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研究生：	吳欣蓓 Wu, Shin-Bei
論文名稱：	氧化壓力改變抗藥性金黃色葡萄球菌之藥物敏感性研究 The study of oxidative stress alters MRSA drug sensitivity
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：	2018
畢業學年度：	106
語文別：	中文
論文頁數：	31
中文關鍵詞：	抗藥性、雙氧水、感染、肺感染、抗藥性金黃色葡萄球菌
外文關鍵詞：	drug-resistance, H2O2, infection, lung infection, Methicillin-resistance Staphylococcus aureus
相關次數：	點閱：150 下載：2
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背景我們最近發現了光動力療法(PDT)的氧化壓力會將抗藥性金黃色葡萄球菌(MRSA)改變成不具抗藥性，因此我們要問一個問題：是否任何氧化壓力都有此效果？

假說低濃度的光動力療法及H2O2所產生的氧化壓力可以選擇性殺菌並改變其抗藥性。

材料和方法從病人分離出來的12株MRSA，用不同濃度的H2O2處理後，存活下來的不同菌株，觀察是否變得比較無抗藥性，並分析它的生理特徵，包括生長速率、生物膜形成，以及用RT-PCR和Western blot去找出可能改變的基因和相關蛋白，並進一步在動物實驗肺感染模式進一步生體上驗證。

結果將MRSA以不同的低濃度(0.1-0.3%) H2O2作用後，可以有效地抑制1至7個logs的菌量。0.1%~0.15％的H2O2處理後12株臨床菌株中的9株（75％）對苯唑西林和頭孢西丁更敏感，3株保持不變。存活的細菌生長得更快且細菌形成的生物膜更少。在3個代表性菌株中重複暴露於0.1%~0.15％ H2O2達5次並不會誘導對抗生素的耐受性。相反的，MRSA對苯唑西林、頭孢西丁、萬古黴素(vancomycin)和多粘菌素B (polymyxin B)都變得更敏感。該機制部分與mec複合體缺失或突變有關，存活下來的細菌對小鼠的肺部感染模型獲得更進一步的證實。

結論低劑量的H2O2能夠有效殺死MRSA，而且存活下來的細胞對多種抗生素變得更敏感。未來在臨床上也許有潛力成為對抗MRSA的輔助或新療法。

Background. Recently, we found methicillin-resistant Staphylococcus aureus (MRSA) became more susceptible to oxacillin after photodynamic therapy (PDT). We asked whether oxidative stress other than PDT can affect bacterial drug susceptibility.

Hypothesis. Oxidative stress generated by low concentration H2O2 can effectively kill bacteria and alter drug susceptibility in MRSA.

Methods. Twelve MRSA clinical isolates were treated with different concentrations of H2O2. Bacterium growth rate, biofilm formation, drug susceptibility to oxacillin and cefoxitin were investigated. The possible related gene and proteins were evaluated with qRT-PCR and Western blot. In vivo pulmonary infection model in mice was used to validate its efficacy in vivo.

Results. Co-cultured MRSA with different low concentrations (0.1- 0.3%) H2O2 effectively inhibits MRSA from 1 to 7 logs. Nine out of 12 strains (75%) of MRSA become more suceptible to oxacillin and cefoxitin after 0.1% - 0.15% H2O2 treatments and 3 remained no change. The survived cell grew faster and less bacterial biofilm formation by the bacteria. Repeated exposure to 0.1% - 0.15% H2O2 in 3 representative strains up to 5 times did not induce adaptation to antibiotics. In contrast, the survived cells become more susceptible to vancomycin and polymyxin B in addition to oxacillin and cefoxitin. The mechanism is partly related to mec complex deletion or mutation. Mice infected with survived bacteria are more sensitive to oxacillin in a pulmonary infection model.

Conclusion. H2O2 can effectively kill MRSA in a relative low dose. The survived cells after treatment become more susceptible to multiple antibiotics. It may have potential as an adjuvant or a new therapy against MRSA.

Table of contents
Abstract                                                           II
Acknowledgements                                                  III
Table of Contents                                                    IV
Abbreviation                                                       VII
1.  Introduction                                                   1
2.  Materials and methods
2.1	Bacteria                                                      3
2.2	H2O2 oxidative stress                                            3
2.3	Antibiotic susceptibility tests                                      3
2.4	MRSA grow rate                                               4
2.5	Biofilm formation assay                                         4
2.6	Polymerase Chain Reaction                                       4
2.7	Quantitative Reverse Transcription Polymerase Chain Reaction          5
2.8	In vivo                                                       5
2.9	Statistics                                                      6
3.  Results                                                        
3.1 Low dose H2O2 killed MRSA effectively and may be safe to human 
fibroblast                                                      7
3.2 MRSA become more sensitivity to antibiotics after H2O2 treatment       7
3.3 Repeated H2O2 treatments did not develop adaptive antibiotic resistance   7
3.4 MRSA Grew Faster after H2O2 Treatment                           8
3.5 Reduced biofilm synthesis after H2O2 treatments                      8
3.6 The change of phenotypes maintain for at least 8 generations           9
3.7 H2O2 damage multiple organelles in a bacterium                     9
3.8 Gene expressions after H2O2 treatment                             9
3.9 Mice infected with H2O2-treated MRSA were more susceptable to 
oxacillin                                                    9
4.   Discussion                                                     10
5.   Conclusion                                                     12
6.   References                                                     13
7.   Table
    Table 1. MIC of oxacillin in 3 MRSA strains.                          15
8.   Figures
    Figure 1. The killing effects of different concentration of H2O2 to MRSA.   16
Figure 2. Oxacillin and cefoxitin susceptibility in 3 representative MRSA
 strains after H2O2 treatment.                                19
Figure 3. H2O2 altered oxacillin and cefoxitin susceptibility in MRSA.      20
Figure 4. Repeated exposure to H2O2 increased oxacillin susceptibility in MRSA-H.                                              21
Figure 5. MRSA grow faster after exposure to H2O2. MRSA-H 
exposed to 0.15% repeatedly.                               22
Figure 6. The biosynthesis of biofilms affected by H2O2 treatment.         23
Figure 7. MRSA became more susceptible to oxacillin after subcultured.    25
Figure 8. The antibiotics susceptibility of MRSA after 0.1%
H2O2 treatment.                                         26
Figure 9. Mec gene complex affected by H2O2 treatment.              27
Figure 10. In vivo bacterial counts after lung infections with oxidative stress-treated MRSA by nose inoculation of the bacteria.      29
9.   Supplementary Tables
Supplementary Table 1. Standards of clear inhibition zone diameter 
required for the definition of drug sensitive Staphylococcus aureus.                   30
Supplementary Table 2. The primers used in this study.              31

                                    

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校內：2023-02-21公開
校外：2023-02-21公開

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