A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries
A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries
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DOI：
10.3791/54829
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发表时间：
2016-12-01
影响因子：
1.2
通讯作者：
Fallarero, Adyary
中科院分区：
文献类型：
--
作者：
Skogman, Malena E.;Vuorela, Pia M.;Fallarero, Adyary
生物膜被认为是现代生物医学最具挑战性的课题之一，它们可能导致超过80%的耐药性感染。生物膜对化疗表现出极高的耐受性，这被认为是多因素的。例如，基质提供了减少抗生素渗透到生物膜中的物理屏障。此外，生物膜内的细胞在表型上是多样的。很可能，生物膜的弹性来自于这些和其他未知机制的组合。所有现有的抗生素都是针对单细胞（嗜酸性）细菌开发的。因此，到目前为止，存在可以选择性地作用于成熟生物膜的非常有限的分子库。这种情况已经推动了药物发现的进步范式转变，其中寻找抗生物膜已经被敦促占据更突出的位置。另一个挑战是，生物膜研究的标准化方法数量非常有限，特别是那些可用于化学文库的大通量筛选的方法。在这里，一个实验性的抗生物膜平台的化学筛选。它使用三种测定来测量生物膜活力（刃天青染色）、总生物量（结晶紫染色）和生物膜基质（使用麦胚凝集素、基于WGA荧光的聚-N-乙酰葡糖胺PNAG级分染色）。所有的测定都是使用金黄色葡萄球菌作为模型细菌开发的。该平台如何可用于初步筛选以及识别的抗生物膜命中的功能表征的例子。该实验序列还允许基于测量的终点对命中进行分类。它还提供了有关其作用模式的信息，特别是长期与短期化疗效果。因此，它对于快速鉴定高质量的命中化合物非常有利，这些化合物可以作为各种生物医学应用的起点。
Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.