Galleria mellonella as an Antimicrobial Screening Model
October 11th, 2024
This study presents a standardized framework for optimizing G. mellonella infection models for use in preclinical antimicrobial assessment. The application of a G. mellonella model as part of a preclinical antimicrobial development pipeline could decrease the number of ineffective compounds progressing to clinical trials.
Our lab seeks to understand how bacteria sense and respond to the environment in human airways. We characterize airway environments and use the information gained in the design of novel models for studying bacterial physiology during airway infection, and in the identification of novel antimicrobial agents. CFTR modulator therapies have greatly improved lung function and quality of life for individuals with cystic fibrosis.
This improvement may cause changes in bacterial expression and factors involved in both virulence and antimicrobial resistance, which may pose challenges in adapting current preclinical infection models. The development of robust cell culture models has been accelerated through the use of air liquid interface, epithelial cell cultures, which were available in both cystic fibrosis and non-CF variants. At the same time, the use of high throughput approaches like transcriptomics to understand bacterial physiology in infection relevant conditions has helped us understand whether models recapitulate infection scenarios.
Developing new antimicrobial agents for treating chronic respiratory infections is challenging. Most preclinical drug screening processes do not fully consider the unique challenges in treating infections in the complex and difficult to access environment of the chronically infected lung. We are working with a network of collaborators to try and standardize a preclinical framework for the development of novel antimicrobial agents for the treatment of chronic respiratory infection.
We aim to use the models generated to accelerate drug discovery and to better understand microbial pathogenesis in an airway environment. Begin by weighing the galleria mellonella larvae. To sterilize the surface of the galleria mellonella larvae in batches of 10 to 20 larvae, place each batch individually in the dish and spray the larvae twice with 70%ethanol.
Using tweezers, roll the larvae to ensure full coverage with ethanol. Then, remove the larvae from the ethanol, place them in a sterile petri dish and leave the dish uncovered to allow them to dry. After two hours, about 90%of the larvae regain activity, separate them into groups based on power calculations detailed in this table.
Begin by preparing the pseudomonas aerogenosa infection stock of known colony forming units per milliliter prior to infection. To pellet the infection stock, centrifuge at 10, 000 G for five minutes at room temperature. Resuspend the pellet in PBS.
Next, prepare three one milliliter microfuged tubes containing either sterile distilled water, ethanol or sterile PBS. Inspect a sterile 100 microliter Hamilton syringe ensuring that the needle tip is sharp and has not developed a hook which could cause significant trauma to the infection site. Now wash the syringe sequentially with distilled water, ethanol and PBS prior to infections, drawing up and discarding the maximum capacity.
Then vortex the resuspended infection inoculum and draw up a maximum of 100 microliters into the Hamilton syringe, sufficient to inject 10 microliters per larvae. Afterward, inject each galleria mellonella larvae with 10 microliters of inoculum in the rear right pro leg. Incubate the infected larvae between 20 degrees Celsius and 42 degrees Celsius, which is an appropriate range for galleria mellonella survival.
Prepare a broad range of doses exceeding the expected therapeutic range of the antimicrobial agent guided by invitro toxicity data. Inject healthy sterile galleria mellonella in appropriate group sizes with the different antimicrobial and PBS vehicle concentrations. To optimize the treatment of bacterial infection in galleria mellonella, prepare a suitable range of antimicrobial agent doses based on preliminary toxicity testing.
Infect groups of larvae with bacteria as previously described. At two hours post-infection, treat the larvae with the prepared antimicrobial agent or vehicle control solution. Monitor galleria mellonella survival at 30 minute intervals or more frequently from the time at which mortality is usually first observed.
Remove any dead larvae and record their time of death. To compare how the timing of treatment impacts mortality, treat the larvae at two, four, six, nine or 12 hours post-infection and monitor their survival at a 30 minute interval. The effective dose of tobramycin varied by strain, five milligrams per kilogram, was optimal for PAO1 and PA14.2.5 milligrams per kilogram for PAK and 10 milligrams per kilogram for IST27-M.
No dose was effective for LESB58. Treatment with five milligrams per kilogram of tobramycin at two, four and six hours post-infection resulted in over 50%survival of plasmodium aeruginosa infected galleria mellonella larvae.
Overview
This study aims to optimize Galleria mellonella infection models for preclinical antimicrobial assessments. By standardizing the framework, the researchers seek to enhance the reliability of these models and reduce the chance of ineffective compounds entering clinical trials.
Key Study Components
Research Area
- Preclinical antimicrobial development
- Infection modeling
- Chronic respiratory infections
Background
- Importance of studying bacterial responses in human airways
- Challenges posed by cystic fibrosis on bacterial virulence and resistance
- Complexities in current preclinical drug screens for chronic lung infections
Methods Used
- Galleria mellonella larvae infection models
- Use of air-liquid interface cell cultures
- High-throughput transcriptomic analysis
Main Results
- Standardized protocols for infection and treatment
- Varied effective dosages of tobramycin based on bacterial strains
- Significant survival rates observed post-treatment with proper timing
Conclusions
- The study demonstrates the potential of G. mellonella models in optimizing preclinical antimicrobial assessments.
- This approach could significantly impact the development of new treatments for chronic respiratory infections.
Frequently Asked Questions
Executive Industry Relevance
Accelerating antimicrobial discovery requires preclinical models that balance biological relevance, throughput, and translational predictivity. The Galleria mellonella infection model offers a standardized, scalable system for early-stage efficacy and toxicity screening, reducing reliance on mammalian models and improving portfolio triage. Integrating this model into antimicrobial pipelines enhances predictive confidence and operational efficiency at critical lead selection inflection points.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables functional interrogation of antimicrobial hypotheses in a living host system.
- Supports biological de-risking by modeling infection dynamics and therapeutic response.
- Facilitates rapid elimination of ineffective compounds before mammalian studies.
- Improves predictive confidence for downstream portfolio advancement.
Screening & Assay Development
- Provides a reproducible, standardized infection model for quantitative efficacy assessment.
- Enables dose-ranging and timing studies to optimize compound selection.
- Supports high-throughput screening with greater biological relevance than in vitro assays.
- Reduces variability and increases assay readiness for lead evaluation.
Translational & Preclinical Research
- Aligns preclinical testing with infection-relevant conditions, especially for respiratory pathogens.
- Bridges the gap between in vitro screens and mammalian models for translational continuity.
- Informs risk-adjusted advancement decisions by providing early in vivo efficacy data.
Pipeline & Workflow Integration
The G. mellonella model fits between in vitro screening and mammalian preclinical studies, enabling early de-risking and prioritization of antimicrobial candidates.
- Discovery Biology: Supports hypothesis testing and pathway clarification in infection-relevant environments.
- Screening: Delivers reproducible, quantitative survival and efficacy readouts for compound comparison.
- Analytics: Enables statistical analysis of dose-response and timing effects on survival outcomes.
- Translational Research: Provides continuity for respiratory infection models and informs biomarker alignment.
- Enterprise Reuse: Offers a scalable, standardized platform adaptable to diverse pathogens and compounds.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in early antimicrobial testing.
- Operational Value: Standardizes workflows, enhances reproducibility, and supports scalable screening.
- Strategic Value: Enables better go/no-go decisions and reduces late-stage biological risk.
- Portfolio Impact: Improves risk-adjusted prioritization and advancement of antimicrobial candidates.
Implementation Considerations
- Requires expertise in infection modeling and statistical analysis of survival data.
- Needs access to standardized larvae, infection stocks, and dosing instrumentation.
- Demands cross-team agreement on model parameters and data interpretation.
- Adaptable across bacterial species and antimicrobial classes with protocol optimization.
- Limitations include differences from mammalian immune responses and pharmacokinetics.
