菌血症為細菌進入患者體內並藉由血液擴散至全身之疾病。一旦患者感染了菌血症，便極易在短時間內引發敗血症，造成多重器官衰竭，最終導致死亡。雖然現今臨床上已有許多技術如紙錠擴散試驗(disk diffusion test)、E-test等能夠檢驗出能抑制患者體內細菌生長之抗生素種類與濃度，但仍需經兩至三天的培養與檢測時間。為了給予患者迅速有效的治療，本研究建立了以布朗運動做為檢測方法的光擴散平台，利用表面修飾螢光微珠來捕捉樣本中的細菌，並藉由分析黏附細菌的螢光微珠的擴散度變化來進行革蘭氏陰、陽、可動、不可動四象限細菌對於六種不同抗生素的藥物敏感性試驗(AST)。基於斯托克斯-愛因斯坦方程式(Stoke-Einstein equation)，微珠直徑與擴散率呈反比，意即當微珠黏附細菌數目增加時，微珠的擴散率便會下降，我們便可藉此得知藥敏試驗結果為抗藥性，該抗生素於最小抑菌濃度(MIC)之下無法抑制細菌生長。在結果方面，本研究利用微量分光光度計與連續稀釋法建立細菌濃度曲線以便在實驗中快速得知細菌濃度。再來，由於我們先前已完成利用光學擴散法檢測三象限細菌的實驗，故於本實驗中我們接續運用此法檢測可動陽性菌-李斯特菌，以證明光學擴散法可實施於不同的特性的細菌檢測。在血液處理方面，本實驗使用離心過濾法，先將大腸桿菌或金黃色葡萄球菌菌液加入全血，配置成107 CFU/mL的菌血，離心160 ×g五分鐘取上清液，再將上清液離心650 ×g十分鐘留下底部沉澱的細菌與少量血球，加入LB培養液混合均勻後通過5 μm過濾器，即可去除99.01%的血球細胞 (經計算，剩餘細胞數量降低至全血的0.09%)，並且大腸桿菌數量與金黃色葡萄球菌數量分別仍存有原樣本的21.7%與28.6%。為了模擬臨床上菌血症的患者檢體，我們在農科院採集的新鮮豬血中分別加入大腸桿菌與金黃色葡萄球菌，並配置出兩種濃度(107 CFU/mL與105 CFU/mL)的菌血。研究結果表示，利用光學擴散法偵測高低濃度的大腸桿菌血與金黃色葡萄球菌血對於六種抗生素的AST試驗皆呈現相同的結果。除此之外，我們還將其結果與臨床常用自動化醫療器材- Biomerieux Vitek2做比較，也得到了一致的成果，證明了光擴散法具有高靈敏度，即使檢體的濃度低至105 CFU/mL仍可獲得具有可信度的報告。相比其他的臨床方法，由於光擴散法不需等待細菌培養的過程，故只需3.5小時即可得出具有靈敏度、可信度的檢驗報告。相信此法日後能夠為菌血症與其他微生物感染疾病的早期治療方案提供另一項快速有效的新解法。

Bacteremia is a severe disease that is bacteria invading the human body and spread all over the body by the blood system. Once a patient developed bacteremia, it might cause multiple organ failure and death. Although there are serval mature techniques that take parts in the clinical field to detect the optimal antibiotic concentrations and types, like disk diffusion test, E-test, and other commercial in vitro diagnostics (IVDs), they need two to three days for cultured and detection.
To provide rapid and accurate diagnosis, we established an optical diffusometry platform based on Brownian Motion, capturing bacteria (including gram-positive, negative, motile, and non-motile strains) in blood samples with modified microbeads and practicing antibiotic susceptibility testing (AST) by analyzing changes in relative diffusivity of microbead-bacteria structure treated with six types of antibiotics with different antibacterial mechanisms. From the Stokes-Einstein equation, the diameter of the microbead is inversely proportional to diffusivity, that is, when bacteria numbers grow, the diffusivity of the microbead will decrease. By this, we can know that the result of AST is resistant, which means the antibiotic cannot inhibit bacteria grows under minimum inhibitory concentration (MIC).
As result, we established an OD600nm calibration curve with a serial dilution method and nanodrop detection to know the bacterial concentration as soon as possible. In previous studies, we have already done the detections except for testing gram-positive, motile bacteria with the optical diffusometry method. Thus, we picked L. monocytogenes, one of the common gram-positive motile strains to prove the method can be universally used to perform Antimicrobial Susceptibility Testing with various bacteria strains. For pre-preparation of whole blood, we used the centrifuge-concentration method. Bacteria broth was added into the blood to make a 107 colony-forming unit / mL (CFU/mL) bacterial blood sample. At first, the sample was gone through centrifuged for 160 ×g, 5 min to separate most blood cells and the supernatant. Then, the supernatant was collected and went through the second round of centrifugation, 650 ×g 10 min, to concentrate the bacteria cells. The pellet from centrifugation was mixed with LB broth and went through a 5 μm filter to remove blood cells and impurities as much as possible before performing optical diffusometry. After the process, 99.01% of the blood cells were removed , yet 21.7% of E. coli and 28.6% of S. aureus remained in the sample. To simulate the clinical blood samples from bacteremic patients, we used fresh porcine blood collected by Agricultural Technology Research Institute, Taiwan, and mixed with the bacteria broth to mimic E. coli bacteremia samples or S. aureus bacteremia samples with two different bacterial concentrations, 107 CFU/mL and 105 CFU/mL. The result showed that the concentration change did not result in different AST results in our optical diffusometry. Furthermore, the AST reports showed good agreement between the optical diffusometry and a commercial automatic clinical instrument, Biomerieux Vitek2. It proved that our system had a high sensitivity for the sample concentration down to 105 CFU/mL. In addition, our research only needs 3.5 h from the sample to the report with sensitivity and reliability. Eventually, we believe this technique can provide a novel solution for AST in the early stage of bacteremia and other infections.

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