困難梭狀芽孢桿菌 (Clostridium difficile, C. difficile)是一隻革蘭氏染色陽性且會產生孢子的厭氧菌，也是導致抗生素相關性腹瀉中最常見的致病菌。困難梭狀芽孢桿菌感染 (Clostridium difficile infection, CDI)可導致廣泛的症狀，從輕度的腹瀉，偽膜性結腸炎到嚴重者導致毒性巨結腸症等。甚至在全世界許多地方的主要流行病中，已出現具有較高死亡率的高毒力困難梭狀芽孢桿菌菌株。CDI現有的治療方法，由於無法克服慢性復發而難以根治，因此，開發其他策略以治療或預防該疾病，是一項迫在眉睫的研究。在本篇研究中，我們評估了可用的臨床藥物在抑制困難梭狀芽孢桿菌的孢子與營養細胞中之作用，試圖在現有的藥物中尋求可作為抗微生物劑的新用途。首先，我們分析了幾種臨床藥物對於困難梭狀芽孢桿菌的最低抑菌濃度 (Minimum inhibitor concentration, MIC)與最低殺菌濃度 (Minimum bactericidal concentration, MBC)，並根據不同的MBC，記錄困難梭狀芽孢桿菌之生長曲線。接著我們將前述所記錄的MBC換算為臨床劑量的體內濃度，以確認臨床藥物在體內的耐受程度。我們評估了上述臨床藥中的一種非類固醇抗炎藥 (Non-Steroidal Anti-Inflammatory Drug，NSAID)-雙氯芬酸鈉 (Diclofenac sodium)，在困難梭狀芽孢桿菌的生物膜形成上所扮演的角色。並利用Microarray分析投以藥物後，困難梭狀芽孢桿菌的基因表達量變化。接著透過Checkerboard的實驗，測試雙氯芬酸與不同抗生素間的交互作用。Phe-Argβ-萘酰胺二鹽酸鹽 (Phe-Arg β-naphthylamide dihydrochloride, PAβN)是一種已知可作為外排幫浦抑制劑 (efflux pump inhibitor)之化合物，因此我們將PAβN加入雙氯芬酸鈉後，測試其是否有助於加強雙氯芬酸鈉之抑菌效果，進而推測雙氯芬酸鈉之抑菌機制與外排幫浦間的關係。最後，微陣列分析顯示，雙氯芬酸鈉治療後影響困難梭狀芽孢桿菌多種基因的表達，包括轉錄調節因子與轉運蛋白等。結論而言，在本項研究中，我們證明了體外實驗中雙氯芬酸鈉能夠抑制困難梭狀芽孢桿菌之生長。預計這些發現將可做為CDI替代療法之治療策略。

Clostridium difficile is the most common cause of antibiotic-associated diarrhea worldwide. Clostridium difficile infections (CDIs) can lead to a wide range of symptoms ranging from mild diarrhea, pseudomembranous colitis to toxic megacolon. The emergence of hyper virulent C. difficile strains with higher mortality rates has resulted in major epidemics in many parts of the world. Current treatment options for CDIs are inadequate due to relapses that could develop into chronic infections. Therefore other strategies must be identified to treat or prevent disease. In this study, we evaluated the role of clinical drugs in inhibiting C. difficile spore and vegetative cells. Based on an extensive literature search, multiple drugs were selected as candidates based on their known antibacterial activity. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each drug against C. difficile strain R20291 were performed, and diclofenac sodium, a nonsteroidal anti-inflammatory drug (NSAID) appeared to have the highest potency. Further analysis with other NSAIDs demonstrated diclofenac sodium was superior compare to others. In addition to the inhibitory effect on vegetative cell growth, biofilm formation and spore survival were also evaluated. The addition of Phe-Arg β-naphthylamide dihydrochloride (PAβN), an efflux pump inhibitor, reduced the MBC of diclofenac sodium against multiple C. difficile isolates. Lastly, microarray analysis revealed that diclofenac sodium treatment affected the expression of multiple genes including transcriptional regulators, transporters, and others. In summary, in this study, we demonstrated that in vitro, diclofenac sodium was able to inhibit C. difficile growth. We expect that these findings could provide alternative adjunt theraputic strategies in the treatment of CDI.

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