In the ongoing battle against antibiotic-resistant bacteria, bacteriophages—viruses that specifically infect and kill bacteria—represent a promising alternative to traditional antibiotics. However, discovering these bacterial viruses, particularly rare ones that evade conventional detection methods, has long been a bottleneck in research. A groundbreaking development from Texas A&M University's Center for Phage Technology changes that. The PRISM platform, or Phage Recovery and Investigation in Single-droplet Microenvironments, enables efficient bacterial virus isolation and the discovery of rare bacteriophages that traditional plaque assays miss entirely.

This innovation arrives at a critical time. The World Health Organization reports that antimicrobial resistance claims over 1.27 million lives annually, with projections reaching 10 million by 2050 if unchecked. Phage therapy could counter this, but the phage 'dark matter'—undiscovered viruses making up 99% of the virome—remains inaccessible due to outdated isolation techniques developed over a century ago. PRISM addresses this by leveraging droplet microfluidics for high-throughput, plating-independent screening.

The Limitations of Traditional Plaque Assays

Conventional phage isolation relies on plaque assays, where phages are mixed with host bacteria in soft agar. Successful infections produce clear zones called plaques after overnight incubation. This method, pioneered by d'Hérelle and Twort in the 1910s, favors 'plaquing' phages—those forming visible lysis zones. Non-plaquing phages, which replicate slowly, adsorb poorly, or cause subtle lysis, go undetected.

Studies show plaque assays recover only a fraction of environmental phages. For Salmonella, traditional methods isolated just six unique phages from wastewater, missing diversity in underrepresented genera like Chivirus. Factors like agar diffusion barriers, spatial separation, and host debris inactivation limit efficiency. Time-consuming subculturing for clonality adds days, hindering rapid discovery.

These shortcomings bias collections toward common, fast-lytic phages, overlooking rare ones crucial for broad-spectrum cocktails against multidrug-resistant pathogens like Salmonella enterica or Escherichia coli.

🧪 Introducing PRISM: A Microfluidics Revolution

Developed by Han Zhang, Justin Boeckman, and colleagues at Texas A&M, PRISM encapsulates single phages with 10-20 host bacteria in picoliter-scale water-in-oil droplets (~65 pL, 50 μm diameter). This confined environment boosts encounter rates via Brownian motion, bypassing agar limitations. Droplets are generated at 100 per second, incubated for 24 hours, and screened for fluorescence indicating lysis (low intensity from growth-inhibited droplets).

Active droplets are sorted using dielectrophoresis at over 100 Hz, dispensed for validation, and sequenced. This plating-independent approach recovers both plaquing and non-plaquing phages, titering poor-plaquers accurately via Poisson statistics: titer = -ln(1 - infection rate) × dilution × droplets/mL.

Step-by-Step: How Bacterial Virus Isolation Works with PRISM

The process unfolds in precise stages:

• Sample Preparation: Environmental samples (e.g., wastewater) concentrated via PEG precipitation, mixed with fluorescently labeled host bacteria like RFP-Salmonella.
• Droplet Generation: Flow-focusing microfluidics creates uniform droplets with Poisson-distributed phages (target λ=0.1 for single occupancy).
• Incubation: 37°C for 16-24 hours; phages infect, lyse hosts, reducing fluorescence.
• Screening and Sorting: Laser excitation detects low-fluorescence 'hit' droplets; dielectrophoretic deflection sorts them.
• Recovery and Validation: Hits plated on agar for plaques/spot tests; lysates sequenced for taxonomy (e.g., via taxMyPhage).
• Characterization: High-MOI assays quantify resistance (surviving droplets) or lysogeny (dual-fluorescence tracking).

From sample to pure phage in under 24 hours—3x faster than traditional methods.

Breakthrough Results: Unlocking Rare Phage Diversity

In tests on Salmonella wastewater, PRISM isolated 15 unique phages versus six by plaque assays. Eleven were PRISM-exclusive, including non-plaquing ones like PI10, PI13 (Chivirus), PI17—species with half the database representation of plaquers. Hit rates: 0.084-0.11%, reproducible across runs.

Taxonomic breadth expanded: seven genera (Chivirus, Kuttervirus, Nonanavirus) versus five. For Rhodococcus phage ReqiDocB7 (poor-plaquer), PRISM titers matched qPCR, outperforming dilutions (10-fold error).The full study details these findings, highlighting PRISM's resolution.

Precision in Titering and Resistance Profiling

PRISM excels at titering: T7 on E. coli showed infection rates aligning with Poisson (12.5% at λ=0.1). Resistance frequencies matched conventions—e.g., 1 in 4.4×10⁵ for T7. Lysogeny for lambda phage: ~1 in 21-23, plating-independent.

This enables rational cocktail design: quantify rare escape mutants, prefer lytic over temperate phages for therapy.

Texas A&M's Center for Phage Technology: Driving Innovation

Housed at Texas A&M University, the Center pioneers phage research since 2011. Funded partly by DARPA, this work by lead authors from the center positions the university as a hub for microfluidics-phage integration. Collaborators like P. de Figueiredo and A. Hargreaves supervise, fostering student training in advanced biotech.Learn more about their programs.

This publication in Science Advances underscores higher education's role in addressing global health crises through cutting-edge tools.

Implications for Phage Therapy Against Superbugs

With 70% of bacteria antibiotic-resistant, phages offer precision antimicrobials. PRISM unlocks rare phages for cocktails targeting biofilms or CRISPR-armed hosts. Clinical trials (e.g., IPATH on P. aeruginosa) succeed with diverse phages; PRISM accelerates sourcing.

• Broader Host Range: Non-plaquers expand therapy options.
• Rapid Cocktails: High-throughput for MDR outbreaks.
• Cost Savings: Reduced labor/materials.

Regulatory nods (FDA compassionate use) pave the way; PRISM supports scalable production.

Challenges and Solutions in Scaling PRISM

Challenges include microfluidics expertise and initial setup costs. Solutions: Open-source designs, LED-based detectors for labs. Adaptable to slow-growers or anaerobes via media tweaks. Ethical: Phage specificity minimizes dysbiosis risks versus broad antibiotics.

Photo by National Institute of Allergy and Infectious Diseases on Unsplash

Future Outlook: Illuminating the Phage Virome

PRISM could screen millions of droplets daily, mapping viromes. Integrations: AI for sorting, multi-host arrays. For academia, it opens postdoc projects in phage genomics. As antibiotic pipelines dry, this platform heralds a phage renaissance, led by universities like Texas A&M.

Researchers worldwide can now access the overlooked 99% of bacterial viruses, transforming microbial ecology and medicine.