Refined methodology for quantifying Pseudomonas aeruginosa virulence using Galleria mellonella.

Larvae of Galleria mellonella (the greater wax moth) are being increasingly used as a model to study microbial pathogenesis. In this model, bacterial virulence is typically measured by determining the 50% lethal dose (LD₅₀) of a bacterial strain or mutant. The use of G. mellonella to study Pseudomonas aeruginosa pathogenesis, however, is challenging because of the extreme sensitivity of larvae to this bacterium. For some P. aeruginosa strains, as few as 1-5 colony-forming units are sufficient to kill G. mellonella, which poses challenges for determining LD₅₀ values. For this reason, some groups have used time-to-death as a measure of P. aeruginosa virulence, but methodologies have not been standardized. We provide a detailed protocol for using the time at which 50% of larvae have died (LT₅₀) at a particular inoculum as a measure of P. aeruginosa virulence. We also describe a quality control metric for enhancing the reproducibility of LT₅₀ values. This approach provides an accurate and reproducible methodology for using G. mellonella larvae to measure and compare the virulence of P. aeruginosa strains.IMPORTANCEPseudomonas aeruginosa is a significant cause of morbidity and mortality. The invertebrate Galleria mellonella is used as a model to determine the virulence of P. aeruginosa strains. We provide a protocol and analytical approach for using a time-to-death metric to accurately quantify the virulence of P. aeruginosa strains in G. mellonella larvae. This methodology, which has several advantages over 50% lethal dose approaches, is a useful resource for the study of P. aeruginosa pathogenicity.