Prophylactic oral Limosilactobacillus fermentum DSM 34872 protects Galleria mellonella from gut-associated pathogens

Background: Lactic acid bacteria are increasingly being studied for their immunomodulatory activity and protective effects against pathogens. Limosilactobacillus fermentum DSM 34872, isolated from human breast milk, was evaluated for its prophylactic potential using Galleria mellonella as an alternative preclinical model. Results: The antimicrobial activity of L. fermentum against Salmonella enterica subsp. enterica serovar Typhimurium or Enterococcus faecalis was first assessed in vitro by the overlay assay. In vivo, G. mellonella larvae were orally administered with L. fermentum via gavage and exposed to pathogens. 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 h after infection. Conclusion: L. fermentum showed antibacterial activity in vitro. In vivo, pre-treatment 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, suggesting controlled immune activation rather than hyperinflammation in L. fermentum pretreated groups. Prophylactic oral administration of L. fermentum protected against lethal bacterial infections in G. mellonella, altering both local and systemic immune responses. These results highlight the potential of L. fermentum as a promising candidate for future in vivo analyses and confirm G. mellonella as an ethical, economical, and reliable preclinical model for preliminary screening, with the aim of subsequent evaluations in accredited clinical models.