Optimization of Experimental Infection of the Animal Model Galleria mellonella Linnaeus 1758 (Lepidoptera: Pyralidae) with the Gram-Positive Bacterium Micrococcus luteus

The aim of this work was to develop an experimental protocol for the infection of <i>Galleria mellonella</i> with Gram-positive bacteria. Some physiological characteristics of these insects are comparable to those of vertebrates, therefore allowing the replacement of mammals in the preclinical phases of drug development. <i>G. mellonella</i> Linnaeus 1758 (Lepidoptera: Pyralidae) is accepted as an alternative model for the study of infectious diseases. Since data on infection procedures with different bacterial strains are scarce and sometimes conflicting, also due to different and non-uniform protocols, we developed an experimental protocol that would allow for controlled and repeatable infections, using the Gram-positive bacterium GRAS (Generally Regarded As Safe) <i>Micrococcus luteus</i>. After analyzing the morphology and defining the growth rate of <i>M. luteus</i>, doses of between 10¹ and 10⁶ CFU/larvae were administered to late-stage larvae. The survival rate of the larvae was monitored up to 7 days and the LD₅₀ determined. The bacterial clearance capacity of the larvae after injection with 10³ and 10⁵ CFU/larvae was assessed by hemolymph bacterial load analysis. The results made it possible to define the growth curve of <i>M. luteus</i> correlated with the CFU count; based on the LD₅₀ (10^3.8 CFU/larvae) calculated on the survival of <i>G. mellonella</i>, infections were carried out to evaluate the immune efficiency of the larvae in bacterial clearance. This protocol, standardized on <i>G. mellonella</i> larvae, could provide a functional tool to study the course of bacterial infections.