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

Standardized &lt;i&gt;Galleria mellonella&lt;/i&gt; 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

239 Views. 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 ...