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

Area of Science:

• Microbiology
• Virology
• Biotechnology

Background:

• Conventional bacteriophage discovery relies on plaque assays, limiting detection to phages forming visible plaques.
• This method overlooks diverse viral populations, including non-plaquing or slow-replicating bacteriophages.
• There is a need for advanced techniques to comprehensively explore bacteriophage diversity.

Purpose of the Study:

• To introduce PRISM (Phage Recovery and Investigation in Single-droplet Microenvironments), a novel droplet microfluidics platform.
• To enable high-throughput, plating-independent isolation, quantification, and characterization of bacteriophages.
• To overcome limitations of conventional methods in bacteriophage discovery and analysis.

Main Methods:

• PRISM utilizes droplet microfluidics to encapsulate phage-host interactions in single-droplet microenvironments.
• The platform employs fluorescence-based detection and sorting for high-throughput analysis.
• Phage-host interactions are analyzed at single-phage resolution within water-in-oil microdroplets.

Main Results:

• PRISM successfully recovered both plaquing and non-plaquing Salmonella phages from environmental samples, including those missed by plaque assays.
• The platform enabled accurate titering of poor-plaquing phages, such as Rhodococcus phage ReqiDocB7.
• Plating-independent determination of phage resistance and lysogeny frequency closely matched conventional estimates.

Conclusions:

• PRISM offers enhanced resolution and accuracy for bacteriophage analysis.
• The platform can detect non-plaquing or slow-replicating phages, expanding access to overlooked viral populations.
• PRISM represents a powerful tool for comprehensive exploration of bacteriophage diversity.