sterilization and selection of galleria mellonella larvae for the assessment of novel antimicrobials

Standardized Galleria mellonella Models for Preclinical Antimicrobial Testing

Galleria mellonella (the greater wax moth) larvae are used extensively across the biological sciences as infection models for microbial species, and for toxicity testing of novel drug compounds1,2. They have the potential for considerable utility in a preclinical antimicrobial testing pipeline, as they are high throughput, replicate integral in vivo characteristics of human infection, and reduce reliance on mammalian models, in line with the principles of reduction, refinement, and replacement that govern ethical use of mammalian species in research.
Developing new antibiotics requires extensive preclinical testing in vitro and in vivo models prior to clinical validation3. Only a few novel agents with promising preclinical data packages ever translate through to the clinic, and one contributor to this high attrition rate is a failure of preclinical screens to capture the complexities of infection environments4. These issues contribute not only to a low translation rate of antimicrobial agents to the clinic, but also to an increased use of experimental vertebrate animals during late-stage preclinical screening. To improve the preclinical assessment of novel antimicrobials and to reduce the usage of expensive, time-consuming, complex, and ethically problematic murine in vivo models, better early-stage drug screening tools are needed that reduce the number of unpromising compounds progressing through to testing in vertebrate systems.
G. mellonella has a short lifecycle of 8 weeks, comprised of four life stages: egg, larvae, pupae, and adults, of which the larval form is utilized in this protocol1. G. mellonella are easy to maintain throughout an experiment without the requirement for specialist equipment or a dedicated animal research facility. There is no requirement to seek ethical approval for their use, and researchers may breed the organism in-house to improve experimental quality2,5,6,7. The G. mellonella immune system closely resembles that of the mammalian innate immune system, with the ability to respond to &#39;self&#39; and &#39;non-self&#39; stimuli8. Haemocytes are responsible for pathogen-associated molecular pattern recognition and subsequent phagocytosis, playing a role that is functionally analogous to that of neutrophils in humans9. G. mellonella encodes three types of toll-like receptors that have been identified by sequence homology to humans, and produce complement-like proteins, which recognize non-self material and form localized melanization complexes following the activation and polymerization of phenoloxidase into melanin10. This can serve as a visual read-out of larval health during infection experiments, as the cuticle is darkened by melanization. It should be noted, however, that the melanization axis in insects, which involves phenoloxidase, differs substantially from the tyrosinase-melanin axis in mammals11,12. Additionally, G. mellonella produces 18 inducible antimicrobial peptides, including lysozyme and defensin homologues13. This similarity, as well as the straightforward larval maintenance procedures and high throughput nature of the model, have made G. mellonella a widely utilized organism in the assessment of novel drugs. In preclinical antibiotic development, G. mellonella has increased utility compared to in vitro models, as they can more accurately model host-pathogen-drug interactions in a complex environment with active immunity.
At present, there is no standardized research-grade supplier of G. mellonella in Europe. Researchers must instead purchase G. mellonella larvae from bait shops or maintain their own colony. Whilst methods for maintaining an in-house G. mellonella colony have been described and can increase experimental consistency5,6,7, this option is likely to be attractive only to those who use the larvae frequently. As such, this protocol focuses on the experimental setup following the purchase of larvae from a live bait supplier. While more accessible, this method increases experimental complexity and can introduce additional variability into assays due to inconsistencies in the health of the larvae at the point at which they are received from suppliers. For academics, industry, and regulators to accept and adopt G. mellonella testing as part of a preclinical antimicrobial development pipeline, a standardized system for optimization and assessment of antimicrobial efficacy is necessary.
This study optimizes the experimental design of a G. mellonella infection model for antibiotic development. While G. mellonella infection models have been described14,15, the present methodology documents additional steps to mitigate the additional complexity introduced by the inconsistency of supply and provides a framework for the assessment of novel antimicrobials. As a test case, G. mellonella was infected with the WHO priority one pathogen Pseudomonas aeruginosa, and treatment with an aminoglycoside agent (tobramycin) was optimized. This framework, illustrated in Figure 1, provides a foundation for future preclinical antimicrobial screening studies with novel agents.

Author Spotlight: Characterizing Airway Environments to Advance Model Systems and Antimicrobial Discovery in Cystic Fibrosis and Chronic Respiratory Infections

Galleria mellonella as an Antimicrobial Screening Model

Our lab seeks to understand how bacteria sense and respond to the environment in the 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.

Sterilization and Selection of Galleria mellonella Larvae for the Assessment of Novel Antimicrobials

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.

sterilization-selection-galleria-mellonella-larvae-for-assessment

Research

JoVE Journal

Biology

To combat the rising global issue of antibiotic resistance, the accelerated development of novel antibiotics is essential. Current preclinical antimicrobial development yields a significant number of leads that prove unsuitable either prior to or during clinical trials. To increase the efficiency of preclinical development, relevant, standardized, accessible, and cost-effective models must be developed. Galleria mellonella (greater wax moth) larvae are widely used as an infection model to assess microbial virulence, conduct drug toxicity testing, and serve as a preliminary means of evaluating the in vivo efficacy of novel antimicrobial compounds. These infection models have greater biological relevance than many in vitro screens of comparable throughput and decrease reliance on mammalian models when used as a pre-screen for antimicrobial testing. This protocol describes a standardized methodology for the optimization of G. mellonella infection models, which can be applied to bacterial species and antimicrobial therapeutics of choice. Using the WHO priority pathogen Pseudomonas aeruginosa as an exemplar, we outline steps that can be undertaken to develop a reproducible model of infection and therapeutic testing. This includes recommendations on experimental setup, sample preparation, and infection and treatment protocols. Integration of this model within preclinical antimicrobial development pipelines would decrease reliance on mammalian models, reduce the number of ineffective compounds reaching clinical trials, and ultimately increase the efficiency of preclinical antimicrobial development.

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.

To combat the rising global issue of antibiotic resistance, the accelerated development of novel antibiotics is essential. Current preclinical ...

Optimizing Galleria mellonella Infection Models for Use in Preclinical Antimicrobial Assessment

Begin by preparing a Pseudomonas aeruginosa infection stock of known colony forming units per milliliter prior to infection. To pellet the infection stock, centrifuge at 10, 000 x g for five minutes at room temperature. Resuspend the pellet in PBS.Next, prepare three one milliliter microfuge 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 larva. Afterward, inject each Galleria mellonella larvae with 10 microliters of inoculum in the rear right proleg. 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 in vitro 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 2, 4, 6, 9 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 mucoid. 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.