• antibiotic alternatives
• antimicrobial resistance
• bacteriophages
• honey bees
• Paenibacillus larvae

American Foulbrood (AFB), a highly contagious and lethal disease caused by the Gram-positive bacterium Paenibacillus larvae, represents a major threat to global honey bee health and agricultural productivity. Its worldwide prevalence and the bacterium’s ability to produce highly resistant spores, which remain viable for decades, significantly contribute to its environmental persistence and the difficulty in managing this infection in apiaries. Both the Animal Health Law (including its implementing and delegated acts) and the WOAH Terrestrial Animal Health Code list AFB as a disease of concern for international trade, thereby requiring stringent surveillance and control strategies. In Italy, upon suspicion of disease presence in apiaries, mandatory reporting to Public Veterinary Services is required. Following clinical and laboratory confirmation, a 3 km protection zone is established, prohibiting the movement of hives and other materials. Disinfection measures for hives and equipment are also enforced. Unlike the older Veterinary Police Regulation (RPV), the new Animal Health Law adopts a more flexible, risk-based approach, delegating disease management decisions to the Public Veterinary Services and allowing for diverse control strategies. While the destruction of affected colonies remains an available option, it is no longer mandatory for all confirmed outbreaks. Conversely, the use of antibiotics remains prohibited, as no authorized treatments are registered for honey bees in the European Union. This precautionary decision is further supported by studies demonstrating the presence of residues in hive products for up to seven months post-treatment and the detection of resistance genes in both treated and untreated colonies, and even in flowers within bee flight range. Conventional antibiotic treatments in beekeeping offer only palliative care. They fail to eradicate the infection, negatively affect honeybee gut health and potentially predisposing bees to other fungal infections like nosemiasis, and exacerbate the critical issue of antimicrobial resistance (AMR). Recognized as one of the most severe contemporary threats, AMR constitutes a formidable One Health - One Welfare burden, given its profound impacts on economics, human, animal, and environmental health, as well as food security. This challenge demands a collaborative, multisectoral strategy to curb antibiotic misuse and to identify sustainable alternatives, including for the beekeeping sector. This study explores bacteriophage therapy as a targeted, efficacious, and environmentally valuable solution for AFB. We report the successful isolation of three novel bacteriophages (rough, smooth, and mosaic variants) from homogenates of honeybee abdomens exhibiting symptoms compatible with AFB. In vitro assessment demonstrated their potent lytic activity, measured by a significant increase in Plaque Forming Units (PFU) and a corresponding decrease in Colony Forming Units (CFU) within bacterial cultures within 120 minutes for all phage variants. These bacteriophages displayed a broad host spectrum, effectively lysing 20 of 119 Paenibacillus larvae field strains from diverse international origins (Italy, Argentina, USA, Sweden). Importantly, they exhibited long-term stability at 4°C for eight months and no observed toxicity to honeybee larvae, even at high concentrations, highlighting their safety profile. Morphological analysis by Transmission Electron Microscopy confirmed their classification into the Myoviridae family, and genomic sequencing enabled the differentiation of mosaic and smooth phage variants. The promising in vitro results suggest their utility in creating phage cocktails to expand host range and make them valuable for future in vivo application. Further studies are needed to fully evaluate the prophylactic and therapeutic potential of these bacteriophages in apiaries. This research contributes to a future shift in AFB management, promoting more sustainable practices and mitigating AMR, which is vital for both pollinator welfare and global health.