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研究生:林佑軒
研究生(外文):You-Hsuan
論文名稱:利用表面增強拉曼散射技術快速診斷甲氧苯青黴素抗藥性金黃色葡萄球菌
論文名稱(外文):Using surface enhanced Raman scattering (SERS) for rapid diagnosis of methicillin resistant Staphylococcus aureus (MRSA)
指導教授:林奇宏林奇宏引用關係
指導教授(外文):Chi-Hung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生醫光電工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:72
中文關鍵詞:表面增強拉曼散射拉曼散射甲氧苯青黴素抗藥性金黃色葡萄球菌
外文關鍵詞:surface enhanced Raman scatteringRaman scatteringmethicillin resistant Staphylococcus aureus
相關次數:
  • 被引用被引用:2
  • 點閱點閱:240
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  • 下載下載:29
  • 收藏至我的研究室書目清單書目收藏:0
抗生素抗藥性細菌對於人類之威脅是長久以來難以控制之課題。目前最常見的抗生素抗藥性細菌便是甲氧苯青黴素抗藥性金黃色葡萄球菌(Methicillin Resistant Staphylococcus aureus,MRSA)或稱為多重抗藥性金黃色葡萄球菌(Multi-Drug Resistant S. aureus)。醫院內部病患集體血液感染的病源有60%皆為MRSA,並且其對多種beta-lactam類抗生素具有抗藥性,故為了能有效治療且防止MRSA的擴散,快速診斷細菌抗生素抗藥與否在臨床上非常重要。然而,傳統分辨MRSA及抗生素敏感型金黃色葡萄球菌(Methicillin Susceptible S. aureus)主要是藉由細菌在抗生素刺激下生長的狀態做為指標,其需要冗長培養時間,依菌種生長之速度需要三天至數週。本研究利用表面增強拉曼散射技術(Surface Enhanced Raman Scattering,SERS)配合高訊號增強能力之奈米銀粒子底板,建立快速診斷MRSA之平台。結果顯示,以rhodamine 6G確認奈米銀粒子底板的訊號增強能力,奈米銀粒子底板可在10秒的積分時間便能得到訊雜比兩萬的明顯訊號,除此之外,同一片奈米銀粒子底板上不同區塊或不同片的奈米銀粒子底板皆能取得高重複性及穩定性之金黃色葡萄球菌拉曼光譜。在分辨MRSA及MSSA上,利用間隔五分鐘偵測一次細菌拉曼光譜的方法,觀察菌體外壁化學結構在含有抗生素cefoxitin或oxacillin環境下動態變化,非抗藥性金黃色葡萄球菌在一小時內,光譜會出現劇烈的變化,而MRSA光譜則穩定不變,將觀察起始點的光譜與每個時間觀察到的光譜做相似度分析,可發現MSSA存在相似度低於0.7的光譜,MRSA相似度則皆高於0.85,若以相似度0.7作為判斷細菌抗藥性與否的標準,檢測臨床金黃色葡萄球菌之抗藥性,雖然施加oxacillin僅有50%的正確性,但若施與cefoxitin則呈現100%的正確性。另外,本研究發現MRSA臨床菌株之標準光譜呈現兩種不同的類型,第一種類型的光譜具有655 raman shift的訊號強度高於732 raman shift的特性,此種類型的光譜與MSSA光譜655 raman shift的訊號強度低於732 raman shift的特性相反,故可以與MSSA有明顯的區分,而另外一種類型的MRSA光譜655 raman shift 的強度低於732 raman shift,故其無法與MSSA有明顯的區分。PBP2a及beta-lactamase是使得MRSA具有抗藥性的兩種重要蛋白,經由西方墨點法及nitrocefin紙錠分析後,發現第一種光譜類型的MRSA菌體外壁含有較第二種光譜類型MRSA多的PBP2a,但beta-lactamase的活性則是第二光譜類型的MRSA較第一種類型的MRSA高,除此之外,若以表面增強拉曼散射技術連續時間觀察MRSA在同時含有beta-lactamase抑制劑potassium clavulanate及抗生素oxacillin的環境下的動態變化,MRSA中第二種類型之光譜便會出現劇烈的變化而第一類型之光譜則仍然呈現穩定不變,所以結果顯示兩種不同光譜特性的MRSA極可能代表兩種不同的抗藥機制。本研究之結果不僅可提供臨床醫師相較於傳統檢驗更快且更多資訊的診斷技術外,對於目前仍然無法解釋細菌表面增強拉曼散射訊號在生物分子所代表之意義,可給予許多重要的訊息。
Methicillin resistant Staphylococcus aureus (MRSA), the most popular strain of antibiotic resistant bacteria or multi-drug resistant Staphylococcus aureus, is resistant to various beta-lactam antibiotics and responsible for 60% nosocomial sepsis infection. It is clinically crucial to rapidly determine the antibiotic susceptibility of S. aureus isolates because this determination is paramount for both treatment and control measures. However, conventional methods for determining S. aureus antibiotic susceptibility depend mostly on measuring proliferation rates upon drug treatments, which inevitably take time, from days for fast-growing bacteria to weeks for slow-growers. We report here a novel way to differentiate MRSA and multi-drug sensitive Staphylococcus aureu (MSSA) using surface enhanced raman scattering (SERS) with a SERS-active substrate made of an array of silver nanoparticles. The result shows the signal to noise ratio of SERS spectra is about twenty thousand photo counts, which can be easily detected within only 10 seconds. Besides, the SERS spectra obtained from different clusters of S. aureus on the same substrate or S. aureus on different substrates of different lots are highly reproducible and stable. Using the steady SERS system, we can differentiate MSSA and MRSA by observing the changes of “chemical features” based on bacterial envelop upon antibiotic treatment. The results of sequential SERS recordings of MRSA and MSSA upon the treatment of oxacillin or cefoxitin show there were no obvious changes in the SERS profile of MRSA but obvious changes in the SERS profile of MSSA within 2 hours. The correlation coefficients between the spectrum of initial and individual time during antibiotic treatment were higher than 0.85 on MRSA and lower than 0.7 on MSSA. We set the correlation coefficient 0.7 as a criterion for determining antibiotic susceptibility of S. aureus isolated from clinical samples. The highly discriminatory readouts show the accuracy is 100% upon the cefoxitin treatment, although there was merely 50% upon the oxacillin treatment. Furthermore, there were two different spectral patterns of MRSA. The spectrum of MRSA Type I has higher intensity at 655 raman shift than that of 732 raman shift. Contrary to the type I spectrum of MRSA, the spectrum of MRSA type II has lower former but higher latter. Penicillin binding protein 2a and ��-lactamase both are the key protein leading antibiotic resistance. Based on the results of western blotting and nitrocefin assay, we found MRSA of type I spectrum express higher amount of PBP2a and lower activity of beta-lactamase than MRSA of type II. Interestingly, sequential SERS recordings of MRSA with the exposure to both oxacillin and beta-lactamase inhibitor show there are no obvious change in type I spectrum of MRSA but obvious change in type II spectrum of MRSA. These results suggest that SERS can not only potentially be developed as a rapid, reliable and non-growth-based method for rapid diagnosis of MRSA, but also differentiate the major antibiotic resistant mechanisms of MRSA and offer some information for explaining the biological meaning of the peaks on SERS spectrum.
中文摘要..............................................i
英文摘要..............................................ii
目次..................................................iii
第一章 研究目的.......................................1
第二章 研究背景.......................................3
2.1 金黃色葡萄球菌背景介紹............................3
2.1.1金黃色葡萄球菌的生理特性.........................3
2.1.2金黃色葡萄球菌的外壁構造.........................3
2.1.3 金黃色葡萄球菌致病物質..........................5
2.1.4 金黃色葡萄球菌感染之臨床病症....................5
2.1.5 甲氧苯青黴素抗藥性金黃色葡萄球菌(MRSA)..........6
2.1.5 MRSA的臨床檢驗方法..............................8
2.2 拉曼散射.........................................10
2.2.1 拉曼散射歷史...................................10
2.2.2 散射種類.......................................11
2.2.3 拉曼散射原理...................................12
2.2.4 拉曼光譜.......................................13
2.3 表面增強拉曼散射.................................15
2.3.1 表面增強拉曼散射歷史...........................15
2.3.2 表面增強拉曼散射原理...........................15
2.3.3 表面增強拉曼散射在生物醫學發展之近況...........17
第三章 材料與方法....................................19
3.1 奈米銀底板訊號增強效果測試.......................19
3.2 拉曼光譜儀.......................................20
3.3 偵測MRSA及MSSA表面增強拉曼散射光譜...............22
3.4 表面增強拉曼散射技術平台觀察菌體對抗生素之反應...23
3.5 拉曼光譜資料處理.................................24
3.6 拉曼光譜相似度分析...............................27
3.7 西方墨點法分析PBP2a蛋白表現量....................27
3.7 Nitrocefin紙錠觀察beta-lactamase活性及表現量.....28
第四章 研究結果......................................29
4.1 奈米銀底板訊號增強效果............................29
4.2 奈米銀底板增強細菌光譜訊號穩定測試...............30
4.3 MRSA及MSSA表面增強拉曼散射光譜...................31
4.4 表面增強拉曼散射技術偵測MRSA及MSSA於抗生素環境下之動態
變化.............................................32
4.5 MRSA之PBP 2a表現量與表面增強拉曼散射光譜之關係...33
4.6 MRSA之beta-lactamase表現量與表面增強拉曼散射光譜之關係
.................................................34
4.7 表面增強拉曼散射技術動態觀察beta-lactamase抑制劑
Potassium clavulanate對於MRSA在抗生素環境下之影
響...............................................35
4.8 以表面增強拉曼散射技術應用於臨床金黃色葡萄球菌抗藥性
鑑定之專一性及敏感性.............................36
第五章 討論..........................................37
5.1 建立抗藥性金黃色葡萄球菌快速診斷平台之重要性......37
5.2 表面增強拉曼散射應用於生物樣品檢測之潛力..........37
5.3 以高穩定度和靈敏度拉曼光譜儀及奈米銀粒子底板建立快
速診斷抗藥性金黃色葡萄球菌平台之可行性............38
5.4 表面增強拉曼散射檢測平台對於臨床檢驗之助益及市場產值.38
5.5 利用表面增強拉曼散射技術快速分析抗藥性金黃色葡萄球菌主要
抗藥機制對於臨床治療之重要性.........................39
5.6 Cefoxitin及Oxacillin在表面增強拉曼散射系統分辨MRSA之效
果...................................................40
第六章 結論..............................................41
附圖.....................................................42
參考文獻.................................................68
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