Toward routine implementation of phage testing: an inter-laboratory comparison of a disk-based susceptibility testing...

In the context of an emerging post-antibiotic era, phage therapy is gaining increasing attention. Standardized approaches for phage susceptibility testing (PST) are crucial to enable objective and effective phage selection for personalized treatment, yet no standardized reference method currently exists. To overcome this impediment, a standardized disk-based PST protocol was validated in-house (intra-laboratory comparison) involving 28 participants, followed by an inter-laboratory comparison across 12 laboratories in Germany with 39 participants. Preselection testing revealed high concordance between Disk-PST and Spot test, supporting the selection of strains with consistent phage susceptibility profiles for the interlaboratory comparison. An Escherichia coli phage, a Staphylococcus epidermidis phage, and a negative control (SM buffer) were tested on four bacterial strains (two E. coli and two S. epidermidis strains). Testing was blinded and included two technical and two biological replicates per condition. The plaque assay was performed at the coordinating laboratory (University Medical Center Rostock) and served as the reference standard. The in-house validation showed high reliability and strong agreement with the plaque assay, requiring only minor methodological adjustments prior to the inter-laboratory trial. The inter-laboratory comparison confirmed the robustness of the method, showing almost perfect agreement with the plaque assay across all participating laboratories. In the rare event of false-positive or inconclusive results, phage cross-contamination, misinterpretation of microcolonies within lysis zones, and limited information on negative controls due to the blinded test setting were causative. In accordance with its high inter-laboratory reliability, the PST-disk method seems to be highly suitable for easy implementation in routine laboratory settings. Further investigations are necessary to correlate phage test results with clinical therapy outcomes-an issue that should be addressed in future clinical trials.IMPORTANCEThe rapid global spread of multidrug-resistant bacteria has intensified the need for effective alternative therapeutic strategies, including bacteriophage therapy. Because phage activity is highly strain-specific, accurate and standardized phage susceptibility testing (PST) is essential for safe and effective clinical implementation, yet standardized protocols are currently lacking. In this nationwide, blinded inter-laboratory study, we evaluated a routine-diagnostic-adapted single-disk PST method and demonstrated its excellent agreement with the gold-standard double agar layer plaque assay. Importantly, this approach represents a significant improvement over existing PST methods by providing a standardized, reproducible, and easy-to-implement assay with high potential for widespread application in clinical routine microbiology laboratories.