Rapid assessment of phage for combating antimicrobial resistance in Enterobacter cloacae using a novel insect model

Antibiotics, drugs that can be used to treat and prevent bacterial infection, have revolutionized our ability to treat infectious disease and prevent infection during surgery. Unfortunately bacteria can become resistant to antibiotics by evolving. They can undergo genetic changes that enable them to tolerate or degrade antibiotics. We believe that there is a need to consider a whole raft of innovative solutions to resistance. One important possible solution is to increase our use of "biological" solutions to treat infections, such as the bacteria-killing viruses known as bacteriophage. While bacteriophage remedies have their limitations, they can be combined with antibiotics in a way that can improve treatment efficacy and also help slow the rate of the evolution of resistance. Evolution experiments require time and potentially many different hosts. While phage has much unexploited potential, currently it is difficult and expensive demonstrate the effectiveness of these tools by doing evolution of resistance experiments with laboratory mammals.

For this proposal, we have specifically developed an insect system to investigate the evolution of resistance to antibiotics in the bacterium Enterobacter cloacae. This species is particularly difficult to treat because of its naturally occurring resistance to many penicillin-based antibiotics. It is an important cause of bladder and kidney infections as well as infections associated with intensive care. Our insect model captures many of important aspects of infections in vertebrates but has the added benefits of being easy to manipulate, free of ethical oversight, and very cost effective. With this model we can vary antibiotic consumption, the presence of resistance plasmids and numerous important conditions that will allow us to mimic different real world scenarios with large numbers of individual hosts.
We will use experimental evolution to assess a range of ways of deploying bacteriophages to slow the evolution of resistance. This will include the use of virus to specifically kill those bacteria carrying genetic elements that confer resistance to antibiotics. We will investigate how to best to deploy bacteriophage in combination with phage, and how best to use phage to combat different forms of resistance. We will also look at important factors that might affect the value of phage in resistance management, such as the diversity of the bacteria in the gut.
With the insect experiments we can find out what happens in weeks or months, if we change a range of control measures. If we would do this in the real world, it would be harder, more expensive and potentially take years. Such an experimental system will never be perfect, but it will give us a good idea what will happen, cheaply and quickly.
While the insect models cannot entirely solve the problem of antibiotic resistance, we believe that they could provide an important new tool. With it we can rapidly look at the effectiveness of a large range of possible resistance management options on a population scale, and pick out the best ones, which can later be tried out in hospitals or in animal health.

This proposal will exploit a validated experimental evolution system based on a marked insect adapted strain of the enteric commensal Enterobacter cloacae. It will address how best to deploy phage therapy for the purpose of preserving the efficacy of antibiotics. Experiments will track the spontaneous de-repression of ampC beta-lactamase or the frequency of an IncF plasmid that confers resistance to a wide range of antibiotics. Repeated infection (passage) experiments will measure bacterial clearance and density, transmission, the frequency of antibiotic and phage resistance and the effect of phage on conjugation rates of evolved clones.

We will use experimental evolution to assess a range of ways of deploying bacteriophages to slow the evolution of resistance. This will include the use of plasmid-dependent MS2 phage that specifically targets F-plasmid carrying bacteria as well a general lytic phage. We will investigate how to best to deploy antibiotics combination with phage, and will test the prediction that prior treatment with phage is best for limiting spontaneous resistance, while simultaneous or post-antibiotic phage treatment will be more effective at minimizing the transmission of plasmid-encoded resistance.

We can rear gnotobiotic insects that are infected with our focal E. cloacae strain and also establish aliquots of a pooled diverse enteric community that can be used to orally infect insects with more diverse enteric microbial communities. We predict that the presence of diverse community might impose between-species competition that could increase the efficacy of phage. We will assess the impact of a diverse microbiota on the efficacy of phage-based resistance management using either a general or a plasmid specific phage. In addition to assessing clearance and evolution of resistance in our focal strain we will confirm differences in microbial diversity between treatments using 16S metagenomics.

In 2014 the World Health Organization stated that a post antibiotic era, "in which common infections and minor injuries can kill is very real possibility for the 21st century". The aim of this proposal is to test experimentally the use of bacteriophage in slowing the spread of antimicrobial resistance. Currently we are in a catch-22 situation where the absence of controlled clinical data on the value of phage therapy is a barrier to reforming legislation restricting the clinical use of phage, while it is also difficult to get these data without a favourable legislative environment. Insect models could help bridge the gap here and allow use to conduct experiments with a naturalistic infection model without legal or ethical restrictions.
This work could ultimately have benefits for the wider public in terms of the increased availability of effective anti-bacterial medicines. Benefits to the public would have to be realized through two important avenues. First, data showing that bacteriophage can effectively slow the evolution of resistance could have impacts on pressure groups, clinicians and industry bodies interesting in increasing the therapeutic use of phage. Secondly, our research will allow policy-makers, government bodies and ultimately public health officials and clinicians to make more informed decisions on resistance management and the costs and benefits of relaxing legislation currently limiting the use of phage.
Outside of these very direct lines of impact the study of resistance and experimental evolution also provides an educational opportunity for the public at large. Increasing awareness of the risks of resistance may ultimately encourage more responsible and sustainable use of antibiotics, such as reduced pressure on unwarranted prescription of antibiotics and better adherence to clinical advice.
The PDRA on this project will benefit from working in an interdisciplinary research team. The PDRA will gain skills in microbiology, gnotobiotic rearing, entomology, resistance management, public engagement and computational statistics.