In vivo functional Screening on Innate Immunity of Lactic Acid Bacteria in Galleria mellonella preclinical model: Comparative Analysis of Lactiplantibacillus plantarum and Lentilactobacillus kefiri

Antonio Guarnieri¹Marilina Falcone¹Natasha Brancazio¹Farwa Mukhtar¹Addis Temie Worku¹Marco Alfio Cutuli¹Vincenzo Pio Iacovino²Sonia Ganassi²Antonio De Cristofaro²Roberto Maria Antonio Di Marco³Giulio Petronio Petronio^1*

• ¹Department of Medicine and Health Sciences Vincenzo Tiberio, University of Molise, Campobasso, Italy
• ²Universita degli Studi del Molise Dipartimento Agricoltura Ambiente e Alimenti, Campobasso, Italy
• ³Universita degli Studi di Catania Dipartimento di Scienze del Farmaco e della Salute, Catania, Italy

Introduction: Among probiotics, Lactic Acid Bacteria modulate host immunity via strain-specific molecular patterns. The invertebrate model Galleria mellonella offers conserved innate immune pathways and is increasingly applied for preclinical screening of probiotic functions. Methods: We evaluated the immunomodulatory activity of Lactiplantibacillus plantarum ATCC 14917 and Lentilactobacillus kefiri DSM 10551 in G. mellonella. Larvae were injected with 10⁶ CFU/larva of each strain, and survival and health indices were monitored for 72 h. The temporal transcriptional response of ten innate immunity-related genes, including Toll and IMD signalling, Toll receptor, cytokine-like ligand, and antimicrobial effectors, was assessed via qRT-PCR over 3–24 hours, complemented by correlation and hierarchical clustering to identify co-expression modules and strain-specific transcriptional patterns. Results: Both strains were non-toxic and induced strain-dependent gene expression patterns. L. plantarum induced a stronger and more sustained activation of immune signalling pathways and effector responses, whereas L. kefiri was characterised by an earlier and prolonged activation of stress-related and tissue-protective mechanisms. Correlation and clustering analyses revealed distinct co-expression modules that reflect modulation of the Toll and IMD pathways. Discussion: These findings suggest that G. mellonella could serve as a cost-effective in vivo model for functional screening of Lactobacillus spp. with immunomodulatory potential and possible translational relevance to human innate immunity.