How wax moth larvae are providing a new system for identifying antimicrobial compounds
The world is consumed with stories about the unfolding COVID-19 coronavirus, which surfaced in China in December and has now spread to more than 60 countries. But inarguably a bigger danger to public health are antibiotic-resistant bacteria and fungus, which infects 2.8 million annually in the US. Clearly we need more antibiotics capable of combating these bugs, and we need more cost-effective ways to produce them.
One way researcher are attempting to meet these twin goals is through insects, specifically the wax moth larvae (Galleria mellonella) infection model system. Galleria mellonella are widely used to study host-pathogen interactions and virulence and are now increasingly used to determine the pharmacokinetics/pharmodynamics, efficacy and toxicity of novel antimicrobial compounds.
Clio Andreae, PhD, a microbiologist with Charles River’s Portishead site in the UK, is an expert in these models. Her lab works with galleria moths, and she offers timely insights into why they are so valuable in finding new antimicrobial compounds.
What differentiates your line of galleria moths from others that are out there, particularly the TruLarv product from Bio Systems Technology? Are there particular features of the way it’s raised or bred that make it unique?
CA: We only use TruLarv produced by Biosystems Technology which are research grade larvae that are bred free of antibiotics and hormones and are surface sterilized prior to their use.
We have worked with Biosystem Technology, for many years using their TruLarv product in our own labs at Charles River Portishead. Before the development of TruLarv, Galleria mellonella used in research were purchased from pet shops, which often produced very variable results due to differences in their size, age, weight and as they were bred using antibiotics. As a result, Dr. Olivia Champion and Professor Titball (founders of Biosystems Technology) developed the TruLarv system which provides a much more robust model system, with less variability within treatment groups seen when compared to those larva purchase from pet shops. Biosystems Technology are our primary supplier and they provide training for use of the Galleria in research.
What efforts are being made to improve the galleria model? Are there plans to make genetic or other alterations to it, or to come up with particular lines that could be used for more specific purposes?
CA: As we don’t produce our own Galleria mellonella in house all modifications to the larval model system will come directly from Biosystems Technology. Biosystems Technology have several plans in the pipeline that they have made us here at Charles River aware of, so watch this space for future developments with the model system.
Here at Charles River it is our aim to provide a robust Galleria mellonella model system for drug discovery projects on ESKAPE pathogens by determining the LD100 and LD50 of various pathogen strains. We plan to work closely with Biosystems Technology to select the appropriate bacterial strains for testing within the Galleria model system.
Can you describe the bioluminescent bacteria system in a bit more detail? Why was it developed, and what research applications is it particularly suited for? Are there any specific examples of it being used that I might look at where researchers used it, such as a published paper?
CA: The use of bioluminescent bacteria enables in-life determination of bacterial burden, reducing the number of animals required for an infection study. Charles River has several luminescent bacterial strains, obtained from Perkin Elmer, that have been engineered to encode a lux operon from Photohabdus luminescens that have been successfully used in murine and Galleria mellonella studies. These luminescent bacterial strains are particularly useful when determine efficacy of novel compounds following infection with a bacterial pathogen, while reducing the number of animals and insects required.
Determining bacterial burden in infected larva requires the addition of various satellite groups for each time point tested, requiring a lot more larva for the study. I have developed the use of bioluminescent bacteria to analyze bacterial burden within live Galleria mellonella using a GloMAX luminescent plate reader, without the need for any additional satellite groups. The idea for our initial study was to determine whether the larva could be used as an ‘in vivo’ model for antibiotic time-kill curves, to determine the efficacy of antibiotics in vivo. For this study we used luminescent Pseudomonas aeruginosa Xen41 purchased from Perkin Elmer and determined the bacterial burden by CFU/mL enumeration and luminescence measurements using a GloMAX at various time points following treatment with ciprofloxacin. Our Galleria time-kill curve analysis have shown comparable luminescence and CFU/mL results, both showing a dose response with increasing antibiotic concentrations, with the decline in luminescence seen being directly comparable with the drop in bacterial burden within individual larvae. The results of this experiment were presented at ECCMID and ELRIG as posters.
Use of a luminescent plate reader enable rapid determination of bacterial burden in infected larvae, allowing for more high-throughput analysis of in vivo antibiotic efficacy.
How much potential does galleria have as a model system? Will it replace mammalian systems in some cases, or will it be complementary?
CA: The continuing spread of antimicrobial resistance and current lack of novel drugs to combat known bacterial infections has driven the development of novel assays to improve compound efficacy testing. The use of Galleria mellonella larvae as preclinical in vivo model system to study host-pathogen interactions, virulence, toxicity and the efficacy of novel compounds, and more recently the Microbiome, is gaining momentum and interest in both the academic and pharmaceutical research space.
The potential for this model system to reduce the number of animals used in microbial drug discovery research is growing. Not only does this model system provide a cost-effective and easy to use model system it is also more ethical, with the use of the larva not requiring a Home-Office license due to their limited ability to feel pain. The main aim of the use of this model system is as an early pre-screening model system for novel antimicrobial agents, with its use predicted to reduce the number of mammalian models used by 80% (YouTube TEDx talk: How we can save mammals including ourselves).
I believed the Galleria model to be complementary to the mammalian model systems, aligning to the 3Rs by its role in partial replacement and reduction of animals used in subsequent studies. The similarities of the innate immune system seen in Galleria to that seen in mammalian models and the ability for the larva to be incubated at 37OC make the larva an ideal early in vivo model system for bacterial pathogen drug discovery projects.
(Charles River will be sponsoring a workshop on Galleria mellonella on May 15-16 in Milan, Italy.)