High-Throughput Spot Assay for Isolation of Bacteriophages Against Multiple Host Strains from Environmental Samples.

Phage therapy has emerged as a promising option for treating infections caused by multidrug-resistant bacteria, offering an alternative to conventional antibiotics. However, conventional isolation methods are laborious and tend to favor dominant host-phage pairs, limiting the diversity of recoverable phages. Expanding the collection of phages is therefore a critical step toward advancing phage-based therapies. To achieve effective isolation outcomes, it is essential to systematically screen environmental samples against a broad range of host strains under standardized conditions. The efficiency of phage recovery depends on several factors, including enrichment strategies, host range coverage, and infection dynamics. To address these challenges, we have developed a streamlined protocol that integrates liquid culture enrichment, polyethylene glycol (PEG)-mediated concentration, high-throughput spot screening on multiple host bacterial lawns, and subsequent streak purification for clonal isolation. This approach enables simultaneous screening of environmental samples against multiple pathogens in a single experimental setup, improving isolation efficiency. Notably, it enhances the recovery of phages with diverse host ranges. Our integrated workflow can reduce manual effort and material consumption and shorten the isolation period compared with conventional methods. This robust and evidence-based method accelerates the discovery of phages with potential applications in therapy and biocontrol. We anticipate that this protocol will prove particularly useful for building phage biobanks, preparing personalized phage therapeutics, and conducting large-scale environmental phage mining. Ultimately, it will help expand the repertoire of characterized phages for combating antimicrobial-resistant bacterial infections.