PRISM: A unified platform for phage isolation and characterization from single-droplet microenvironments.

Abstract

The exploration of bacteriophage diversity remains constrained by reliance on conventional plaque assays, which bias discovery toward phages that form visible plaques. We present PRISM (Phage Recovery and Investigation in Single-droplet Microenvironments), a droplet microfluidics-based, plating-independent platform for high-throughput isolation, quantification, and characterization of bacteriophages. By encapsulating phage-host interactions in water-in-oil microdroplets at single-phage resolution and coupling them with fluorescence-based detection and sorting, we demonstrate PRISM's capabilities across several key applications: (i) recovery of both "plaquing" and "nonplaquing" <i>Salmonella</i> phages from environmental samples, where nonplaquing phages were missed by conventional approaches; (ii) accurate titering of poor-plaquing phages, such as <i>Rhodococcus</i> phage ReqiDocB7; and (iii) plating-independent determination of phage resistance and lysogeny frequency, with results closely matching conventional estimates. This highlights PRISM's enhanced resolution and accuracy, as well as its ability to detect nonplaquing or slow-replicating phages, underscoring the platform's power to access viral populations overlooked using conventional methods.