小分子合成物對於會引起心內膜炎的鏈球菌的生物膜形成之抑制作用

Abstract

感染性心內膜炎（infective endocarditis）是一種高致死率、高復發率的心血管感染疾病，主要由一些口腔中的鏈球菌例如:轉糖鏈球菌(Streptococcus mutans)所引發。這些會引起心內膜炎的致病株可在受傷的瓣膜上形成生物膜(biofilm)，並與血小板、纖維蛋白(fibrin)及發炎細胞堆疊成贅疣（vegetation）。目前治療的潛在問題是這些在贅疣中形成生物膜的致病株具有高抗藥性。先前實驗室曾利用體外篩選模式(in vitro)模擬體內產生贅疣的狀況，證實血小板對於轉糖鏈球菌生成生物膜是重要的、以及所生成的生物膜對於抗生素具有更高的抗性，因此有必要進一步尋找一些小分子合成物來輔助抗生素抑制生物膜形成。本篇研究藉由體外生成生物膜的方法，並搭配結晶紫(crystal violet)的染色，篩選出CYY-X022, 47, 48, 58, 60五種小分子合成物能夠抑制細菌形成生物膜，而CYY-X011, 12, 22, 47, 52, 55, 56則可以干擾由細菌和血小板所形成的生物膜的生成。進一步測定細菌生長曲線，發現CYY-X022, 47, 48, 58, 60能有效地抑制細菌生長。利用血小板凝集計(aggregometer) 分析血小板活性以及測定細菌和血小板之間交互作用的狀況，發現CYY-X011, 12, 22, 47, 52, 55, 56能夠藉由干擾細菌和血小板之間的接觸來抑制血小板凝集，進而抑制由細菌和血小板所形成的生物膜。在活體 (in vivo) 實驗中，利用實驗室先前所建立的感染性心內膜炎大鼠模式(experimental streptococcal endocarditis rat model)，將CYY-X022, 47, 52, 55, 56分別注入大鼠體內，發現能夠降低贅疣中生物膜生成的狀況。除此之外，當抗生素搭配小分子一同使用，利用共軛焦顯微鏡的觀察以及菌落的計數，發現抑制效果比給予單一抗生素處理還要好。本篇研究結果篩選出具有抑制生物膜生成的小分子合成物，希望對於未來在感染性心內膜炎的治療上能夠提供重要的資訊，並且能夠在其他臨床的研究上也有更多的應用。

Infective endocarditis (IE) is a cardiovascular disease with high mortality rate and usually caused by oral streptococci (such as Streptococcus mutans) infection. The characteristic of IE is the formation of vegetations, fibrin-platelet clots with the embedded bacteria forming biofilm, which is refractory to routine antibiotic treatment. Previously, our data reported that platelets play important roles in vegetation formation and could enhance the resistance of streptococcal biofilm to antibiotics. Therefore, to search novel prophylactic agents that specifically target the platelet-associated biofilm will provide effective strategy for the successful control of IE. In this study, 76 different synthetic molecules have been screened in vitro by using biofilm formation assay and crystal violet staining. We found that CYY-X022, 47, 48, 58 and 60 could eliminate homotypic bacterial biofilm formation, and CYY-X011, 12, 22, 47, 52, 55 and 56 could interfere with platelet-associated biofilm formation. Among these effective synthetic molecules, CYY-X022, 47, 48, 58 and 60 could inhibit the bacterial growth , and CYY-X011, 12, 22, 47, 52, 55 and 56, alternatively, could interfere with the binding of S. mutans and platelets, as well as inhibiting the streptococci-induced platelet aggregation according to the results of platelet aggregation test. Consistent with the in vitro data, the vegetation size was decreased by intravenous administration of CYY-X022, 47, 52, 55 and 56 in the experimental streptococcal endocarditis rat model. Moreover, antibiotics combined with the targeted small molecules in vitro could effectively inhibit the biofilm formation based on the results of confocal laser scanning microscope observation and the measurement of survival bacteria. Taken together, the data suggest that these synthetic small molecules could eliminate streptococcal biofilm formation by inhibiting the bacterial growth or the binding of S. mutans and platelets. For clinical treatment on infective endocarditis, these targeted small molecules may have promising applications and offer another strategy.