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研究生:楊燿慈
研究生(外文):Yao-TzuYang
論文名稱:以微珠式光擴散儀發展適用於多樣性抗生素與菌株之快速藥物敏感試驗
論文名稱(外文):Developing rapid antimicrobial susceptibility testing for universal bacteria and antibiotics based on bead-based optical diffusometry
指導教授:莊漢聲莊漢聲引用關係
指導教授(外文):Han-Sheng Chuang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物醫學工程學系
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:72
中文關鍵詞:表面修飾微珠快速檢測細菌藥敏試驗光學擴散法布朗運動抗藥性細菌革蘭氏陰性菌革蘭氏陽性菌抗生素
外文關鍵詞:functionalized microbeadsrapid detectionantimicrobial susceptibility testingoptical diffusometryBrownian motiondrug-resistant bacteriaGram-negative bacteriaGram-positive bacteriaantibiotic
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細菌藥物敏感性試驗(AST)的快速化是治療感染和預防細菌抗藥性的有效措施之一;然而,日漸增多的抗生素以及細菌種類已經形成了快速診斷的障礙。為了突破此局面,此研究以光擴散儀觀測修飾微珠和細菌之間的相互作用造成布朗運動的變化,當與細菌結合時,微珠的有效直徑增加,因此導致了隨時間的擴散率下降。根據斯托克斯-愛因斯坦方程式(Stoke-Einstein equation),擴散率與微珠直徑成反比,若擴散率下降表示細菌數目增加並與微珠結合,進一步得出細菌藥敏試驗結果為具有抗藥性。結果表明,基於微珠擴散率的趨勢變化,此研究可以在三小時內完成藥敏試驗,即使加上檢體前處理,從取得檢體到取得檢驗結果總時長也僅需要四小時,這比冗長的傳統方式從取得檢體後進行細菌培養再進行紙碇擴散法藥敏試驗要快了許多。本研究選定涵蓋了革蘭氏陽性/陰性和可動/不可動等特徵的四種細菌菌株,即大腸桿菌、綠膿桿菌、克雷伯氏菌肺炎和金黃色葡萄球菌,與六種涵蓋了五個不同殺菌機制之抗生素結合使用,以了解細菌和抗生素之間的相互作用如何造成微珠的擴散性的變化。儘管各種抗生素擁有不同機制,但在兩小時的測量期的最後40分鐘,對於敏感和抗藥的組別,都能得出各自的擴散係數變化趨勢以供判斷。此外,使用光擴散儀測得的結果也與從商業自動化儀器以及傳統人工法獲得的結果一致。最後,我們進行了單盲臨床樣品測試,本技術於靈敏度、特異性和準確性分別達到92.9 %,91.4 %與 91.8 %。總體而言,本研究開發的光學擴散法達到了快速化藥敏試驗(〈 4 h),微小化樣品量(15 μL),需求初始細菌濃度低(10^5 CFU / mL)。為將來針對微生物疾病的早期治療提供了一種快速且有效的新選擇。
The rapid antimicrobial susceptibility testing (AST) is an effective measure in the treatment of infections with an appropriate concentration which preventing bacterial drug resistance. However, diverse antibiotic types and bacterial characteristics have formed complicated barriers to rapid diagnosis. To counteract the limitations, we investigated the interactions between antibiotic-treated bacteria and functionalized microbeads in a self-developed optical diffusometry. The microbead diameter increased when conjugating with bacteria, hence resulting in a time-dependent diffusivity change. According to the Stokes-Einstein equation, the diffusivity is inversely proportional to the microbead diameter. The yielded data were then sorted and analyzed to delineate a pattern for prediction of antimicrobial susceptibility. The results showed that rapid AST based on the dependent trend of microbead diffusivity could be obtained within 4 h, which was faster than the lengthy bacterial culture. Four strains of bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus, which cover the domains of Gram-positive/negative and motile/non-motile in combination with six antibiotics associated with five antiseptic mechanisms were investigated for understanding how interactions between bacteria and antibiotics may alter the microbead diffusivity. Despite different inhibitory effects caused by various antibiotics, similar trends in diffusivity alteration for all susceptible and resistant cases in the last 40 min of the 2-h measurement period were deduced. In addition, our AST results using the optical diffusometry also showed good agreement with those acquired from commercial instrument as well as conventional culture, with the sensitivity, specificity, and accuracy of the system achieving 100 %, 90.1 %, and 96.4 %, respectively. At last, we conducted a single-blinded clinical test, in which 6 out of 7 samples were accurately diagnosed with our technique. Overall, the developed optical diffusometry showcased rapid AST with only a small volume (20 μL) of sample and a low initial bacterial count (105 CFU/mL) for all antibiotics and bacterial strains. The technique provides a promising way to achieve early therapy against microbial diseases in the near future.
CONTENT
摘要 II
ABSTRACT III
ACKNOWLEDGEMENT V
CONTENT VI
LIST OF FIGURES IX
LIST OF TABLES XI
LIST OF ABBREVIATIONS XII
1 INTRODUCTION 1
1.1 Motivation and Overview 1
1.2 Bacteria Strains in this Research 3
1.2.1 Gram-Positive and Gram-Negative Bacteria 4
1.2.2 Motile and Non-motile Bacteria 5
1.2.3 Aerobic, Anaerobic and Facultative Anaerobic Bacteria 5
1.3 Mechanisms of Bacterial Inhibition and Morphological Change from Antibiotics 6
1.4 Rapid AST 7
1.4.1 The State-of-the-Art Techniques 8
1.4.2 Optical Diffusometry 8
1.5 Aims and Contribution of this Thesis 10
2 MATERIALS AND METHODS 11
2.1 Reagents 11
2.2 Experimental Setup and Microchip Fabrication 13
2.3 Functionalization of Probe Microbeads 15
2.4 Performance between Optical Diffusometry and Vitek2 17
2.4.1 Bacterial Culture 17
2.4.2 Preparations of Antibiotics 17
2.4.3 Optical Diffusometry/BacilluSense 18
2.4.4 Vitek2 19
2.5 Evaluation of Clinical Samples using Optical Diffusometry and Vitek2 20
2.5.1 Transportation of Clinical Samples 20
2.5.2 Optical Diffusometry/BacilluSense 20
2.5.3 Vitek2 21
2.6 Deduction of Diffusive Pattern from Bacterial Motility and Morphological Changes 22
3 RESULTS AND DISCUSSION 25
3.1 Bacterial Growth in PMMA Chip 25
3.2 Determination of an Optimal Threshold for Rapid AST 28
3.2.1 Receiver Operating Characteristic Curve 29
3.3 AST Results of Optical Diffusometry and Vitek2 31
3.3.1 Comparisons of the Six Antibiotics on E. coli between Optical Diffusometry and Vitek2 31
3.3.2 Comparisons of the Six Antibiotics on S. aureus between Optical Diffusometry and Vitek2 33
3.3.3 Comparisons of the Six Antibiotics on P. aeruginosa between Optical Diffusometry and Vitek2 34
3.3.4 Comparisons of the Six Antibiotics on K. pneumoniae between Optical Diffusometry and Vitek2 36
3.4 Evaluations of Clinical Samples using Optical Diffusometry and Vitek2 38
4 CONCLUSIONS 42
5 FUTURE WORK 44
REFERENCES 45
APPENDICES 50
APPENDIX A 50
APPENDIX B 53
APPENDIX C 72
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