<b>Prophylactic Oral </b><b><i>Limosilactobacillus fermentum </i></b><b>DSM 34872 Administration Protects </b><b><i>Galleria mellonella </i></b><b>from Gut-Associated Pathogens</b>

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