Comparison of agar-based and checkerboard methods to assess phage-antimicrobial synergy in Pseudomonas aeruginosa

Multidrug-resistant (MDR) Pseudomonas aeruginosa requires innovative therapeutic approaches. In this study, two lytic bacteriophages (BAUNPA1, BAUNPA3) were isolated from wastewater in Türkiye, and the combined efficacy of these phages with antibiotics (ciprofloxacin, colistin, meropenem, tobramycin) or antimicrobial peptides (murepavadin, pexiganan) was evaluated in vitro.Phages were characterized morphologically by electron microscopy and genotypically by restriction fragment length polymorphism, and their different identities were confirmed. Biological evaluation showed rapid replication (latent period: 30 min; burst sizes: 189 and 43 PFU/cell), lytic activity against clinical MDR isolates, and stability under physiological conditions (pH 6-8; temperature ≤ 50 °C). Synergy was assessed using modified double-layer agar and checkerboard assays under simultaneous and sequential applications. The checkerboard assay showed that synergy was significantly enhanced under phage-first application. BAUNPA1 showed strong synergy with meropenem (FICI ≤0.5) and colistin, while BAUNPA3 showed synergy with murepavadin and pexiganan. Agar-based methods have shown variable results, as plaque morphology does not always correlate quantitatively synergy. This study provides a comparative in vitro framework for assessing phage-antimicrobial interactions and, for the first time, reports reproducible synergy between intact lytic phages and murepavadin or pexiganan, highlighting the importance of methodological approach and application timing in synergy assessments.