BACTERIOPHAGE-ANTIBIOTIC COMBINATION THERAPY AGAINST PSEUDOMONAS AERUGINOSA IMPLANT-RELATED INFECTIONS IN GALLERIA MELLONELLA

Introduction & Aim Implant-related infections are increasing due to population aging and the growing use of medical implants. Although Gram-positive bacteria are the most common causative pathogen, Pseudomonas aeruginosa is of particular interest because it is associated with acute, polymicrobial, and difficult-to-treat infections. Its multidrug resistance, virulence, and ability to form biofilms highlight the need for new antimicrobial strategies. Bacteriophage (‘phage’) therapy represents a promising approach because of its specificity for target bacterial strains or species, with limited effects on host cells and the microbiota. Bacteriophages bind to bacterial receptors, inject their genetic material, replicate intracellularly, and are released through bacterial lysis. Therefore, bacteriophages, alone or in combination with antibiotics, may inhibit bacterial growth and biofilm formation. In this study, we aimed to investigate the antimicrobial efficacy of bacteriophages, alone and in combination with antibiotics, against P. aeruginosa implant-related infections using in vitro approaches and an in vivo Galleria mellonella model. Methods Bacteriophages were screened against P. aeruginosa strains using the double-layer agar method. The antimicrobial activity of antibiotics alone and in combination with bacteriophages was assessed using checkerboard assays, including minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) analyses. In addition, in vivo evaluation of bacteriophages in combination with antibiotics in G. mellonella larvae against planktonic, implant-associated hematogenous, and early-stage biofilm infections was assessed. Results Initial double-layer agar screening showed strain-dependent susceptibility of P. aeruginosa to the tested phages, indicating differences in phage host range. In checkerboard assays, selected phage-antibiotic combinations showed enhanced antimicrobial activity compared with single treatments, with ciprofloxacin emerging as a promising combination partner. In the G. mellonella planktonic infection model, phage treatment delayed larval death, while combination treatment with ciprofloxacin further improved larval survival. Conclusions These findings support further investigation of phage therapy as an adjuvant strategy against P. aeruginosa implant-related infections, particularly in combination with ciprofloxacin. The G. mellonella model provides a cost-effective, rapid, and practical system for evaluating therapeutic efficacy and optimizing phage-antibiotic combinations. Acknowledgements This project has received funding from the European Union's Horizon Europe research and innovation programme under the Marie Skłodowska-Curie Actions Doctoral Networks, grant agreement No. 101226717 — SHIELD.