Using double-layer agar and checkerboard assays to detect phage cocktail synergy with selected antimicrobials in Pseudomonas aeruginosa

The increasing prevalence of multidrug-resistant Pseudomonas aeruginosa highlights the need for alternative therapeutic strategies. This study evaluated the potential of a commercial bacteriophage cocktail combined with antimicrobial agents and compared two practical methods for screening phage-antimicrobial synergy. A lytic bacteriophage isolated from the PYO phage cocktail was characterized by host range, size, pH and thermal stability, and lytic activity against the reference strain PAO1. Synergy was assessed with nine antimicrobial agents, including conventional antibiotics, efflux pump inhibitors, and essential oil compounds, using agar-based plaque assays and broth microdilution checkerboard assays. Plaque diameter and plaque number were evaluated at sub-MIC concentrations, whereas synergistic interactions were determined by the fractional inhibitory concentration index (FICI). Statistical significance was analyzed using one-way ANOVA followed by Dunnett's multiple comparison test, and the association between plaque parameters and FICI values was explored using Spearman's rank correlation. The isolated phage exhibited a burst size of approximately 259 PFU per infected cell and remained stable over a broad pH and temperature range, with significant reductions under highly acidic, highly alkaline, and high-temperature conditions. In agar-based assays, colistin, meropenem, and cinnamaldehyde significantly increased plaque diameter and/or plaque number (p < 0.05-0.001), depending on the bacterial strain tested. Checkerboard analysis identified the same four combinations as synergistic (FICI ≤ 0.5). Spearman's rank correlation indicated a relationship between plaque parameters and FICI values. Overall, both methods identified the same synergistic combinations, suggesting that agar-based plaque assays may serve as a practical preliminary screening tool for phage-antimicrobial combinations, although further validation is required.