Precision of a phage susceptibility spot assay assessed with 154 clinical <i>Staphylococcus aureus</i> isolates

Lytic phages are a promising consideration to combat recalcitrant bacterial infections. To advance the possibility of using phage therapy in the clinical routine, standardized phage susceptibility testing methods are needed. Here, staphylococcal phage K was tested against 154 periprosthetic joint infection-associated <i>Staphylococcus aureus</i> isolates using a double overlay spot assay in technical and biological triplicates. Individual plaques were assessed using a morphology scoring system ranging from 0 (no plaques) to 4 (large, clear plaques), with scores ≥2 considered indicative of infectivity. Overall, 107 isolates exhibited scores ≥2. The intra-day and inter-day coefficients of variation were 0.9 and 2.5, respectively. Plaque-forming unit per milliliter (PFU/mL) titers correlated with morphology scores (Spearman's ρ = 0.8, <i>P</i> < 0.0001). The results suggest high precision of the described spot assay assessing <i>S. aureus</i> susceptibility to phage K.IMPORTANCEStandardized laboratory assays for assessing phage susceptibility are a prerequisite for clinical implementation of phage therapy. No assay has been denoted a reference standard. Criteria that can be used to assess assay precision and suitability for routine testing are intermediate precision and repeatability. Here, staphylococcal phage K was tested using the double overlay spot assay against 154 periprosthetic joint infection-associated <i>Staphylococcus aureus</i> isolates in technical and biological triplicates. In addition to measuring phage titers, plaque-forming units were assessed with a scoring system. Results indicate high intermediate precision and repeatability of the method described.