Barua Niloy, Herken Ashlee M, Melendez-Velador Natalie, Platt Thomas G, Hansen Ryan R
Tim Taylor Department of Chemical Engineering, Kansas State University, 1701A Platt Street, Manhattan, Kansas 66506, USA.
Division of Biology, Kansas State University, 1717 Claflin Road, Manhattan, Kansas 66506, USA.
Biomicrofluidics. 2024 Feb 29;18(1):014107. doi: 10.1063/5.0188270. eCollection 2024 Jan.
Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens.
发现抑制病原体生长的新型细菌菌株有助于改进生物防治和益生菌策略。传统的基于平板的共培养方法用于探究微生物相互作用，可能会阻碍这一发现，因为这些方法本质上是低通量、劳动密集型且定性的。我们报告了一种第二代光寻址微孔装置，该装置旨在在不断增加的病原体压力下，迭代筛选细菌菌株文库中存在的候选生物防治剂与病原体之间的相互作用。微孔（0.6皮升体积）以可控的细胞比例在文库菌株和病原体之间提供独特的共培养位点。在连续筛选迭代过程中，文库菌株面临数量不断增加的病原体挑战，以定量鉴定含有抑制病原体数量最多的菌株的微孔。然后使用环形图案的365纳米光烧蚀可光降解水凝胶膜，并依次从装置中释放抑制性菌株以进行回收。对每个回收菌株的病原体抑制作用进行验证，随后进行全基因组测序。为了证明这种方法的快速性，该装置用于筛选一个由293个成员组成的生物变种1农杆菌菌株文库，以寻找对植物病原体丁香假单胞菌15955有抑制作用的菌株。一次迭代筛选就发现了9个新的抑制性菌株。相比之下，基于平板的方法未从该文库中发现任何抑制性菌株（n = 30个平板）。这里开发的新型病原体挑战筛选模式能够从细菌菌株文库中快速选择和回收有效抑制病原体生长的菌株，将这种微孔技术平台扩展到对益生菌、生物防治剂和能够抵御已知或新出现病原体的抑制性分子进行快速、经济高效且可扩展的筛选。
