Virulence assesment of Enterococcus strains using Galleria mellonella as host model

MARTINO, G; OSORIO, J; ESPARIZ, M; MAGNI, C; BLANCATO, VS

San Miguel de Tucuman

Simposio; IV Simposio Internacional de Bacterias Lácticas (SIBAL); 2013

CERELA

Enterococi are present as non-starter lactic acid bacteria in a wide variety of cheeses, mostly produced from raw milk. It has been shown that these microorganisms have a positive influence during the ripening process of cheese since they contribute to the development of organoleptic properties; though, their presence in food remains controversial. These microorganisms can be potentially hazardous to human health due to their pathogenicity or their ability to produce toxic compounds like biogenic amines. Using insects as model organisms to study host-microbe interactions provides numerous advantages (e.g. ethical status, low rearing costs and convenient experiments). Recently, the greater wax moth, Galleria mellonella, has emerged as a reliable model host to study the pathogenesis of numerous human pathogens. A correlation between the capacity of a pathogen to kill G. mellonella and virulence in mammalian models has been established. Indeed, the innate immune systems of Galleria larvae and mammals share a high degree of structural and functional homology. In this work we present the selection of optimal growth conditions for G. mellonella in our laboratory and the optimization of the inoculation technique in order to use the insect model to evaluate virulence of Enterococcus strains. As a first step, several growth mediums were analyzed. The best growth of G. mellonella larvae was obtained with a medium composed of bee´s wax and pollen. Once the colony was established, the inoculation protocol was optimized by varying inoculum volume and CFU per larvae. In initial dose?response experiments, different inocula of E. faecalis JH2-2 were injected in G. mellonella and it was observed that increasing the cell number inoculated (6,85x106, 1,35x107, 3,9x107 and 9,75x107 CFU/larva) resulted in progressively decreasing survival of the infected larvae. Survival curves were constructed by the Kaplan-Meier method and compared by the Log-rank test. Injection with Lactococcus lactis IL1403 strain resulted harmless to G. mellonella since no larvae were killed post-injection, indicating that this bacterium is an appropriate non-pathogenic control for the experiment. Afterwards, we compared the survival of G. mellonella injected with different strains of enterococci. We found a reproducible dose?response for each strain, and that at lower CFU/larva (approx. 7x106) E. faecalis JH2-2 resulted more virulent than the E. faecium strains assayed. But at higher CFU/larva (approx. 3x107) all of the strains were equally lethal for G. mellonella. We also found that differential survival could be observed when analyzing wildtype and mutant strains of E. faecalis JH2-2. These results indicate that the G. mellonella model could be employed to evaluate the virulence of different strains and could be an important tool to confirm the safe use grade of a particular lactic acid bacterium.