A Label-free, Ultra-High-Throughput Bacteriolysis Droplet Screening Platform (KillerDrop)

Some viruses called 'phages' have developed, over millions of years, strategies to invade bacteria in which they multiply and kill them from inside. We can use and develop similar strategies in the laboratory to find new ways to kill bacteria. This is important because the rapid emergence and spread of antimicrobial resistance has evolved into a major healthcare threat. However, the development of new phage-inspired therapies depends on our understanding of how phages interact with bacteria. For instance, some phages will infect bacteria but not kill them. Current methods to observe these interactions are very slow and hard to interpret because they do not track the fate of every bacteria. What we need are novel techniques that can track bacteria individually when in presence of lytic agents. In this project, we will encapsulate bacteria and phages in tiny water-in-oil microdroplets and record images so that we count every single bacterium that is killed by phages. This will help us better understand how and in what time bacteria get lysed. The proposed research will provide a tool to understand these interactions for up to a million simultaneous experiments, enabling us to have a complete picture of environmental conditions required for cell lysis.

The key proteins that help phages kill bacteria are called lysins. They are enzymes that can degrade bacterial membranes until bacteria burst open. Importantly, these enzymes can be modified in the laboratory to make them more efficient at killing bacteria, in a process called directed evolution. However, this process is slow, tedious and costly, often limited to testing a handful of changes in the gene sequence encoding the enzymes and, furthermore, is focused on a relatively small pool of previously characterised enzymes. There is a clear need for technologies that allow us to make huge numbers (millions) of changes to the enzymes and screen them rapidly. Such technologies should also isolate the very best enzymes so that we can test their properties as therapeutic agent in isolation.

The new technology we propose will allow us to express thousands of fragments of DNA (molecules encoding enzymes) into lysins all at once. We will then use cutting edge micro-plumbing to combine all these proteins with bacteria. Using state-of-the-art optical and electronics instrumentation, we will build a new platform for detecting and counting the number of lysed bacteria. This innovative and challenging combination of technology will allow us to screen huge numbers of variant lysins and search for DNA sequence encoding the most active ones.

The power of the technology proposed will allow us to take high-throughput measurements surpassing current approaches. Specifically, the new technology that will be utilised for detecting bacteria lysis will be around 1000 times faster than current technologies used. Our proposed project will unlock the door to a range of new approaches for both investigating how bacteria and phages interact, understand how lysin evolution relate to their lytic function and the development of new antimicrobial drugs.

In this project, we aim to build a technology platform for high-throughput imaging and sorting of bacteria co-encapsulated with bacteriophages or lytic enzymes in micro-droplets. The platform will enable imaging at single cell resolution, allowing us to unravel bacteria-phage interactions and quantify phenotypic variations. This will be achieved by building novel real-time image acquisition and processing architectures. The throughput and resolution of this droplet-based imager will surpass currently available techniques and allow us to precisely count the number of lysed/unlysed bacteria within each compartment. In addition, we will introduce dual field-of-view imaging so that bacteria imaging can be performed in conjunction with droplet sorting, confirming the isolation of the correct droplets. By combining this novel technology with synthetic biology approaches for expressing diverse DNA templates, we will build a new tool for high throughput investigation of lytic enzymes function.

This project will enable advances in several fields including healthcare, biotechnology and microfluidic engineering. The key potential beneficiaries are therefore biotech firms developing novel antimicrobial compounds and assays, or looking to evolve lysins as bacteriolytic agents for specific applications. This proposal will provide them with a tool to evolve such enzymes. Likewise, the elucidation of bacteria-phage interactions may provide novel strategies to tackle antibiotic resistant bacterial strains. This will feed into a growing industry seeking to develop alternative therapies and targets to tackle antimicrobial resistance.

The second beneficiaries are engineering companies looking to commercialize hardware kits enabling screening of bacteria at high-throughput. In particular, microfluidic hardware innovations will be of interest for several biotechnology companies, who are keenly interested in high-throughput selection using miniaturised microfluidic formats.

Such approaches, with further development, will also have knock on benefits for how we approach studying phage interactions with bacterial strains, providing new platform technologies that are likely to reduce either the amount of bacteria/phages needed for every tests and the time it takes to achieve results.

We anticipate that these technologies will ultimately be complementary to classical microtiter plate screens and therefore represent a step change in throughput. As such, we will try to identify market niches and opportunities for licensing and commercialization of the technology coordinated by the department of Innovation, Impact and Business at Exeter. This may create jobs and new streams of research. If adopted, potential exists for huge savings in solutions and materials commonly utilised in more traditional, less efficient applications, indicating potential market value and representing an important ecological benefit and further advancement for sustainable microbiology.

The skills, methods and results generated in this project will be directly important for companies and research institutions that engage in high-throughput screening for bacterial lysis assays. Staff development is also an objective of the project: companies will look for skilled staff to introduce the novel approach of screening bacteriolysis in droplets using microfluidic devices. Thus, the trained personnel including the recruited PDRA will acquire new skills in microfluidic technologies. The crossing of boundaries between optics, micro-engineering and directed evolution is one of the unique features of this training experience. The skills and capabilities achieved as part of this project will increase the availability of highly skilled workers in the UK that will undoubtedly be an advantage in a knowledge-based economy. The researcher trained in this project will be in a position to make a valuable and practical contribution to the continued growth of the biotechnology sector in the UK.

Finding efficient treatments to tackle antimicrobial resistance is crucial: the global costs of AMR to health services and associated productivity losses in the EU is estimated to be $1.5 billion per year and the antibiotics market is thought to reach $63 billion by 2026. Other industries will benefit from the advances made in this project and we anticipate high impact in these areas: high-throughput screening ($21billion), analysis instrumentation ($10 billion), enzymes ($19 billion).