Prophylactic Oral Limosilactobacillus fermentum DSM 34872 Administration Protects Galleria mellonella from Gut-Associated Pathogens

Lactic acid bacteria are increasingly being studied for their immunomodulatory activity and protective effects against pathogens. Limosilactobacillus fermentum DSM 34872, a strain isolated from human breast milk, was evaluated for its prophylactic potential in the alternative preclinical in vivo model of Galleria mellonella. The antimicrobial activity of L. fermentum was first assessed using the overlay assay. Subsequently, larvae were orally administered L. fermentum via gavage and exposed to Salmonella typhimurium or Enterococcus faecalis. Survival rates, melanization score, and expression of immune-related genes (dorsal, relish, gallerimycin, gloverin) were evaluated in both the gut and hemolymph using RT-qPCR at 4 and 24 hours after treatment. L. fermentum showed antibacterial activity in vitro. In vivo, pretreatment with L. fermentum significantly increased survival and delayed mortality. The melanization score supported the in vivo survival results. Gene expression analysis revealed modulation of key immune markers in both the gut and hemolymph, with reduced antimicrobial peptide expression in L. fermentum pretreated groups. Prophylactic oral administration of L. fermentum protected against lethal bacterial infections in G. mellonella, enhancing both local and systemic immune responses. These results highlight the potential of L. fermentum as a valid candidate for future in vivo analyses and confirm G. mellonella as an ethical, economical, and reliable preclinical model for initial screening, with the aim of subsequent evaluations in accredited clinical models.