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

Simple Summary: In recent years, the use of alternative animal models to vertebrates for the study of infectious processes and antimicrobial drug development has become a major challenge in experimentation. Insects, in particular Galleria mellonella, may represent a good model for preclinical studies, as their response to infections allows for the preliminary selection of molecules with biological activity in a potential sepsis event. However, discordant data are often reported in the literature, and this is often due to the different methods implemented in many laboratories. The aim of this work was therefore to develop a standard protocol for infection with a Gram-positive bacterium, as we consider it important to apply these unified methodologies in order to obtain reproducible data. Our results made it possible to define a correct growth curve of Micrococcus luteus and, in parallel, an infection methodology that would provide consistent and repeatable data. We are therefore confident that this work can be a support for preclinical studies on model insects, as a link between the development of new drugs and its availability for patients. The aim of this work was to develop an experimental protocol for the infection of Galleria mellonella 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. G. mellonella 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) Micrococcus luteus. After analyzing the morphology and defining the growth rate of M. luteus, doses of between 10(1) and 10(6) 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 10(3) and 10(5) CFU/larvae was assessed by hemolymph bacterial load analysis. The results made it possible to define the growth curve of M. luteus correlated with the CFU count; based on the LD50 (10(3.8) CFU/larvae) calculated on the survival of G. mellonella, infections were carried out to evaluate the immune efficiency of the larvae in bacterial clearance. This protocol, standardized on G. mellonella larvae, could provide a functional tool to study the course of bacterial infections.