Refined methodology for quantifying <i>Pseudomonas aeruginosa</i> virulence using <i>Galleria mellonella</i>

Larvae of <i>Galleria mellonella</i> (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 <i>G. mellonella</i> to study <i>Pseudomonas aeruginosa</i> pathogenesis, however, is challenging because of the extreme sensitivity of larvae to this bacterium. For some <i>P. aeruginosa</i> strains, as few as 1-5 colony-forming units are sufficient to kill <i>G. mellonella,</i> which poses challenges for determining LD₅₀ values. For this reason, some groups have used time-to-death as a measure of <i>P. aeruginosa</i> 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 <i>P. aeruginosa</i> 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 <i>G. mellonella</i> larvae to measure and compare the virulence of <i>P. aeruginosa</i> strains.IMPORTANCE<i>Pseudomonas aeruginosa</i> is a significant cause of morbidity and mortality. The invertebrate <i>Galleria mellonella</i> is used as a model to determine the virulence of <i>P. aeruginosa</i> strains. We provide a protocol and analytical approach for using a time-to-death metric to accurately quantify the virulence of <i>P. aeruginosa</i> strains in <i>G. mellonella</i> larvae. This methodology, which has several advantages over 50% lethal dose approaches, is a useful resource for the study of <i>P. aeruginosa</i> pathogenicity.