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系統識別號 U0026-2708201811243700
論文名稱(中文) 以熱擴散平台監測致病菌及其對抗生素的反應
論文名稱(英文) Monitoring of Pathogenic Microorganisms and Their Responses to Antibiotics on a Diffusometric Platform
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 106
學期 2
出版年 107
研究生(中文) 紀邵文
研究生(英文) Shao-Wen Chi
電子信箱 zoo53071ox@gmail.com
學號 p86041231
學位類別 碩士
語文別 英文
論文頁數 78頁
口試委員 口試委員-張憲彰
口試委員-李汪洋
口試委員-吳炳慶
指導教授-莊漢聲
中文關鍵字 光學擴散法  粒子免疫分析  細菌  抗體  抗菌藥敏試驗  布朗運動  抗生素  萬古黴素 
英文關鍵字 optical diffusometry  bead-based immunoassays  bacteria  antibody  antimicrobial susceptibility testing  Brownian motion  antibiotic  vancomycin 
學科別分類
中文摘要 針對細菌感染的成功治療,取決於抗菌藥物敏感性測試(AST)。快速有效的抗生素篩選可以及時挽救許多生命。然而,一般傳統的AST通常需要超過24小時來確定結果,導致患者死亡率高。此外,多種微生物感染加劇了這種情況。因此,本文開發了結合光學擴散測定法和基於珠子的免疫測定的技術,以檢測微生物並實現快速AST。布朗運動是受環境溫度,液體粘度和粒度影響的顆粒的隨機運動。在恆定溫度和液體粘度下,擴散係數與粒徑成反比。因此,布朗運動將會隨著細菌結合顆粒而產生變化。本技術的測量系統簡單地由螢光顯微鏡和數碼相機所組成。我們使用的演算法-空間互相關算法,用於量化來自一系列連續粒子圖像的布朗運動的程度。
在前半部分,用兩種共培養的細菌綠膿桿菌和金黃色葡萄球菌探討該系統。粒子的免疫測定用於檢測其相對應抗原的存在,並相對於細菌濃度的擴散率變化與我們理論預測的一致。考慮到快速AST,將細菌與慶大霉素在TSB中在37℃下混合2小時。每20分鐘記錄三色粒子圖像,以便能夠同時檢測雙重微生物。三種熒光顆粒中的兩種用不同的病原體特異性抗體功能化,而第三種粒子用來作為對照組。最終的結果表明該系統能夠在兩小時內分別顯示抗生素對共培養的綠膿桿菌和金黃色葡萄球菌的有效性。
在第二部分中,我們使用萬古黴素修飾的顆粒來試驗三種不同的革蘭氏細菌。革蘭氏陰性菌大腸桿菌,綠膿桿菌和革蘭氏陽性菌的金黃色葡萄球菌。最終的實驗報告顯示萬古黴素修飾的粒子,對於無論是革蘭氏陽性還是革蘭氏陰性,都可以產生接合。因此,如果將萬古黴素修飾粒子與我們的光學擴散分析和粒子的免疫分析結合使用以檢測未知病原體,在臨床試驗中患者樣本的病原體是未知的,這不僅可以解決病原的特異性問題,還可以直接進行抗生素治療,有效縮短檢測時間。
在隨後的臨床試驗中,在擴散測量平台上測量77個尿液樣本以評估大腸桿菌的感染。然而本技術與瓊脂上一般傳統的細菌培養相比,使用抗體修飾粒子的診斷準確率為86.1%,而使用萬古黴素粒子更好,它的準確率達到80%。總體而言,本系統能夠同時檢測多種微生物,節省時間(< 2小時),小樣品體積(5μL)和低微生物初始細菌數(105 CFU / mL)監測。本研究提供了在不久的將來快速治療多種微生物疾病的實際意義。
英文摘要 Successful treatments against bacterial infections depend on antimicrobial susceptibility testing (AST). A rapid and effective antibiotic screen can save numerous lives in time. However, conventional AST usually requires more than 24 h to determine an outcome, resulting in high patient mortality. In addition, multi-microbial infections exacerbate the situation. A technique combining optical diffusometry and bead-based immunoassays was therefore developed herein to detect selected microorganisms and achieve rapid AST. Brownian motion is a random movement of particles subject to ambient temperature, liquid viscosity, and particle size. With constant temperature and liquid viscosity, the diffusion coefficient is inversely proportional to the particle diameter. Accordingly, the Brownian motion will tend to vary in response to the bacterium-binding particles. The measurement system was simply composed of a fluorescent microscope and a digital camera. A spatial cross-correlation algorithm was used to quantify the degree of Brownian motion from a series of consecutive particle images. In the front part, the system was evaluated with two co-cultured bacteria, Pseudomonas aeruginosa and Staphylococcus aureus. Bead-based immunoassays were used to detect the presence of their corresponding antigens. The diffusivity changes with respect to the bacterial concentrations were consistent with the theoretical predictions. Considering rapid AST, bacteria were mixed with gentamicin in TSB at 37 °C for 2 h. Triple color particle images were recorded every 20 min to enable simultaneous detection of the dual microorganisms. Two of the three fluorescent particles were functionalized with dissimilar pathogen-specific antibodies while the third one was used as a control. The final result suggested that the system was able to respectively indicate the effectiveness of antibiotic on the co-cultured Pseudomonas aeruginosa and Staphylococcus aureus within 2 h. In the second part, we used vancomycin modified particles to experiment with three types of bacteria, Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and Gram-positive bacteria of Staphylococcus aureus. The final result showed that whether Gram-positive or Gram-negative bacteria, vancomycin-modified particles can be combined. Therefore, we consider that the pathogens of patient samples are unknown in clinical trials if vancomycin-modified particles are used in conjunction with our optical diffusion assays and bead-based immunoassays to detect unknown pathogens. This can not only solve the specific problem but also directly give antibiotic treatment to effectively reduce the detection time. In subsequent clinical trials, 77 urine samples were measured on the diffusometry platform to evaluate the infection of E. coli. However, compared with the conventional bacterial culture on agar, the diagnostic accuracy of using antibody-modified particles is 86.1%. Using vancomycin-modified particles to detect which is better, the accuracy is 80%. Overall, the system was capable of multiplexed, time-saving (< 2 h), small sample volume (5 µL), and low initial bacterial count (105 CFU/mL) monitoring for microorganisms. This study provides a practical sense of rapid therapy against polymicrobial diseases in the near future.
論文目次 摘要 .................................................I
ABSTRACT .......................................III
致謝 .................................................V
CONTENTS ........................................VI
LIST OF FIGURES ........................................IX
LIST OF TABLES .......................................XII
LIST OF ABBREVIATIONS ..............................XIII
CHAPTER 1 INTRODUCTION .........................1
1.1 Motivation and Overview .........................1
1.2 Bead-Based Immunoassay .........................4
1.3 Brownian Motion .................................5
1.4 Microbiology of Bacteria .................6
1.4.1 Gram-Positive and Gram-Negative Bacteria .........7
1.4.2 Antibiotic Resistance .........................8
1.5 Antimicrobial Susceptibility Testing .........9
1.6 Aims and Contribution of the Thesis ........11
CHAPTER 2 MATERIALS AND METHODS ................13
2.1 Reagents ................................13
2.2 Optical Diffusometric Platform ................15
2.3 Evaluations of Diffusometry ................16
2.3.1 Cross-Correlation Algorithm ................16
2.3.2 Diffusivity Calculation ........................17
2.4 Bead-Based Sensing ........................17
2.4.1 Separation of Different Color Particles ........18
2.4.2 Single Color Particles ........................18
2.4.3 Multiple Color Particles ................19
2.5 Equivalent Volume Diameter ................20
2.6 Quantification of Bacteria by Diffusometry ....21
2.6.1 Motility of Bacteria ........................21
2.7 Preparation of Samples ........................22
2.7.1 Bacterial Culture ........................22
2.7.2 Preparation of Clinical Samples ................22
2.8 Interactions Between Vancomycin Modified Particles and Bacteria ........................................23
2.9 Determination of AST ........................25
2.10 Rapid Antimicrobial Susceptibility Testing ....26
CHAPTER 3 RESULTS AND DISCUSSION ................27
3.1 Antibody-Modified Particles ................27
3.1.1 Detection of Single Bacteria ................27
3.1.2 Escherichia coli ................................29
3.1.3 Pseudomonas aeruginosa ........................30
3.1.4 Staphylococcus aureus ........................31
3.2 Detection of Multiple Bacteria ................32
3.2.1 Quantification of the Modified Particles ........32
3.2.2 AST with Gentamicin ........................33
3.3 Vancomycin-Modified Particles ................36
3.3.1 Validations of the Modified Particles ........36
3.3.2 Aggregation ................................38
3.4 Experimental Results of vancomycin Modified Particles ........................................40
3.4.1 Escherichia coli ................................41
3.4.2 Pseudomonas aeruginosa ........................42
3.4.3 Staphylococcus aureus ........................44
3.5 Interactions between Beads and Interferences ...46
3.5.1 Proteins and Cells ........................47
3.5.2 Proteins, Cells, and Bacteria ................49
3.6 Clinical Samples ........................56
3.6.1 Antibody-Conjugated Particles ................56
3.6.2 Vancomycin-Conjugated Particles ................57
CHAPTER 4 CONCLUSION ........................63
CHAPTER 5 FUTURE WORK ........................65
REFERENCES ........................................66
APPENDICES ........................................72
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