由於長期濫用抗生素，使許多微生物產生多重抗藥性。有些細菌能產生乙內醯胺酶 (beta-lactamase) ，用以破壞乙內醯胺類抗生素 (beta-lactam antibiotic) ，因此科學家發展出乙內醯胺酶抑制劑 (beta-lactamase inhibitor)，可與乙內醯胺酶產生不可逆的結合，使其無法破壞乙內醯胺類抗生素。乙內醯胺酶抑制劑與乙內醯胺類抗生素共同使用，單獨使用則無抗菌效果。但最近發現乙內醯胺類抑制劑 sulbactam 可抑制鮑氏不動桿菌生長。鮑氏不動桿菌 (Acinetobacter baumannii) 為一伺機性感染人類之病原菌，也是院內感染的主要菌種之一。此生長抑制作用的機制可能與青黴素結合蛋白 (penicillin-binding protein, PBP) 結合有關，但機制尚未明瞭。因此，本研究目的為探討 sulbactam 抑制鮑氏不動桿菌 ATCC 19606 生長的作用機制。首先我們由 sulbactam 對鮑氏不動桿菌產生之抑菌圈中篩選具有抗 sulbactam 能力之逆突變株 (sulbactam-resistant strain, SRS)，並測試 sulbactam 對這些菌株之最低抑制濃度，結果顯示 SRS 抗 sulbactam 程度與野生株相比明顯上升。相較於 ATCC 19606，SRS 生長速度較慢，且菌體形狀較長，推測 sulbactam 在鮑氏不動桿菌的抑菌機制可能與細胞壁生合成有關。接著，我們分別測試了野生株與抗sulbactam株的 PBP與 sulbactam 的親和力，結果並無顯著差異。由於綠膿桿菌 (Pseudomonas aeruginosa) 的生長不受 sulbactam 影響，因此我們將綠膿桿菌 PAO1 的 pbp1a、pbp1b 及 pbp3，分別互補至鮑氏不動桿菌 ATCC 19606，並測試其對 sulbactam 之抗性。實驗結果顯示，表現綠膿桿菌 PBP 之鮑氏不動桿菌，其抗 sulbactam 的能力沒有改變。最後，我們比對鮑氏不動桿菌 ATCC 19606 及 SRSs 的 pbp1a、pbp1b 及 pbp3 之序列，結果顯示此三個基因之序列在 SRS 菌株中並無改變。因此，我們推論可能有除了pbp1a、pbp1b 及 pbp3 以外的基因參與sulbactam 抑制鮑氏不動桿菌 ATCC 19606 生長之機制。綜合上述的實驗結果，可協助日後對抑制鮑氏不動桿菌感染之相關研究。

Over the past several decades, the overuse of antibiotics leads to the development of multidrug resistance in pathogens. Many bacteria can resist beta-lactam antibiotics by disrupting the beta-lactam ring of the drugs with -lactamase. To counter the effects of -lactamase, scientists have developed -lactamase inhibitors which protect beta-lactam antibiotic from degradation by irreversible binding with -lactamase. Beta-lactamase inhibitors do not exhibit strong antibacterial activity if given alone. Interestingly, it was noted recently that sulbactam, a -lactamase inhibitor, can inhibit growth of Acinetobacterium baumannii, a human opportunistic pathogen, although the mechanism remains not clear. To explore the antibacterial mechanism of sulbactam, this study first isolated sulbactam resistant strains (SRSs) of A. baumannii ATCC 19606, and then determined their minimal inhibitory concentration of sulbactam. These sulbactam-resistant strains grew slower and appeared longer. Comparison of sulbactam affinity to penicillin-binding proteins between ATCC 19606 and SRSs showed no difference. Introduction of pbp1a, pbp1b and pbp3 gene of Pseudomonas aeruginosa PAO1, a sulbactam resistant bacterium, into A. baumannii ATCC 19606 individually did not confer the recipient sulbactam resistance. Sequence analysis of pbp1a, pbp1b and pbp3 gene of A. baumannii ATCC 19606 and its SRSs also revealed no difference. It conclusion, this study is unable to identify a direct role of PBP1a, PBP1b and PBP3 in growth inhibition of A. baumannii by sulbactam.

摘要 I
英文摘要 III
目錄 V
表目錄 VII
圖目錄 VIII
壹、 前言 1
貳、 材料與實驗方法 5
2.1. 菌株與生長環境 5
2.2. 紙錠擴散感受性實驗以及突變株篩選 5
2.3. 最小抑制濃度 (Minimal inhibitory concentration, MIC) 6
2.4. 生長曲線 6
2.5. 革蘭氏染色 6
2.6. 染色體DNA萃取 7
2.7. 聚合酶連鎖反應 8
2.8. 質體構築 8
2.9. 勝任細胞製備 9
2.10. 熱休克轉型法 10
2.11. 接合作用 10
2.12. 從鮑氏不動桿菌分離與細胞膜結合的PBP 12
2.13. 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳 (SDS-PAGE) 13
2.14. 檢測 sulbactam 對PBP的親和性 14
參、 結果 15
3.1. Sulbactam 可抑制鮑氏不動桿菌 ATCC 19606 的生長 15
3.2. Sulbactam 對 SRSs 的最小抑制濃度較ATCC 19606高 15
3.3. SRSs的生長速率較 ATCC 19606 慢且菌體外型較長 16
3.4. Sulbactam 與PBP 結合的情況在ATCC 19606和 SRSs 相比沒有差異 17
3.5. 表現 PAO1 的 pbp 之 ATCC 19606 不具抗 sulbactam 的能力 17
3.6. SRSs 的 pbp1a 、pbp1b 以及 pbp3 基因序列與 ATCC 19606 相比沒有突變 18
肆、 討論 19
伍、 參考文獻 21

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