Abstract
Campylobacter jejuni and Campylobacter coli are the main agents of campylobacteriosis, a globally prevalent foodborne illness predominantly linked to the consumption of contaminated poultry products. The increasing antimicrobial resistance in Campylobacter requires innovative control strategies throughout the poultry production chain. Bacteriophages, highly specific bacterial viruses, represent a promising biocontrol approach capable of selectively targeting Campylobacter without disrupting the natural microbiota. However, early-stage validation in intermediate models, such as Galleria mellonella, is essential to ensure safety and efficacy before application in poultry, as has been established for other zoonotic pathogens. This study evaluated the in vitro and in vivo efficacy of a novel four-phage cocktail targeting Campylobacter. In vitro assays showed that the phage cocktail successfully lysed all 13 strains tested, and each individual phage displayed a broad lytic spectrum, with most strains being susceptible to multiple phages. In vivo virulence screening in G. mellonella revealed marked strain-dependent virulence, with only five of 13 strains reducing larval survival below 50%. Phage efficacy in vivo was optimized using C. jejuni CJE065, the most virulent strain in the model. The phage cocktail applied at MOI 10 increased the G. mellonella survival from 25.5% in untreated controls to 57.5% (p < 0.001), whereas lower MOIs provided only transient protection. Phage-antibiotic therapy combining phage cocktail and either erythromycin or ciprofloxacin further enhanced larval survival rates, reaching up to 88.8 and 83.8%, respectively (p < 0.001). Overall, these findings support the potential use of this phage cocktail as an early-stage intervention against Campylobacter and highlight G. mellonella as a suitable intermediate model for optimizing phage-based treatments while reducing the need for vertebrate models.