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研究生：	廖淑貞 Liao, Shu-Zhen
論文名稱：	光動力治療改變金黃色葡萄球菌傷口分離株對甲氧苯青黴素的感受性 Photodynamic therapy can alter methicillin susceptibility in Staphylococcus aureus wound isolates
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：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	34
中文關鍵詞：	抗藥性、循血綠、抗藥性金黃色葡萄球菌、光動力療法
外文關鍵詞：	drug-resistant, indocyanine green, methicillin-resistant Staphylococcus aureus, photodynamic therapy
相關次數：	點閱：190 下載：1
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細菌抗藥性已經成為一個全球臨床及公共衛生的問題，methicillin抗藥性金黃色葡萄球菌(MRSA) 是臨床上重要的皮膚病原菌之一，並且有可能進展至菌血症而致死。光動力療法經由適當波長激發感光劑來產生許多活性氧化物，可以破壞細菌的多種標的，因此已漸漸成為抗生素一種新的輔助替代療法。最近我們發現MRSA在光動力治療後會改變細菌的抗藥性，但作用機制未明。在此研究中，我們想要探索為何光動力療法可以改變細菌的抗藥性。
我們首先分類三株對光動力療法感受性不同的臨床菌株去探究抗藥性變化的濃度效應，藉由測定細菌的生長速率與細胞壁厚度來分析光動力療法如何改變細菌的生理特性，並探討光動力療法後基因表達(DNA、RNA)及相關蛋白的改變。光動力療法存活下的MRSA生長變得較快速、細胞壁變得較薄，這些存活者有趨向藥物敏感的趨勢而且具有濃度效應，失去抗藥性的機轉與 mec A複合體的基因遺失有部分的相關性。
總括來說，光動力療法透過抗藥基因的剔除，將細菌從抗藥性的表型轉變偏向敏感性表型，可能具有成為抗生素的輔助療法的潛力。

Antibiotic resistance has become a serious clinical and public health problem globally. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens causing skin infections and may be life threatening if progresses to bacteremia. Photodynamic therapy (PDT) damages multiple targets in a bacterium by free radicals which are generated after exciting a photosensitizer with a specific wavelength of light and has becoming a new adjuvant/ alternative for antibiotics. We found PDT altered drug sensitivity in MRSA recently. However, the mechanism is unknown. In this study, we aim to explore how PDT overcome drug resistance in MRSA.
We first classified 3 MRSA strains isolated from skin wounds with different sensitivities towards PDT to study the dose-dependent effects on drug sensitivity. Bacteria growth rate and cell wall thickness were analyzed to determine how PDT changed bacteria physiologically. Gene regulations after PDT were determined by DNA, RNA and the related protein expressions. PDT-survived MRSA grew faster and had a thinner cell wall. They had a trend to march toward a methicillin-sensitive S. aureus (MSSA) phenotype and was dose-dependent. The loss of drug resistant mechanism was partly related to mecA complex gene deletion.
In conclusion, PDT skewed MRSA toward a drug sensitive phenotype via drug resistant gene deletion and may have the potential to become an adjuvant treatment for MRSA.

Abstract  	                                          I
Acknowledgement	                                        III
Abbreviations                                           VII
1.	Introduction	                                  1
2.	Materials and methods                  	          3
2.1	 Bacterial isolates	                          3
2.2	 Photodynamic therapy system     	          4
2.3	 Photoinactivation of MRSA	                  4
2.4 	 Growth curves                                    5
2.5 	 Antibiotic susceptibility testing                5
2.6	 Transmission electron microscopy	          6
2.7 	 DNA fragmentation analysis	                  6
2.8 	 Polymerase chain reaction	                  7
2.9   Quantitative reverse transcription polymerase chain reaction	                                          7
2.10	 Western blot                                     8
2.11	 Statistics                                       9
3.	Results	                                          9
3.1	 MRSA grew faster after PDT                       9
3.2	 The MRSA cell wall become thinner after PDT      9
3.3	 MRSA were driven toward a drug sensitive phenotype by PDT                                                    9
3.4	 Gene alterations and deletions after PDT        10
3.5	 Reduction of PBP2a by PDT                       10
4.	Discussion	                                 11
5.	References	                                 14
6.	Table                                     	 18
Table 1. The alteration of MIC value of oxacillin after PDT                                             18
7.	Figures	                                         19
Figure 1. Bacterial growth curves after photodynamic therapy.	                                         19
Figure 2. Changes of cell wall thickness of MRSA-L after photodynamic therapy.                                    20
Figure 3. Changes of drug sensitivity of MRSA after photodynamic therapy.	                                 21
Figure 4. Genotyping of three clinical isolate after one photodynamic therapy.                                    23
Figure 5A. MecA complex deletion after photodynamic therapy in MRSA-L.	                                         24
Figure 5B. No changes of mecA complex after photodynamic therapy in MRSA-M. 	                                 25
Figure 5C. No changes of mecA complex after photodynamic therapy in MRSA-H.                                       26
Figure 6. Quantitative changes of mecA mRNA of three clinical MRSA isolates after photodynamic therapy.	 27
Figure 7. PBP2a protein expression in three clinical MRSA isolates after photodynamic therapy.                     28
8.	Supplementary Tables	                         29
Supplementary Table 1. The MRSA inhibition zone diameter and MIC value according to CLSI criteria.	         29
Supplementary Table 2. The PCR primer used in this study.30
9.	Supplementary Figures	                         31
Supplementary Figure 1. The mechanism of photodynamic therapy.                                                 31
Supplementary Figure 2. The photodynamic therapy system. 32
Supplementary Figure 3. The photodynamic inactivation effects on three clinical MRSA strains.	                 33
Supplementary Figure 4. Mec gene typing of three MRSA clinical isolates.	                                 34
                                    

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