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

<meta charset="utf8"></head><body>Modelos estandarizados de Galleria mellonella para pruebas preclínicas de antimicrobianos

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.

<meta charset="utf8"></head><body>Autor destacado: Caracterización de los entornos de las vías respiratorias para avanzar en los sistemas modelo y el descubrimiento de antimicrobianos en la fibrosi

Galleria mellonella como modelo de cribado antimicrobiano

Esterilización y selección de larvas de Galleria mellonella para la evaluación de nuevos antimicrobianos

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

Research

JoVE Journal

Biology

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

262 vistas. Comience pesando las larvas de Galleria mellonella. Para esterilizar la superficie de las larvas de Galleria mellonella en lotes de 10 a 20 larvas, coloque cada lote individualmente en el plato y rocíe las larvas dos veces con etanol al 70%. Con unas pinzas, enrolle las larvas para asegurar una cobertura total con etanol.Luego retire las larvas del etanol, colóquelas en una placa de Petri estéril y deje la placa descubierta para permitir que se sequ...

Video: Esterilización y selección de larvas de Galleria mellonella para la evaluación de nuevos antimicrobianos

Galleria mellonella as an Antimicrobial Screening Model

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.