Refined methodology for quantifying Pseudomonas aeruginosa virulence using Galleria mellonella
Christopher M R Axline1,2, Travis J Kochan1, Sophie Nozick1
1Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Abstract:
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.
Insights
This study introduces a standardized time-to-death (LT50) protocol for measuring Pseudomonas aeruginosa virulence in Galleria mellonella larvae. This method offers a reproducible alternative to the challenging 50% lethal dose (LD50) approach for studying bacterial pathogenesis.
Area of Science:
- Microbiology
- Infectious Diseases
- Pathogenesis Research
Background:
- Galleria mellonella larvae are a common model for microbial pathogenesis.
- Measuring Pseudomonas aeruginosa virulence using 50% lethal dose (LD50) is difficult due to extreme larval sensitivity.
- Existing time-to-death methods for P. aeruginosa virulence lack standardization.
Purpose of the Study:
- To provide a detailed protocol for using time-to-50%-death (LT50) to measure P. aeruginosa virulence in G. mellonella.
- To introduce a quality control metric for improving LT50 reproducibility.
- To establish an accurate and reproducible method for comparing P. aeruginosa strain virulence.
Main Methods:
- Larval infection with specific P. aeruginosa inoculum.
- Monitoring and recording larval mortality over time.
- Calculating the time at which 50% of larvae succumb (LT50).
- Implementing a quality control metric for data validation.
Main Results:
- A detailed protocol for LT50 measurement was established.
- A quality control metric was described to enhance reproducibility.
- The LT50 approach provides accurate and reproducible virulence quantification.
- This method overcomes challenges associated with LD50 determination for P. aeruginosa.
Conclusions:
- The standardized LT50 protocol offers a reliable method for assessing P. aeruginosa virulence in G. mellonella.
- This approach is advantageous over LD50 for studying P. aeruginosa pathogenicity.
- The methodology serves as a valuable resource for P. aeruginosa virulence research.
