Isolation characterization of lytic bacteriophages and In Vitro investigation of their synergistic interaction with various antimicrobial agents against multidrug-resistant Pseudomonas aeruginosa

Multidrug-resistant Pseudomonas aeruginosa is a critical public health concern due to increasing treatment challenges. In this study, two lytic bacteriophages (BAUNPA1 and BAUNPA3) isolated from wastewater in the Balıkesir region were evaluated in vitro in combination with various antibiotics [colistin (COL), ciprofloxacin (CIP), meropenem (MEM), tobramycin (TOB)] and antimicrobial peptides [murepavadin (MUR), pexiganan (PEX)]. The phages were characterized morphologically (TEM) and genetically (RFLP). Biological properties of both phages were assessed, revealing a 30-minute latent period, an optimal MOI value of 10, stability between pH 6–8 and up to 50 °C, and burst sizes of approximately 189 for BAUNPA1 and 43 for BAUNPA3. In host range analysis, BAUNPA1 and BAUNPA3 showed lysis rates of 34% and 60%, respectively. Among fifty P. aeruginosa isolates obtained from clinical samples, COL and MUR were the most effective agents with the lowest MIC₅₀/MIC₉₀ values (0.5/1 µg/mL). Phage–antimicrobial agent combinations were tested on five selected clinical isolates using modified double-layer agar methods (disk diffusion, incorporation of sub-MIC antibiotic/peptide into bottom or soft agar) and checkerboard assays under simultaneous and sequential application models. The disk diffusion method yielded limited increases in plaque size, whereas the addition of sub-MIC antibiotics/peptides to the bottom or soft agar led to significant increases in both plaque number and size. Incorporation into bottom agar was more effective in increasing plaque size, while incorporation into soft agar caused greater increases in plaque number. The most notable increase in plaque size was observed with MEM, while MEM, COL, and CIP contributed to increased plaque numbers. In checkerboard assays, simultaneous application generally resulted in synergistic interactions with CIP, COL, and MEM, whereas in the sequential model (phage first), synergistic effects were also observed with MUR and PEX (FICI ≤ 0.5). This thesis presents a comparative analysis of lytic phage combinations with clinically used antibiotics and antimicrobial peptides across different in vitro models, contributing to the development of novel therapeutic strategies against resistant P. aeruginosa infections.