P14 – Galleria mellonella- a high throughput inexpensive model to screen for the presence of lethality factors in the stool of animals and humans

Author(s):
Alexander Zaborin, Jennifer DeFazio, Olga Zaborina, John Alverdy, University of Chicago

Background: There is an unmet need to identify the factors present in the human gut that drive lethal gut- derived sepsis. Here we propose to use Galleria mellonella larvae as an inexpensive, high throughput screening platform to determine whether lethality factors are present in stool samples of critically ill humans. In G. mellonella larvae, there is a hemocoelic compartment filled with hemolymph that is functionally comparable to the peritoneal fluid of mammals. In the hemocoel, hemocytes and humoral immune factors are located with granulocytes being are the most abundant phagocytic cells analogous to neutrophils. Humoral factors include signaling cascades initiated by hemocytes that lead to melanization. Melanization precedes death when the hemocoelic cavity is exposed to pathogens or their secreted toxins.

Hypothesis: We hypothesized that G. mellonella is a reliable intraperitoneal infection model that can discriminate between stool filtrates from healthy vs septic host.

Methods: Four sources of samples were screened in Galleria mellonella via direct intrahemocoelic injection: filtered stool contents from healthy volunteers; filtered stool samples from septic critically ill human harboring well characterized human pathogen communities (HPCs); filtered cecal contents of septic- appearing and non- septic appearing mice intestinally inoculated with the characterized HPC; and filtered contents from the above HPC grown under low and high phosphate conditions, a critical factor for microbial virulence expression. Larvae were observed for melanization and death over 24 hours.

Results: Filtered stool samples from 5 healthy volunteers caused no mortality, whereas filtered stool samples from 4 patients dying of severe sepsis caused >50% melanization and subsequent death in larvae (n=20, p<0.001).
Filtered cecal contents of 5 septic mice but not cecal contents of 5 healthy appearing mice intestinally inoculated with the HPC resulted in 40% melanization and subsequent death in larvae (n=20, p<0.01).

Filtered contents from the HPC grown in low phosphate media caused 45% melanization and mortality in larvae (n=20, p<0.01) whereas there was no mortality when the HPC was grown in high phosphate media.

Conclusions: The G. mellonella model can narrow down the search for gut factors that drive inflammation and lethality in a stringent and high throughput manner.