Insects as models to study the impact of antibiotics and microbiota therapies on the human gut microbiome: reducing the use of animals in research

Project background

Gut microbiota are complex microbial communities that colonise the human gut (which becomes colonised by the beneficial bacteria) soon after birth. These bacteria play important roles in both health and disease, and microbiota disturbance in newborn babies is linked to increased risk of chronic inflammatory diseases, allergies and infection. Antibiotic treatment, which is highly prescribed during the first few years of life, has been suggested as the most significant disturbance to the developing microbiota. It is also possible that exposing developing microbiota to high levels of antibiotics may contribute to the presence of antibiotic-resistant bacteria and transferable resistance genes. Studies such as these to improve the understanding of the impact of antibiotics on the developing microbiota are commonly performed in rodent models.

Why we funded it

This PhD Studentship aims to replace the use of rodent models in gut microbiota studies using wax moth (Galleria mellonella) larvae. There are a number of advantages to using G. mellonella larvae for gut microbiota research, for example, G. mellonella possess an innate immune system with many similarities to that of humans and the larvae can be fed artificial food supplemented with antibiotics and bacteria (which is non-toxic to the larvae).

There has been a significant increase in microbiota studies and a subsequent increase in the number of animals required for these studies. In 2006, over 48,000 studies were registered in PubMed using mice in microbiota studies, this rose to nearly 73,500 studies in 2016. Maxwell and Hall estimate that each experiment requires at least ten mice and each study consists of between one and ten experiments. There are approximately 30 groups in the Norwich Research Park performing related work suggesting that the use of G. mellonella could replace up to 500 mice annually in the Research Park alone.

Research methods

Initially, the duration of antibiotic treatment necessary to produce larvae without an established gut microbiome will be determined by collecting longitudinal faecal and gut samples from treated and untreated larvae. Attempts will then be made to stably colonise G. mellonella with microbiota samples obtained from infants so the gut microbiota resembles that of a developing human. This colony of G. mellonella will have samples taken at regular intervals to determine when a stable population of bacteria has been established. Once stability has been reached, the G. mellonella will be treated with antibiotics as before. DNA samples will then be taken and deep sequencing analysis performed using shotgun metagenomics to analyse the effect of antibiotics on the microbiome. The data will be correlated to specific antibiotics so that the impact on the presence of commensal bacteria and antibiotic resistance genes/bacteria can be determined. Finally, the ability of introduced beneficial bacteria, such as found in probiotics, to restore the microbiota after being disturbed by antibiotics will be determined by analysing colonisation potential and the impact on the wider microbiota.

If Galleria can be established as a model for the infant gut microbiome then it can be used for a range of other experiments testing the effect of various treatments on the infant gut microbiome.

The human gut contains microbial communities (gut microbiota) that have profound effects on health and well-being, including immune development, metabolism and infection resistance. Disturbances in these beneficial microbial communities are linked to numerous diseases; it is critical to understand the composition and dynamics of the microbiota so that we may positively modulate this ecosystem to promote health. Of particular importance is the gut microbiota of newborn babies. Disturbances in this developing and unstable community appears to have significant short- and longer-term impacts on overall composition, and is linked to increased risk of chronic inflammatory diseases, allergies and infection. Importantly, early life microbiota can be influenced by numerous factors, with antibiotic treatment suggested as among the most significant, leading to an immediate reduction in microbial abundance and species diversity. Antibiotics are highly prescribed during the first few years of life and the impact that the various antibiotic regimens have on the early life microbiota over time is unclear. Furthermore, exposing the early life gut microbiota to significant levels of antibiotics may create an important reservoir of resistant strains and of transferable resistance genes, the so called resistome, which may correlate with the increasing incidence of antibiotic-resistant and more infective pathogens, which is directly relevant to the current global antimicrobial resistance (AMR) threat. After these antibiotic-induced disturbances several approaches could potentially be used to restore the microbial ecosystem into one able to promote health, including targeted dietary modulations and/or bacterial therapies such as probiotics. However, to understand the impact of these factors and to probe specific mechanisms underpinning microbiota changes is extremely challenging in humans, particularly infants, especially performing long-term longitudinal studies. Rodent models, e.g. mice, are routinely used mammalian models that in principle can be used for microbiota studies, however there are significant ethical and cost issues.

Galleria mellonella, the Greater wax moth, presents significant advantages as a surrogate for animal models, and it has already been used in toxicity, microbial virulence and antibiotic susceptibility trials. We propose to develop G. mellonella as a surrogate system to study human gut microbiota, particularly that of infants. Among the advantages of G. mellonella are: low cost/no ethical issues; its innate immune system shares similarities with those of mammals; it can feed on artificial food that can be dosed with antibiotics/probiotics; bacteria can be isolated directly from the gut; antibiotics can be easily injected into the body cavity with minimal distress to the insect; longitudinal studies are feasible.

Our preliminary work has shown that G. mellonella larvae can be cleared of gut bacteria, with appropriate administration of antibiotics, and human-relevant bacteria can be substituted. Our study will establish whether feeding larvae on food supplemented with antibiotics leads to alterations in the microbiome profile and whether this altered microbiome persists over generations (in preliminary work we have shown that G. mellonella will persist for at least 6 generations on artificial food). We will also investigate whether feeding with beneficial bacterial species, such as Bifidobacteria, will result in microbiome. Key aims will be to investigate antibiotic resistance acquisition in gut bacteria, determining the degree and nature of horizontal and longitudinal transmission, and determining the nature of the resistance.

Taken together we aim to establish whether the use of G. mellonella larvae for microbiome work can refine experimental design and additionally reduce and replace the use of rodent models in this rapidly developing and important human health research area.