β-lactam類抗生素 3
3-1. Carbapenems 3
4. β-lactam類抗生素的抗藥機制 4
4-1. 輸出幫浦的表現 (Efflux pumps) 4
4-2. 運輸孔蛋白變異 (Porin mutation) 4
4-3. 改變藥物標的結構 5
4-4. 產生beta-lactamases 5
4-4-1. AmpC 6
4-4-2. Extended-spectrum β-lactamases (ESBL) 6
4-4-3. Carbapenemases 6
5. Klebsiella pneumoniae Carbapenemase (KPC) 7
6. 現行抗藥感染菌檢測方式 8
7. 抗體 10
實驗目的 11

第二章 材料與方法 12
1. 克雷伯氏肺炎菌來源 12
1-1. 藥敏測試 12
1-1-1. VITEK 2 Compact 自動化微生物分析儀 12
1-1-2. E-test 13
2. KPC-2基因選殖 13
2-1. 以Colony PCR方式取得KPC-2 基因片段 13
2-1-1. PCR條件 14
2-2. Agarose Gel 14
2-3. 苯酚冷凍DNA分離法 (Phenol Freeze DNA Isolation Method) 15
3. 構築yT&A-KPC-2質體 16
3-1. DNA接合 (Ligation) 16
3-2. 轉形方法 (Transformation Method) 16
3-3. 氯化鋰-酚-氯仿萃取法 (LiCl-Phenol-Chloroform Extraction Method) 17
3-4. 限制酵素鑑定 (Restriction Digestion) 17
4. 構築pQE30-KPC-2質體 18
4-1. KPC-2基因轉殖 18
4-2. 製備勝任E. coli-M15 18
4-3. pQE30-KPC-2轉形至M15勝任E. coli 20
5. 表現KPC-2蛋白 20
5-1. 小量表現 20
5-2. 大量表現 20
6. KPC-2蛋白純化 21
6-1. Bacterial Protein Extraction Reagent (B-PER®) 使用流程 21
6-2. KPC-2-His融合蛋白純化 22
7. 免疫兔多株抗體生產流程 22
7-1. 抗原 (目標蛋白KPC-2) 之製備 22
7-2. 抗血清製備所需材料 23
7-3. 注射流程 24
7-4. 抗血清之處理 24
7-5. 克雷伯氏肺炎菌的蛋白萃取液製備方式 24
7-5-1. 煮沸 (Direct Boil) 25
7-5-2. 超音波震碎 (Sonication) 25
7-5-3. 煮沸及超音波震碎 (Boil and Sonication) 25
8. 西方墨點法 (Western blot) 26
8-1. SDS-膠體電泳 26
8-2. PVDF或硝化纖維膜之預處理 27
8-3. 電泳轉漬 (Electrotransfer) 27
8-4. 抗體之結合 (Binding Antibodies) 28
8-5. 冷光偵測 29
9. 斑點墨點法 (Dot blot) 29

第三章 實驗結果 31
1. 菌落PCR和選殖KPC-2基因 31
2. pQE30-KPC-2表現質體 31
3. 表現的KPC-2蛋白以不溶的Inculstion body存在 31
4. KPC-2蛋白是N-端His-tagged融合的蛋白 32
5. 大量表現KPC-2蛋白及其純化 32
6. SDS-PAGE純化分離KPC-2蛋白及免疫兔子產生抗血清 33
7. KPC-2抗血清的靈敏度 33
8. 西方墨點法分析臨床CRKP的KPC-2蛋白 34
9. 以菌落PCR分析每株CRKP的KPC-2基因和Hind III鑑定 34
10. 西方墨點法及菌落PCR結果相符合 34
11. KPC-2的表現與Imipenem MIC的比較 35

第四章 討論 36
1. 選殖KPC-2基因及建構KPC-2蛋白的pQE30 36
2. 表現的KPC-2蛋白以不可溶的包含體存在 36
3. 大量表現及純化KPC-2蛋白 37
5. SDS-PAGE純化分離KPC-2蛋白及免疫兔子產生抗血清 37
6. KPC-2抗血清的敏感度 37
7. 西方墨點法分析臨床CRKP的KPC-2蛋白以及基因的菌落PCR驗證 38
8. KPC-2表現與MIC關係 38

第五章 結論 40
參考文獻 63
附錄 68
附錄一 臨床克雷伯氏肺炎菌Carbapenem藥敏測試 68
附錄二 實驗流程圖 63
附錄三 以VIM-2抗血清偵測臨床五十株CRKP 71

表目錄
Table 1 西方墨點法與菌落PCR檢測臨床CRKP綜合結果 41
Table 2 西方墨點法與菌落PCR檢測臨床CRKP綜合結果 42
Table 3 KPC-2表現與MIC關係綜合結果 43

圖目錄
Fig. 1 美國1999年至2010年對於Carbapenem抗藥性統計分析 44
Fig. 2 克雷伯氏肺炎菌型態 45
Fig. 3 β-lactam化學結構 46
Fig. 4 Penicillins及Carbapenems結構 46
Fig. 5 Carbapenem類抗生素化學結構 47
Fig. 6 克雷伯氏肺炎菌之KPC-2蛋白結構 47
Fig. 7 KPC於2013年時在世界各地散佈情形 48
Fig. 8 Tn3型轉因子 49
Fig. 9 菌落PCR選殖KPC-2基因 49
Fig. 10 KPC-2基因的核苷酸及胺基酸序列 50
Fig. 11 重組的KPC-2質體及使用Hind III、EcoR I限制酵素鑑定 51
Fig. 12 IPTG誘導KPC-2蛋白表現 52
Fig. 13 純化KPC-2蛋白 53
Fig. 14 西方墨點法及斑點墨點法顯示KPC-2抗體具有高度敏感性 54
Fig. 15 西方墨點法檢測10株以Direct boil方式偵測克雷伯氏肺炎菌之KPC-2蛋白 55
Fig. 16 西方墨點法檢測10株以Direct boil方式偵測克雷伯氏肺炎菌之KPC-2蛋白 56
Fig. 17 西方墨點法檢測10株以Direct boil方式偵測克雷伯氏肺炎菌之KPC-2蛋白 57
Fig. 18 西方墨點法檢測10株以Direct boil方式偵測克雷伯氏肺炎菌之KPC-2蛋白 58
Fig. 19 西方墨點法檢測10株以Direct boil方式偵測克雷伯氏肺炎菌之KPC-2蛋白 59
Fig. 20 西方墨點法檢測10株以Direct boil方式偵測克雷伯氏肺炎菌之KPC-2蛋白 60
Fig. 21 利用Hind III限制酶切鑑定Colony PCR的KPC-2產物 61
Fig. 22 KPC-2表現與MIC關係綜合結果 62
Fig. 23 西方墨點法檢測五十株以Direct boil方式偵測克雷伯氏肺炎菌的VIM蛋白 73

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