Yadav Mukesh K, Go Yoon Y, Kim Shin Hye, Chae Sung-Won, Song Jae-Jun
Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea.
Institute for Medical Device Clinical Trials, Korea University College of Medicine, Seoul, South Korea.
Front Microbiol. 2017 Oct 10;8:1948. doi: 10.3389/fmicb.2017.01948. eCollection 2017.
colonize the human nasopharynx in the form of biofilms. The biofilms act as bacterial reservoirs and planktonic bacteria from these biofilms can migrate to other sterile anatomical sites to cause pneumonia, otitis media (OM), bacteremia and meningitis. Human amniotic membrane contains numerous growth factors and antimicrobial activity; however, these have not been studied in detail. In this study, we prepared amniotic membrane extract and chorionic membrane extract (AME/CME) and evaluated their antibacterial and antibiofilm activities against using an biofilm model and OM rat model. The AME/CME were prepared and protein was quantified using DC (detergent compatible) method. The minimum inhibitory concentrations were determined using broth dilution method, and the synergistic effect of AME/CME with Penicillin-streptomycin was detected checkerboard. The biofilm and colonization of were studied using microtiter plate assay and OM rat model, respectively. The AME/CME-treated biofilms were examined using scanning electron microscope and confocal microscopy. To examine the constituents of AME/CME, we determined the proteins and peptides of AME/CME using tandem mass tag-based quantitative mass spectrometry. AME/CME treatment significantly ( < 0.05) inhibited growth in planktonic form and in biofilms. Combined application of AME/CME and Penicillin-streptomycin solution had a synergistic effect against Biofilms grown with AME/CME were thin, scattered, and unorganized. AME/CME effectively eradicated pre-established pneumococci biofilms and has a bactericidal effect. AME treatment significantly ( < 0.05) reduced bacterial colonization in the rat middle ear. The proteomics analysis revealed that the AME/CME contains hydrolase, ribonuclease, protease, and other antimicrobial proteins and peptides. AME/CME inhibits growth in the planktonic and biofilm states via its antimicrobial proteins and peptides. AME/CME are non-cytotoxic, natural human product; therefore, they may be used alone or with antibiotics to treat infections.
以生物膜的形式定殖于人类鼻咽部。这些生物膜充当细菌储存库，来自这些生物膜的浮游细菌可迁移至其他无菌解剖部位，引发肺炎、中耳炎（OM）、菌血症和脑膜炎。人羊膜含有多种生长因子和抗菌活性；然而，尚未对其进行详细研究。在本研究中，我们制备了羊膜提取物和绒毛膜提取物（AME/CME），并使用体外生物膜模型和OM大鼠模型评估了它们对肺炎链球菌的抗菌和抗生物膜活性。制备AME/CME并使用DC（去污剂兼容）法对蛋白质进行定量。采用肉汤稀释法测定最低抑菌浓度，并通过棋盘法检测AME/CME与青霉素 - 链霉素的协同作用。分别使用微量滴定板法和OM大鼠模型研究肺炎链球菌的生物膜形成和定殖情况。使用扫描电子显微镜和共聚焦显微镜检查经AME/CME处理的生物膜。为了检测AME/CME的成分，我们使用基于串联质量标签的定量质谱法测定AME/CME的蛋白质和肽。AME/CME处理显著（P < 0.05）抑制肺炎链球菌的浮游形式生长和生物膜生长。AME/CME与青霉素 - 链霉素溶液联合应用对肺炎链球菌具有协同作用。用AME/CME培养的生物膜薄、分散且无组织。AME/CME有效根除预先形成的肺炎链球菌生物膜并具有杀菌作用。AME处理显著（P < 0.05）减少大鼠中耳的细菌定殖。蛋白质组学分析表明，AME/CME含有水解酶、核糖核酸酶、蛋白酶和其他抗菌蛋白质及肽。AME/CME通过其抗菌蛋白质和肽抑制肺炎链球菌在浮游和生物膜状态下的生长。AME/CME是无细胞毒性的天然人类产物；因此，它们可单独使用或与抗生素联合用于治疗肺炎链球菌感染。
