LAB-IN-A-TABLET: PHAGE TABLETS ENABLE HIGH-THROUGHPUT SCREENING FOR RAPID IDENTIFICATION OF PERSONALIZED THERAPEUTIC BACTERIOPHAGES

The spread of antimicrobial resistance (AMR) is now considered one of the major challenges globally, taking more than 700,000 lives per year, with an estimated death toll of 10 million people by 2050. 1 As regulatory approval for personalized phage therapy against antibiotic-resistant infections advances, there is a critical need for rapid target identification of therapeutic phages against multi-drug resistant bacterial pathogens.We designed a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in allinclusive solid tablets.Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst.The presence of sugar polymers provides desiccation-tolerance, shelf-stability, and prevents thermal deactivation of sensitive ATP bioluminescence assay reagents at 37C (the optimal temperature for growth of pathogenic clinical bacterial and thus phage lytic action).Timely administration of phage therapy requires rapid screening of large libraries to identify target bacteriophages.Although widely used in research labs, the spot test method is labor-intensive and slow.In spot test, cleared zones are formed on solid medium as a result of phage lysing bacterial cells and the release of progeny phages 1 .Liquid assays can also be used to monitor bacterial lysis as a result of phage infection by tracking the bacteria culture turbidity (optical density) as a result of phage lysis 2 .The main hurdle of monitoring optical density of a bacteria culture is that bacteria cell debris can contribute to turbidity of the culture and affect the results negatively. 3 Monitoring the metabolic activity of phage-infected bacterial cultures using redox-sensitive dyes can be used to analyze cellular respiration. 3However, these metabolic assays are detecting the phage-mediated bacterial cell lysis indirectly.Here, we demonstrate the utility of a single-pill sugar-based ATP bioluminescent assay for fast screening a phage library in a microtiter plate.The feasibility assay in detecting the phage-mediated bacterial cell lysis was shown using three phages including P32, JG004, and PP7 with high, medium and weak lytic strength.Phages alongside luciferin and luciferase were encased in a sugar polymer matrix containing 10 wt% pullulan and 0.5 M trehalose forming single pills having dried under nitrogen followed by storage under vacuum.The assay successfully detected the lytic activity of phages in as fast as 30 minutes, which typically requires a minimum of 15 hours for the current gold standard culture-based method.Encapsulation of all assay components in pullulan and trehalose matrices improved thermal stability of the enzyme luciferase and improved bioluminescence signal intensity by more than 90% as compared to sugar-free preparations (Figure 2).Desiccation tolerance of phage and assay reagents stored under ambient conditions for four weeks was also significantly improved through the incorporation of sugar polymer matrix (Figure 3).We successfully demonstrated a high-throughput screening identifying target phages for multidrug-resistant clinical isolates of E. coli, P. aeruginosa, S. aureus, and Salmonella against in-house phage libraries using all-inclusive ATP bioluminescence assay (Figure 4).