2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/644904摘要：草綠色鏈球菌（Viridans streptococci）為伺機性的感染菌，也是造成亞急性感染性心內膜炎(subacute infective endocarditis)的主要病原菌。在台灣由草綠色鏈球菌轉糖鏈球菌引發菌血症造成亞急性感染性心內膜炎的比率最高，因此本實驗將利用轉糖鏈球菌作為代表菌種。當草綠色鏈球菌藉由傷口進入血流，造成暫時性的菌血症，存在於血流中的細菌會黏附到受損的心臟瓣膜上，形成贅疣（vegetation）進而引發感染性心內膜炎（infective endocarditis）的發生。而在先前學者利用電子顯微鏡觀察贅疣的結構發現，贅疣中是由細菌、血小板與纖維蛋白所構成的緊密包裹的結構，而這樣的結構符合學者針對細菌形成生物膜所造成疾病的臨床特徵。因此以往也認為在贅疣中草綠色鏈球菌是以生物膜的形式存在。而當細菌形成生物膜時，常具有抵抗抗生素與宿主細胞攻擊的能力。因此在臨床治療中，感染性心內膜炎一般建議持續接受靜脈注射抗生素達四到六週以上；一旦發現有難以控制的感染或心臟衰竭現象，除了抗生素治療外，更需要接受瓣膜修補或置換手術，而且手術死亡率也大幅提高。因此發展有效治療感染性心內膜炎的新型療法是有迫切需要的，而這也是本計畫的主要目標。以往學者們認為菌血症持續時間越長，越有利細菌黏附上受傷瓣膜，促使感染性心內膜炎的發生。因此在感染性心內膜炎發生的高危險群病患施行手術前，常會給予低量的抗生素來防止細菌在手術中進入人體造成菌血症，引發感染性心內膜炎的發生。然而對於這種手術前給予抗生素做法，迄今尚未被證明是否確實有防止病患菌血症的發生。先前的研究發現多株草綠色鏈球菌可以主動誘發血小板凝集，在我們先前的研究發現以人類血漿刺激轉糖鏈球菌後，可增加細菌在血液循環中的存活率。此外，我們也發現在利用血漿刺激細菌引發菌血症的老鼠的血液中可以找到類似生物膜結構的血小板與細菌的聚合物，我們推測這樣的類生物膜結構可能幫助細菌藉此躲避宿主免疫系統的攻擊，造成持續性的菌血症發生。因此我們第一部分除了想要釐清手術前給予抗生素是否能有效控制菌血症的發生外，也想要篩選可破壞血漿中細菌與血小板聚合物形成的抗生素，更有利於菌血症的清除。另外一方面，當感染性心內膜炎發生後，細菌在受傷的心臟瓣膜位置常以生物膜的形式存在。因此我們也嘗試篩選可有效破壞或抑制細菌體內生物膜形成之抗生素或天然及合成化合物。先前我們已經發展一套新的體外篩選細菌生物模形成模式，可以有效模擬細菌進入人體受到血液刺激後所形成的細菌生物膜，並且進一步搭配感染性心內膜炎大鼠模式進行動物實驗進行篩選。這種新穎的細菌生物膜篩選模式已進行台灣專利案件之申請(申請號: 098107239 ;申請日期: 2009/3/6)。而在此研究計畫中，我們嘗試藉由此種體外與體內的篩選模式平台，來更進一步篩檢可能有效應用於破壞或治療感染性心內膜炎中細菌生物膜形成的抗生素或小分子藥物，本計劃目標有三大點。1. 評估各種不同抗生素或合併天然及合成化合物預防菌血症之單獨或加成效用。2. 利用已建立之體外模式初步篩選可抑制或破壞細菌生物膜形成之抗生素或自然界小分子化合物及加成效用。3. 在心內膜炎大鼠模式進一步驗證研究目標2中所篩選出之藥物於體內模式之治療效果。本計畫之優勢在於本實驗室已建立良好的體外與體內模式可快速有效篩選抑制或破壞細菌生物膜形成的專利模型。故能有效測試不同抗生素與可能的小分子化合物對細菌生物膜的抑制效果，期望未來可應用於臨床治療解決治療感染性心內膜炎病患需要長期使用抗生素的缺點。所獲得的結果將申請專利並提供其他臨床治療細菌性感染症的應用。<br> Abstract: Viridans streptococci are the major opportunistic pathogens that cause the subacute type of infective endocarditis (IE). In viridans streptococci induced bacteremia, Streptococcus mutans has the highest rate to cause IE in Taiwan and therefore S. mutans is used as the representative species in this project for the IE model system. Bacteremia of viridans streptococci is caused by trauma or dental procedures in the oral cavity and the circulating bacteria can adhere to the damaged valve to form a compact structure, vegetation. Vegetation is composed of the platelets, fibrin and bacteria aggregates that form biofilm structure, within which bacteria become highly resistant to antibiotics and host immune defense. The characteristics of biofilm formation in IE also account for the prolonged usage of four to six week course of high dose antibiotics for the treatment of IE and the higher recurrence rate in IE patients. Therefore, the development of new therapeutic regimen is necessary for treating IE and is the primary goal of this translational research project.The longer bacteremia persist, patients are more susceptible to get IE. It is proposed that the prolonged bacteremia reinforce the bacterial adherence to the damaged valve. Clinically, the prophylaxis use of a high dose antibiotics is always performed to devoid the bacteremia in the patients under surgery. But there is no direct evidence to clarify the in vivo effect of antibiotics prophylaxis in prevention of the bacteremia. Viridans streptococci can actively induce platelet aggregation and we found that stimulation by human plasma can enhance the bacterial survival rate in the blood circulation. In addition, the bacteria-platelet aggregate, which is similar to the biofilm structure, is also detected in the bacteremia caused by plasma-enhaced S. mutans. We hypothesize that bacteria-platelet aggregate may help bacteria to escape from immune surveillance and cause the persistent bacteremia in vivo. Here, we will clarify the effect of antibiotics prophylaxis in controlling bacteremia and identify antibiotics which can disrupt the bacteria-platelet aggregate to effectively devoid and reduce the bacteremia in vivo.On the other hand, for successful treatment or control of IE, the essential element is to disrupt or prevent bacteria to form and survive in biofilm structure during vegetation formation at the damaged valve. In the second part, we will identify antibiotics and natural and synthetic compounds which can efficiently inhibit or disrupt the bacterial biofilm formation. In our previous research, we have developed a new in vitro model, which can mimic the bacterial biofilm formation in the presence of human blood. In addition, we have also established quantitative assay for biofilm formation in situ using an endocarditis rat model to confirm the in vivo effect of potential candidates which can disrupt or inhibit bacteria biofilm formation. Both the in vitro and in vivo biofilm model has been applied for a patent in Taiwan (Application number: 098107239; profiling date: 2009/3/6 ). In this project, we will utilize these in vitro and in vivo models to identify effective antibiotics or natural and synthetic compounds.The specific aims of this two-year proposal are to:1. Test and confirm the effect of antibiotics prophylaxis in controlling bacteremia caused by bacteria after stimulation by human whole plasma2. Identify effective antibiotics or natural and synthetic compounds which can inhibit or disrupt bacterial biofilm formation when administered alone or in conjunction.3. Confirm the effect of the selected antibiotics or natural and synthetic compounds in prevention of the disease in vivo using experimental endocarditis rat model.The advantage of our project is we have a well-setup in vitro and in vivo model which can efficiently identify different antibiotics or natural and synthetic compounds which can lead to the inhibition or disruption on bacterial biofilm. The selected possible candidates can be applied to the clinical usage to help the treatment of patients with infective endocarditis.