Abstract

Objectives: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) causes challenging healthcare-associated infections, many of which arise from superficial sites. This study aimed to develop an effective phage cocktail targeting CRPA, and to evaluate its potential as a topical hydrogel to prevent wound infections using an ex-vivo porcine skin model. Methods: Three phages – EKU1 (Yuavirus), MHN1 (Bruynoghevirus) and Bobae (PhiKZvirus) – were isolated from wastewater and characterized. A cocktail of these phages was formulated against a CRPA strain. Phage-resistant isolates that emerged after exposure to the cocktail were analysed for fitness costs. The phage cocktail was incorporated into hydroxyethyl cellulose (HEC) hydrogel and tested against CRPA on ex-vivo porcine skin. The shelf-life of the gel was observed under storage at different temperatures. Results: EKU1, MHN1 and Bobae have distinct evolutionary lineages. The phage cocktail exhibited synergistic activity against CRPA, with over 5-log [colony-forming units (CFU)/mL] reduction after 24 h at a multiplicity of infection of 1000. Among 12 phage-resistant isolates, 11 exhibited slower growth rates than the wild type, and five of 12 isolates produced significantly less biofilm. On porcine skin, the phage cocktail gel reduced the bacterial load significantly by 1.5 log CFU/mL after 12 h compared with the blank gel (control). Phage titres in the gel stored at 4 °C decreased by 1–1.5 log plaque-forming units/mL over 8 weeks. Conclusions: The phage cocktail showed strong synergistic activity against CRPA with resistance-associated trade-offs. When incorporated into HEC hydrogel, the phage cocktail suppressed CRPA growth on porcine skin significantly, and remained stable under refrigeration for months, supporting its potential as a promising topical prototype for wound infections.