Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Parque Tecnológico de Ciencias de la Salud, Armilla, Granada, Spain.
Antonie Van Leeuwenhoek. 2012 Aug;102(2):361-74. doi: 10.1007/s10482-012-9746-7. Epub 2012 May 5.
Despite the availability of many culture-based antibiotic screening methods, the lack of sensitive automated methods to identify functional molecules directly from microbial cells still limits the search for new biologically active compounds. The effectiveness of antibiotic detection is influenced by the solubility of the assayed compounds, indicator strain sensitivity, culture media and assay configuration. We describe a qualitative high throughput screening system for detecting cell-perturbing molecules from bacterial colonies employing two opposed agar layers sequentially formed in prototype Society for Biomolecular Screening (SBS) plates, named Janus plates. Direct assay of microbial colonies against target organisms in opposed agar layers overcomes some of the limitations of agar overlay methods. The system enables the rapid detection of extracellular cell-perturbing molecules, e.g., antibiotics, excreted directly from environmental isolates. The source bacterial colonies remain separate from the target organism. The growth layer is prepared and grown independently, so environmental strains can be grown for longer intervals, at temperatures and in media that favor their growth and metabolite expression, while the assay layer with pathogens, usually requiring nutrient-rich medium and elevated temperatures, are added later. Colonies to be tested can be precisely arrayed on the first agar surface, thus avoiding dispersion and disturbance of potential antibiotic-producing colonies by overlaying agar with the target strain. The rectangular SBS configuration facilitates factorial replication of dense microbial colony arrays for testing with multiple assays and assay conditions employing robotic colony pickers and pin tools. Opposed agar layers only slightly reduced the effectiveness for detecting growth inhibition from pure antibiotics compared to single-layer agar diffusion assays. The Janus plate enabled an automation-assisted workflow where a lone operator can effectively identify and accumulate bioactive soil bacterial strains within a few weeks. We also envisage the method's utility for functional prescreening colonies of clones from genomic and metagenomic libraries or improved strains originating from mutagenized cells.
尽管有许多基于培养的抗生素筛选方法，但缺乏直接从微生物细胞中识别功能分子的敏感自动化方法，这仍然限制了新生物活性化合物的寻找。抗生素检测的效果受到被检测化合物的溶解度、指示菌株的敏感性、培养基和检测配置的影响。我们描述了一种定性高通量筛选系统，用于通过使用在原型生物分子相互作用筛选协会 (SBS) 板中依次形成的两个相反琼脂层来检测来自细菌菌落的扰乱细胞分子，该系统被命名为双面 Janus 板。在相反的琼脂层中直接检测微生物菌落针对目标生物克服了琼脂覆盖方法的一些局限性。该系统能够快速检测细胞外扰乱分子，例如抗生素，这些抗生素可直接从环境分离株中分泌。原始细菌菌落与目标生物保持分离。生长层是独立制备和生长的，因此可以在有利于其生长和代谢产物表达的温度和培养基中培养环境菌株更长的时间，而具有病原体的检测层，通常需要营养丰富的培养基和升高的温度，稍后再添加。要测试的菌落可以精确地排列在第一层琼脂表面上，从而避免用目标菌株的琼脂覆盖分散和干扰潜在的抗生素产生菌落。矩形 SBS 配置便于使用机器人菌落挑取器和针工具对具有多种检测和检测条件的密集微生物菌落阵列进行因子复制。与单层琼脂扩散检测相比，双面琼脂层略微降低了检测纯抗生素抑制生长的效果。Janus 板使自动化辅助工作流程成为可能，在该工作流程中，单个操作员可以在几周内有效地识别和积累具有生物活性的土壤细菌菌株。我们还设想该方法可用于功能预筛选来自基因组和宏基因组文库的克隆的菌落，或者源自诱变细胞的改良菌株。
