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研究生：	吳恩綺 Wu, En-Chi
論文名稱：	光動力療法對致病菌之抗菌機轉研究-以抗藥性金黃色葡萄球菌為模式 The antibacterial mechanism of photodynamic therapy on bacterial pathogen: oxacillin-resistant Staphylococcus aureus as a model organism
指導教授：	王德華 Wong, Tak-Wah
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學暨分子生物學研究所 Department of Biochemistry and Molecular Biology
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	32
中文關鍵詞：	光動力療法、循血綠、抗藥性、金黃色葡萄球菌
外文關鍵詞：	Photodynamic therapy, indocyanine green, antibiotic-resistant, Staphylococcus aureus
相關次數：	點閱：134 下載：2
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具抗藥性的細菌感染已成為嚴重的全球問題，其中oxacillin-resistant Staphylococcus aurues (ORSA)是造成醫院內嚴重感染的來源之一。本研究利用ORSA作為模型，研究光動力療法克服抗藥性的分子機制與可能性。本實驗利用循血綠作為感光劑，透過光動力療法應用於體外實驗來抑制ORSA生長，並透過人類纖維母細胞檢測此療法的安全性。光動力治療對細菌毒力因子的改變，則透過凝固酶、腸毒素以及蛋白酶檢測，並透過抗生素錠劑測試細菌對藥物的敏感性。實驗過程中加入活性氧分子促進劑(重水和雙氧水)和清除劑(色胺酸和維他命C)來研究氧化壓力是否參與光動力作用，並利用電子顯微鏡觀察光動力療法對細菌造成的破壞。從實驗結果發現，在低濃度(25 g/ml)循血綠存在下，以65.5 毫瓦特/平方公分近紅外光(波長為700至2200奈米)，200焦耳/平方公分照射後，能夠達到兩個log的抑菌效果，而此條件對人類纖維母細胞完全無害。研究中亦發現，光動力療法可顯著降低細菌的毒力因子，包括凝固酶、腸毒素，但卻不影響其蛋白酶；然而此改變可能與光動力療法後活菌量減少有關。有趣的是，光動力療法可促使ORSA恢復對oxacillin的敏感性，而此藥物敏感性的型態至少可以維持十四代。光動力的抑菌效果與活性氧分子有部分相關性。電子顯微鏡的結果則顯示，光動力療法在光照後，馬上造成細菌嚴重的破壞。總括來說，光動力療法也許能夠加強抗生素療效或成為治療ORSA的替代方案。其作用機轉是否與基因改變有關，值得進一步探討。

Antibiotic-resistant bacterial infection has become a severe global problem. Oxacillin-resistant Staphylococcus aureus (ORSA) is one of the major bacterial pathogens causing hospital-acquired bacterial infections. In this study, ORSA was used as a model to study the molecular mechanisms and possibilities of overcoming drug resistance by photodynamic therapy (PDT). PDT with indocyanine green (ICG), a photosensitizer, was used to inhibit the growth of ORSA in vitro. The safety of the regimen was examined on human fibroblasts. Bacterial virulence including the activities of coagulase, enterotoxin and protease was investigated after PDT. The sensitivity to oxacillin was examined by disk diffusion method. Reactive oxygen species (ROS) enhancers (D2O and H2O2) and scavengers (tryptophan and ascorbic acid) were added during PDT to evaluate whether oxidative stress was involved in PDT. The morphology of bacteria after ICG-PDT was observed by transmission electron microscopy (TEM). A 2 log10 growth inhibition of ORSA was observed after 200 J/cm2 infrared irradiation at 65.5 mW/cm2 in the presence of 25 g/ml ICG. This condition showed no phototoxicity in human fibroblasts. PDT significantly reduced bacterial virulence including coagulase and enterotoxin, but not protease. This change might be related to the reduction of viable bacteria after PDT. Interestingly, PDT increased the susceptibility of ORSA to oxacillin. The drug sensitive phenotype persisted for at least 14 passages. The inhibition of cell growth was mediated partly through the ROS generated during PDT. TEM showed severe cell destruction immediately after irradiation. In conclusion, PDT may have potential to become a new alternative or adjuvant therapy against ORSA. Whether gene mutation is involved in the molecular mechanism merits further exploration.

Abstract                                                  Ⅰ
Acknowledgement                                           Ⅳ
Abbreviations                                             Ⅸ
1. Introduction                                            1
2. Materials and methods                                   3
2.1 Bacterial isolates                                     3
2.2 Photodynamic therapy system                            4
2.3 Photoinactivation of ORSA                              4
2.4 Phototoxicity in human fibroblasts                     5
2.5 Bacterial virulence                                    5
2.5.1 Coagulase activity                                   5
2.5.2 Enterotoxins                                         6
2.5.3 Protease activity                                    6
2.6 Effects of ROS scavenger and stabilizer on photodynamic antibacterial effect                                       7
2.7 Antibiotics susceptibilities                           7
2.8 Transmission electron microscopy                       8
2.9 Statistics                                             8
3. Results                                                 8
3.1 Low-dose PDT selectively inhibited bacterial growth in vitro                                                      9
3.2 ICG-PDT reduced bacterial virulence                   10
3.3 Photoinactivation of ORSA involved ROS                10
3.4 PDT enhanced the susceptibility of ORSA/ JD004 to antibiotics                                               10
3.5 PDT may alter ORSA genotype to drug sensitive         11
3.6 Low dose ICG-PDT destructed ORSA/ JD004 immediately after treatment                                           11
4. Discussion                                             12
5. References                                             15
6. Table                                                  18
Table 1. Standards of clear zone diameter required for the definition of drug sensitive Staphylococcus aureus        18
7. Figures                                                19
Figure 1. The photodynamic therapy system.                19
Figure 2. The photodynamic effects on ORSA isolated from a wound.                                                    20
Figure 3. Effects of ICG-mediated PDT on the viability of normal human fibroblasts.                                 21
Figure 4. Effects of ICG-mediated PDT on coagulase activity of ORSA/JD004.                                            22
Figure 5. Effects of ICG-mediated PDT on ORSA/JD004 enterotoxins.                                             23
Figure 6. Effects of ICG-mediated PDT on the protease activity of ORSA/JD004.                                   24
Figure 7. Effects of ROS scavengers and enhancers on photodynamic inactivation of ORSA/JD004.                  25
Figure 8. The antibiotics susceptibility of ORSA/JD004 after one ICG-mediated PDT treatment.                     26
Figure 9. The oxacillin sensitive phenotype of ORSA-T1 Clone 2.                                                  27
Figure 10. Ultrastructure of ORSA/JD004 after PDT.        28
8. Supplementary Data                                     29
Supplementary Figure 1. The photodynamic inhibition effects on the growth of ORSA blood isolates.                     29
Supplementary Figure 2. Effects of ROS enhancers on photodynamic inactivation of blood isolates.              30
Supplementary Figure 3. Effects of the amount of bacteria on coagulase activity of ORSA/JD004.                      31
Supplementary Figure 4. Effects of the amount of bacteria and incubation time on protease activity.                 32
                                    

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校內：2016-08-02公開
校外：2018-08-02公開

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