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

TL;DR

PRISM is a new droplet-based platform that improves the discovery and study of bacteriophages, including those that traditional methods miss.

Contribution

PRISM introduces a plating-independent, high-throughput method for isolating and characterizing bacteriophages at single-phage resolution.

Findings

PRISM recovers both plaquing and nonplaquing Salmonella phages from environmental samples.

The platform accurately titered poor-plaquing phages like Rhodococcus phage ReqiDocB7.

PRISM determined phage resistance and lysogeny frequency without plating, matching conventional estimates.

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” Salmonella phages from environmental samples, where nonplaquing phages were missed by conventional approaches; (ii) accurate titering of poor-plaquing phages, such as Rhodococcus 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.

A droplet microfluidics platform for studying bacteriophages, a tool for exploring overlooked populations in the virosphere.

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