Work

Rapid phage susceptibility determination using a semi-automated bioluminescent ATP assay

Year: 2026

Type: article

Abstract: ABSTRACT Antimicrobial resistance is a major threat to human health. Phage therapy is a promising alternative to antibiotics, yet routine diagnostic tools capable of rapidly determining phage susceptibility are lacking. Our goal was to develop a diagnostic tool providing easy-to-interpret in vitro phage activity results within a clinically relevant time frame. We developed a prototype based on bioluminescence emitted by adenosine triphosphate (ATP) release during phage-mediated bacterial lysis. Ten phages were tested against 20 clinical isolates of four species: Pseudomonas aeruginosa , Staphylococcus aureus , Escherichia coli , and Klebsiella pneumoniae . Overall concordance with double agar overlay (DAO) was fair (72%). The prototype showed high sensitivity (94.5%) at detecting weak killers and high specificity (87%) at identifying strong killer phages. Most discordances involved DAO weak killers classified as active by the prototype. Most of these discordant phage-bacteria couples produced a spot in the spot test, suggesting the assay may detect but not distinguish killing without replication from killing with replication. The method showed high repeatability and reproducibility. A few aberrant species specificity results highlighted the need for routine controls. Results from prospective urine samples were obtained within an average of 5 h. IMPORTANCE The lack of reproducible user-friendly phage susceptibility testing that can timely inform clinical decisions remains a major barrier to the routine clinical use of phages. We developed a high-throughput, semi-automated prototype that delivers easy-to-interpret phage susceptibility results for four bacterial species within 5 h in a “real-life” clinical flow. However, the correlation between in vitro lytic activity and clinical outcomes still needs to be established to meet regulatory requirements for in vitro diagnostic (IVD) medical devices.

Source: Microbiology Spectrum

DOI: 10.1128/spectrum.00782-26