【作者】 李京宝；

【导师】 于文功；

【作者基本信息】 中国海洋大学 ， 药物化学， 2007， 博士

【摘要】 人们一直认为细菌是以单细胞形式存在,传统的抗菌药物研发也是以浮游细菌为靶点。然而近年来,研究发现自然界和宿主体内的细菌绝大多数以生物膜(biofilm)形式存在。生物膜是由细菌和其分泌的胞外基质在物体表面形成的高度组织化的多细胞结构,是细菌产生抗生素耐药和逃避机体免疫系统攻击的主要原因。细菌生物膜无处不在,在很多方面给人类生活带来严重的影响,如细菌的耐药性、生物腐蚀、食品污染等,已成为全球关注的重大科学难题。根据美国健康研究院的调查公告,80%以上的细菌性感染与生物膜有关。因此,细菌生物膜的防治已成为目前医药、材料等多领域研究与开发的前沿和热点。本论文旨在从海洋微生物中筛选能够抑制生物膜形成和/或破坏已形成的生物膜的活性物质,并探讨其结构特征、作用机制以及在生物膜防治方面的应用前景。以铜绿假单胞菌FRD1为试验菌株,用微量离心管生物膜体外模型筛选,发现一株来自海洋的弧菌QY101,其发酵液可以抑制FRD1的生物膜形成,并在一定浓度范围内具有量效关系。进一步试验表明,QY101发酵液中的活性成分与核酸、蛋白类物质无关,而与糖类物质有关。经过丙酮沉淀、透析、离子交换等方法分级纯化后,从QY101发酵液中得到了一种多糖,命名为A101。气相色谱分析结果表明,弧菌胞外多糖A101至少含有4种单糖残基,从光散射结果计算得出该多糖重均分子量约为1000 kD,红外光谱结果显示A101含有硫酸基,乙酰基,氨基等多种活性基团,其溶液有胶体性质并对波长为220nm的紫外光有强烈吸收。该多糖具有广谱的抗生物膜作用,对铜绿假单胞菌、金黄色葡萄球菌和表皮葡萄球菌三种病原菌的20个菌株中的17株有显著生物膜形成抑制作用。为了更加直观和实时地观察A101对生物膜形成的抑制作用,建立了Flow-cell生物膜体外模型。利用该模型,发现A101对FRD1生物膜形成的抑制效果达到95%以上。此外,A101对已形成的FRD1生物膜也具有破坏作用,破坏效果达到85%。最低生物膜消除浓度MBEC试验结果表明, A101可以提高生物膜对抗生素的敏感性,使阿米卡星对生物膜细菌的杀灭作用提高16倍以上。A101并不抑制游离铜绿假单胞菌FRD1的生长,相反地能够促进其生长。唯一碳源实验结果表明A101不能作为碳源被FRD1利用。进一步研究发现,A101可以防止FRD1菌体簇集,使其分散生长,从而能够更好地利用环境营养。这种分散作用也正是A101抗生物膜作用的机制。结合相关研究报道,推测A101可能是封闭了FRD1细胞表面某些菌体赖以相互结合的位点,阻碍了菌体之间的相互作用,从而抑制了生物膜形成;而且,由于A101对菌体表面位点的结合力强于细菌间的结合力,故可以破坏已经形成的生物膜。本文发现了一种弧菌胞外多糖A101,该多糖不仅抑制生物膜的形成,还能破坏已经形成的生物膜;并且其作用具有广谱性,对多种细菌,尤其是临床常见的铜绿假单胞菌、金黄色葡萄球菌、表皮葡萄球菌的生物膜均有作用;A101可以增强抗生素对生物膜细菌的杀灭效果,具有这种作用的多糖在国际上尚属首次报道。A101有望开发成为高效、广谱、安全的抗生物膜制剂,不但在临床细菌生物膜相关慢性感染的治疗、抗细菌生物膜新材料的研发等方面具有重要的应用前景,而且在生物膜形成与放散、细菌细胞间的识别和相互作用等多个领域具有重要的研究价值。更多还原

【Abstract】 In the past, bacterial cells were regarded as individual organisms growing in planktonic populations. However, it is now recognized that many or perhaps most bacteria have a strong propensity to form multicellular, matrix-enclosed assemblies, or biofilms, which are found on surfaces throughout the biological world. Cells within a biofilm have a number of advantages over their planktonic counterparts, including protection against the immune system and predation by protozoa, enhanced ability to transfer genetic information, and enhanced resistance to antimicrobial agents and other stresses. Thus, biofilm formation complicates a variety of chronic infections, including the devastating pulmonary infections that are caused by Pseudomonas aeruginosa in cystic fibrosis patients and other opportunistic infections by this organism. According to a public announcement by US National Institute of Health,“biofilms are medically important, accounting for over 80% of microbial infections in the body”. These problems of sessile organisms predominate in most of the environmental, industrial, and medical problems and cause the interest of microbiologists. Yet bacterial biofilms remain poorly understood and the strategies for their control remain underdeveloped. Therefore, anti-biofilm substances have become the hotspots in medicine, material and other research fields.The aim of this study was to screen active substance from marine bacteria, which can inhibit biofilm formation and/or disrupt established biofilm; and to explore its structural character and anti-biofilm mechanism.Using micro tube bacterial biofilm model, we found that the supernatant of marine Vibrio sp QY101 significantly inhibited the biofilm formation of P. aeruginosa FRD1, and the inhibiting effect was dose-dependent in a certain concentration range. Further study showed that the active ingredient in supernatant did not relate to nucleic acid and protein, but to carbohydrate.A polysaccharide, namely A101, was purified from QY101 supernatant by acetone precipitation, dialysis and ion exchange chromatography. In order to characterize the polysaccharide structurally, many modern techniques were used which include elemental analysis, gas chromatography, infrared spectroscopy, light scattering and so on. As a result, exopolysaccharide A101 contains at lest 4 monosaccharide residues and sulfuric acid, amino and acetyl group. The average molecular weight of A101 is determined to be 1000 kD. A101 solution has colloidal character and strong absorption at 220nm. It significantly inhibited the biofilm formation of P. aeruginosa, Staphylococcus aureus and S. epidermidis.In order to monitor biofilm more directly and in real-time manner, the flow cell model was established. The development of flow cell-grown biofilm of P. aeruginosa FRD1 was investigated by confocal laser scanning microscopy and the structural development of the biofilms was quantified by the computer program COMSTAT. The exopolysaccharide A101 inhibited P. aeruginosa FRD1 biofilm formation by more than 90% and disrupted its established biofilm by 85%. In contrast to amikacin alone, the combination of amikacin and A101 increased the susceptibility of FRD1 biofilm by more than 16 times in minimum biofilm elimination concentration (MBEC) test. Our result suggested that A101 could significantly enhance the activities of antibiotics against biofilms.A101 did not inhibit planktonic FRD1 cell growth. On the contrary, it promoted cell growth without being used as carbon source. In addition, A101 prevent bacterial cells from aggregating, made them grow dispersedly, and bettered utilizing the environmental nutrition. This function of A101 may explain its anti-biofilm effect. Based on our results, we hypothesized that the mechanism of A101 anti-biofilm is mediated by blocking interaction between the cells.We demonstrated here an exopolysaccharide extract from marine Vibrio sp QY101 which can inhibit the biofilm formation and disrupt the established biofilm. This exopolysaccharide A101 prevented the formation of biofilms by a wide range of bacteria, particularly clinical infective P. aeruginosa, S. aureus, and S. epidermidis. Combined with exopolysaccharide A101, antibiotic was strengthened in killing bacterial cells in biofilm. This is the first report about a polysaccharide having these functions. A101 is expected to develop as an effective, broad-spectrum and safe anti-biofilm agent. It not only has wide application in the treatment of clinical biofilm-related infections and development of novel anti-biofilm materials, but also has important value in biofilm research fields, such as the formation and detachment of biofilm, the recognition and interaction among bacterial cells.更多还原