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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 邱浩傑(Hao-Chieh Chiu) | |
dc.contributor.author | Yi-Ru Chen | en |
dc.contributor.author | 陳逸洳 | zh_TW |
dc.date.accessioned | 2021-06-16T13:00:52Z | - |
dc.date.available | 2018-09-24 | |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-08-08 | |
dc.identifier.citation | 1. Gordon, R.J. and F.D. Lowy, Pathogenesis of methicillin-resistant Staphylococcus aureus infection. Clin Infect Dis, 2008. 46 Suppl 5: p. S350-9.
2. Goss, C.H. and M.S. Muhlebach, Review: Staphylococcus aureus and MRSA in cystic fibrosis. J Cyst Fibros, 2011. 10(5): p. 298-306. 3. Diekema, D.J., et al., Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe, and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997-1999. Clin Infect Dis, 2001. 32 Suppl 2: p. S114-32. 4. Appelbaum, P.C., Microbiology of antibiotic resistance in Staphylococcus aureus. Clin Infect Dis, 2007. 45 Suppl 3: p. S165-70. 5. Johnson, A.P., A. Pearson, and G. Duckworth, Surveillance and epidemiology of MRSA bacteraemia in the UK. J Antimicrob Chemother, 2005. 56(3): p. 455-62. 6. Schaefler, S., et al., Emergence of gentamicin- and methicillin-resistant Staphylococcus aureus strains in New York City hospitals. J Clin Microbiol, 1981. 13(4): p. 754-9. 7. Liu, C., et al., Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: executive summary. Clin Infect Dis, 2011. 52(3): p. 285-92. 8. Chang, S., et al., Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene. N Engl J Med, 2003. 348(14): p. 1342-7. 9. Tsiodras, S., et al., Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet, 2001. 358(9277): p. 207-8. 10. Mangili, A., et al., Daptomycin-resistant, methicillin-resistant Staphylococcus aureus bacteremia. Clin Infect Dis, 2005. 40(7): p. 1058-60. 11. Poon, H., M.H. Chang, and H.B. Fung, Ceftaroline fosamil: a cephalosporin with activity against methicillin-resistant Staphylococcus aureus. Clin Ther, 2012. 34(4): p. 743-65. 12. Wang, J., et al., Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature, 2006. 441(7091): p. 358-61. 13. Anesini, C. and C. Perez, Screening of plants used in Argentine folk medicine for antimicrobial activity. J Ethnopharmacol, 1993. 39(2): p. 119-28. 14. Casero, C., et al., Achyrofuran is an antibacterial agent capable of killing methicillin-resistant vancomycin-intermediate Staphylococcus aureus in the nanomolar range. Phytomedicine, 2013. 20(2): p. 133-8. 15. de Jonge, M.R., et al., A computational model of the inhibition of Mycobacterium tuberculosis ATPase by a new drug candidate R207910. Proteins, 2007. 67(4): p. 971-80. 16. <Performance Standards for Antimicrobial. Susceptibility Testing; Twenty-Second Informational Supplement.pdf>. 17. Pankey, G.A. and L.D. Sabath, Clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram-positive bacterial infections. Clin Infect Dis, 2004. 38(6): p. 864-70. 18. Vuorio, R. and M. Vaara, The lipid A biosynthesis mutation lpxA2 of Escherichia coli results in drastic antibiotic supersusceptibility. Antimicrob Agents Chemother, 1992. 36(4): p. 826-9. 19. Chiu, H.C., et al., Pharmacological exploitation of an off-target antibacterial effect of the cyclooxygenase-2 inhibitor celecoxib against Francisella tularensis. Antimicrob Agents Chemother, 2009. 53(7): p. 2998-3002. 20. Chiu, H.C., et al., Development of novel antibacterial agents against methicillin-resistant Staphylococcus aureus. Bioorg Med Chem, 2012. 20(15): p. 4653-60. 21. Schenk, S. and R.A. Laddaga, Improved method for electroporation of Staphylococcus aureus. FEMS Microbiol Lett, 1992. 73(1-2): p. 133-8. 22. Funes, S., et al., Independent gene duplications of the YidC/Oxa/Alb3 family enabled a specialized cotranslational function. Proc Natl Acad Sci U S A, 2009. 106(16): p. 6656-61. 23. van Bloois, E., et al., The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential. J Biol Chem, 2005. 280(13): p. 12996-3003. 24. Imhof, N., A. Kuhn, and U. Gerken, Substrate-dependent conformational dynamics of the Escherichia coli membrane insertase YidC. Biochemistry, 2011. 50(15): p. 3229-39. 25. Palmer, S.R., et al., YidC1 and YidC2 are functionally distinct proteins involved in protein secretion, biofilm formation and cariogenicity of Streptococcus mutans. Microbiology, 2012. 158(Pt 7): p. 1702-12. 26. Samuelson, J.C., et al., YidC mediates membrane protein insertion in bacteria. Nature, 2000. 406(6796): p. 637-41. 27. Xie, K. and R.E. Dalbey, Inserting proteins into the bacterial cytoplasmic membrane using the Sec and YidC translocases. Nat Rev Microbiol, 2008. 6(3): p. 234-44. 28. Kalle, A.M. and A. Rizvi, Inhibition of bacterial multidrug resistance by celecoxib, a cyclooxygenase-2 inhibitor. Antimicrob Agents Chemother, 2011. 55(1): p. 439-42. 29. Amaral, L., et al., Review. Comparison of multidrug resistant efflux pumps of cancer and bacterial cells with respect to the same inhibitory agents. In Vivo, 2007. 21(2): p. 237-44. 30. Chen, H.J., et al., Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother, 2010. 54(12): p. 4985-91. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61307 | - |
dc.description.abstract | Chapter I
發展針對金黃色葡萄球菌的新穎小分子化合物 耐甲氧苯青霉素之金黃色葡萄球菌(Methicillin-resistant Staphylococcus aureus , MRSA)具有能對抗多種抗生素的能力,在用藥方面增加困難度,在現今已成為了臨床治療上的很大威脅,因此開發新的抗生素是很重要的議題。我們自陽明大學的蕭崇瑋老師取得了157個化合物,分別對MRSA以及鮑氏不動桿菌(A. baumannii) 進行抗生素感受性試驗,篩選出14個具有潛力的化合物,再接著對細胞進行毒性測試,最後篩選出選擇性最高的前兩名,分別為SC78、SC80。 為了明白化合物的效果與應用性,我們將SC78、SC80與其他細菌進行測試,發現對45株自醫院分離出來的MRSA菌株與其他非金黃色葡萄球菌的革蘭氏陽性細菌有抑制效果,但在革蘭氏陰性細菌則不具有抑制能力。再來,我們將SC78、SC80與其他臨床上常用的抗生素比較效果,在MRSA與抗萬古霉素腸球菌(VR-E)這兩株多重抗藥細菌身上,SC78的效果明顯比其他抗生素為佳。 除此之外,為了瞭解SC78與SC80的作用特性,我們也進行殺菌效力評估試驗(time-kill assay),發現4倍、8倍MIC濃度的SC78與SC80可以在24小時之內殺死99.9%的細菌,但是2倍MIC濃度的SC78與SC80則無此現象,顯示在高濃度的情況下為殺死細菌的效果,而在低濃度則為抑制細菌生長的效果。 另一方面,為了評估SC78是否有在臨床應用的潛力,我們將SC78給予受到MRSA感染的小鼠進行動物實驗,雖目前以此條件尚無法看到小鼠有治癒的現象,但即使如此,SC78這個先導藥物在抗生素的開發上仍具有相當的潛力。 Chapter II 以全基因體的策略去尋找由Celecoxib衍生的新穎抗金黃色葡萄球菌之化合物的作用機制 耐甲氧苯青霉素之金黃色葡萄球菌(Methicillin-resistant Staphylococcus aureus , MRSA)具有能對抗多種抗生素的能力,使得在用藥方面增加困難度,於現今已成為臨床治療上很大的威脅,因此開發新的抗生素是相當重要的議題。在先前的研究中,本實驗室以非類固醇類的抗發炎藥物希樂葆(Celecoxib)為基礎,合成並篩選出兩個可以有效抑制MRSA生長的化合物,暫命名為化合物-36(Cpd-36)與化合物-46(Cpd-46)。首先為了測試化合物的效果與應用性,本實驗使用革蘭氏陽性細菌例如葡萄球菌、腸球菌以及MRSA的標準菌株與35株台大醫院臨床分離的菌株;革蘭氏陰性細菌如大腸桿菌、沙門氏菌及孢氏不動桿菌和Cpd-36與Cpd-46進行抗生素感受性試驗,實驗結果發現革蘭氏陽性細菌對本實驗的藥物皆具有感受性,但在革蘭氏陰性菌上則沒有觀察到抑制效果。 為了提供資訊以利於合成更有效的化合物,我們想要瞭解化合物的作用機制,因此我們將化合物與金黃色葡萄球菌長期培養,挑選出對化合物具有抵抗能力的抗藥菌株,再抽取其全基因組去氧核醣核酸(genomic DNA),利用全基因定序(next generation sequencing,NGS)去尋找並分析金黃色葡萄球菌的抗藥株與野型株在哪些基因上具有變異。 根據全基因定序的結果顯示,存在數個可能具有突變位點的基因,接著再使用傳統定序方式確認,最後找出一個最可能的yidC2基因,並發現突變株的YidC2蛋白其胺基酸確實有產生變化,因此我們推測此蛋白的改變和抗藥性的上升有關。同時我們將具有抗藥性的yidC2基因利用質體送入野型金黃色葡萄球菌當中,發現帶有抗藥基因的金黃色葡萄球菌,對於化合物的感受性會下降,此基因確實為化合物影響細菌的重要標的。 先前有研究指出,若細菌的YidC2蛋白失去功能,會影響其將氫離子排出菌體外的能力,因此對酸性環境會較敏感。本研究將化合物與金黃色葡萄球菌先行培養,再將細菌移入酸性環境中,發現細菌生長情形會下降,故證實此藥物會與YidC2蛋白作用。 另外,為了評估Cpd-36與Cpd-46是否有在臨床上實際應用的潛力,我們使用小鼠以腹腔注射感染MRSA後,再口服給予Cpd-46,觀察藥物是否可以降低因MRSA所造成的感染,或減緩感染情況並降低致死率,實驗結果顯示,Cpd-46應可以被腸胃道吸收,進而達成殺菌效果,推測Cpd-46應該具有應用在臨床上的潛力,因此值得去發展以Cpd-46為基礎的其他化合物,提高藥物的作用能力,進而發展更有效的抗耐甲氧苯青霉素之金黃色葡萄球菌的藥物。 | zh_TW |
dc.description.abstract | Chapter I
Development of Novel Small-Molecule Anti-Staphylococcal Agents Methicillin-resistant Staphylococcus aureus (MRSA) possesses multi-drug resistance and increase difficulty in treatment, thus it has become a major threat in clinical treatment nowadays. We have tested a small compound library with antibiotic susceptibility testing against MRSA and A. baumannii. The results indicated that fourteen compounds show inhibitory efficacy against MRSA and A. baumannii. Further, we applied these fourteen compounds in MTT assay to test for cell toxicity, and finally we selected two best compounds, SC78 and SC80. In order to understand the efficacy and the application of the candidate compounds, we tested SC78 and SC80 against the other bacteria, the result showed that SC78 and SC80 inhibit the growth of 45 clinically-isolated MRSAs and other non-Staphylococcus bacteria, but they are ineffective against Gram negative bacteria. Also, we have compared SC78 and SC80 to common antibiotics used in clinical treatment, the efficacy of SC78 is more effective than other antibiotics against MRSA and Vancomycin-Resistant Enterococci (VR-E). To further understand the action mechanism of SC78 and SC80, we proceeded time-kill assay and discovered that four times and eight times the MIC of SC78 and SC80 killed 99.9% bacteria within 24 hours, but two times the MIC of SC78 and SC80 could not cause the same outcome, thus this indicated that it is bactericidal antibiotics in high dose and bacteriostatic antibiotics in low dose. On the other hand, in order to estimate whether SC78 has potential in clinical application, we have given SC78 to mice infected by MRSA for animal experiment. Although there is no significant recovery in this condition, but SC78, the lead compound which inhibits the growth of MRSA efficaciously, has a lot of potential in the research on antibiotics. Chapter II Genome-wide Identification of the Action Mechanism of Novel Celecoxib Derived Agents against Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) possesses multi-drug resistance and increase difficulty in treatment, and the development of novel antibiotics is an important issue. In our previous study, our laboratory had successfully developed two potent anti-MRSA drugs, named Compound-36 (Cpd-36) and Compound-46 (Cpd-46). To examine the effects and applications of the compounds, we test the susceptibility of several Gram positive bacteria, including Staphylococci, Enterococci, standard MRSA strains as well as 35 clinically-isolated MRSAs. Gram negative bacteria such as E. coli, Salmonella, and A. baumannii were also tested. The results showed that Gram positive bacteria tested are susceptible to these two drugs. In contrast, for the Gram negative bacteria tested, they are not susceptible to the drugs. To further understand the action mechanism of these two drugs, we cultured Staphylococcus aureus in the presence of compounds for a long period of time to select resistant mutant strains. The genomic DNA of resistant strains were sequenced by using Next-generation Sequencing (NGS) technique to identify the potential mutations associated with compounds resistance. The NGS result has exclusively pointed out several mutation spots, and some of them are located within essential genes. We used conventional sequencing method to confirm and identified one possible mutated gene, yidC2. From the protein product of the yidC2 gene, we found that amino acid alterations are indeed present; therefore we presumed that there is a relationship between the change of the YidC2 protein and the rise of resistance. We transformed plasmid carrying mutated yidC2 gene into wild-type S. aureus, and found that the resistance against compounds was increased, thus we suppose that the yidC2 gene is the target of compounds. In previous study, YidC2 has been reported to influence the ability of bacteria to exclude hydrogen. If YidC2 protein lost its function, bacteria will become sensitive to acidic enviroment. Our result indicated that while S. aureus were treated with Cpd-46 and Cpd-36, the growth of S. aureus in acidic environment was reduced. It implicated that the compounds might act on the YidC2 protein. On the other hand, to evaluate whether Cpd-36 and Cpd-46 have potential for clinical application, we infect mice with MRSA via intraperitoneal injection followed by orally administrated of Cpd-46. The result showed that oral Cpd-46 can suppress the MRSA and reduce the mortality rate, proving the feasibility of this drug in infection treatment. Altogether, Cpd-46 represent a promising lead agent to develop more potent anti-MRSA agents by targeting to YidC2. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T13:00:52Z (GMT). No. of bitstreams: 1 ntu-102-R00424009-1.pdf: 5755068 bytes, checksum: 4c36cb8dc439d07a4cf90310160dfee5 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | Contents I
Chapter I: V Development of Novel Small-Molecule Anti-Staphylococcal Agents V 中文摘要 VI Abstract VIII 1. Introduction 1 1.1 Staphylococcus aureus 2 1.2 Methicillin-resistant Staphylococcus aureus 2 1.3 Treatments of S. aureus and MRSA 3 1.4 Development of novel antibiotics against MRSA 3 1.5 Research aims 5 2. Materials and Methods 6 2.1 Bacteria and culture condition 7 2.2 Media 7 2.3 Chemicals 8 2.4 Antibacterial assays 8 2.5 Cell culture 9 2.6 MTT cell viability assay 10 2.7 Time-kill assay 10 2.8 In vivo infection assay 11 3. Results 13 3.1 Compounds screening 14 3.2 Susceptibility test and cytotoxicity assay 14 3.3 In vitro activities of SC78 and SC80 against MSSA and MRSA 15 3.4 Spectrum of antibacterial activity of SC78 and SC80 15 3.5 Compounds 36 and 46 are bactericidal agents against S. aureus 15 3.6 Estimate the efficacy of SC78 in vivo 16 4. Discussion 17 4.1 SC compounds were efficacious against MRSA 18 4.2 The IC50 value of HT-29 is higher than other cell lines 18 4.3 The animal experiment showed indifferent results on the curability of SC78 18 4.4 The time-kill kinetics of SC80 were different from other antibiotics 19 4.5 SC78 and SC80 are inefficacious against Gram negative bacteria 19 5. References 21 6. Tables 25 Table 1. Anti-Staphylococcus (MIC) versus antiproliferative (IC50) activities of test agents 26 Table 2. In vitro activities of SC78 and SC80 against S. aureus 27 Table 3. Antibacterial activity spectrum of SC78 and SC80 against Staphylococcal species 27 Table 4. (a) and (b). Spectrum of antibacterial activity of SC78 and SC80. 28 Table 5. SC78 and SC80 are inefficacious against Gram negative bacteria 29 7. Figures 30 Figure 1. Flow chart of compounds screening 31 Figure 2. Time-kill assay 32 Figure 3. Estimate the efficacy of SC78 in vivo 34 Chapter II: 35 Genome-wide Identification of the Action Mechanism of Novel Celecoxib Derived Agents against Staphylococcus aureus 35 中文摘要 36 Abstract 38 1. Introduction 41 1.1 Research aims 42 2. Materials and Methods 44 2.1 Antibiotics and compounds 45 2.2 Genomic DNA extraction 45 2.3 Plasmid DNA extraction 46 2.4 Competent cell preparation 47 3. Results 49 3.1 Spectrum of antibacterial activity of Cpd-36 and Cpd-46 50 3.2 Cytotoxicity assay of Cpd-36 and Cpd-46 against K562 cell lines and HEK293 cell lines 50 3.3 Cpd-36 and Cpd-46 are unable to bind the drug target in Gram negative bacteria 51 3.4 Both Cpd-36 and Cpd-46 can effectively inhibit the growth of MRSA and clinically-isolated MRSAs 52 3.5 Select the compounds resistant strains 52 3.6 Using NGS to find out the existence of point mutation in both wild-type and mutant strains 53 3.7 Using Sanger sequencing to confirm whether the point mutations are true 53 3.8 The drug target of S. aureus NCTC 8325 may be YidC2 protein 54 3.9 Mutant strain has point mutation on yidC2 gene 55 3.10 Effect of plasmid-expressed YidC2 protein in wild-type S. aureus 56 3.11 To validate whether YidC2 protein is the drug target 57 3.12 Estimate the efficacy of Cpd-46 in vivo 57 4. Discussion 59 4.1 The S. aureus is difficult to generate resistance against the compounds 60 4.2 The mutant amino acids are locate in the functional region of YidC2 protein 60 4.3 To forecast how compounds conjugate the YidC2 protein 60 4.4 Comparison of the YidC amino acid sequence between different bacteria strains 61 4.5 Cpd-36 and Cpd-46 are real antibiotics that can inhibit the growth of bacteria 61 4.6 Cpd-46 can efficaciously reduce the infection caused by MRSA in mouse model 62 5. References 64 6. Tables 70 Table 1-a. Spectrum of antibacterial activity of Cpd-36 and Cpd-46 71 Table 1-b. Spectrum of antibacterial activity of Cpd-36 and Cpd-46 72 Table 2-a. MTT cell viability assay 73 Table 2-b. MTT cell viability assay 73 Table 3. Cpd-36 and Cpd-46 can’t bind the drug target in Gram negative bacteria 74 Table 4. In vitro activities of Cpd-36 and Cpd-46 against clinical MRSA isolates 74 Table 5. Point mutation of Cpd-9 resistant strain and Cpd-36 resistant strain 75 Table 6. All primers used in the studies. 76 Table 7. Single-nucleotide mutation and corresponding change in amino acid 77 7. Figures 78 Figure 1. The structure of Celecoxib 79 Figure 2. The structural modification of Celecoxib to get better efficacy compounds 79 Figure 3. The structure of (a) Cpd-9 and (b) Cpd-36 79 Figure 4. The structure of Cpd-46 79 Figure 5. The structure of the YidC2 protein and the mutant amino acids 80 Figure 6. Effect of plasmid-expressed YidC2 protein in wild-type S. aureus 81 Figure 7. To validate whether YidC2 protein is the drug target 82 Figure 8. Exam the efficacy of Cpd-46 in vivo 83 Figure 9. The point mutations locate in YidC2 conserve region 85 8. Supplementary 86 | |
dc.language.iso | en | |
dc.title | 發展抗金黃色葡萄球菌的新穎小分子藥物 | zh_TW |
dc.title | Development of Novel Small-Molecule Agents against Staphylococcus aureus | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧麗珍(Lee-Jene Teng),蕭崇瑋(Chung-Wai Shiau),顏伯勳(Bo-Shiun, Yan) | |
dc.subject.keyword | 金黃色葡萄球菌,抗甲氧苯青霉素之金黃色葡萄球菌,抗藥性,抗生素之新藥開發, | zh_TW |
dc.subject.keyword | S.aureus,MRSA,novel development of antibiotics,resistance of antibiotics, | en |
dc.relation.page | 87 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2013-08-08 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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