Cells are compartmentalized by membranes, which provide a barrier to the external environments of the cell and its organelles. They are dynamic structures consisting mostly of protein and lipid. They contain an important subset of proteins, integral membrane proteins, which reside within a lipid bilayer and are responsible for a variety of essential cellular processes, such as sensation, cellular regulation, and metabolism - making them essential for all life, as well as key drug targets. This project aims to uncover the structural dynamics of membrane proteins involved in antimicrobial resistance (AMR) of bacteria. AMR is recognised by the WHO and United Nations as a global health emergency. The traditional model for the development and marketing of new chemical antibiotics against drug-resistant bacteria has disintegrated due to the high cost ($1.5 billion per drug). The projects' focus is on multidrug efflux membrane protein systems which are known to play major roles in bacterial antibiotic resistance, specifically the resistance-nodulation-division (RND) efflux pumps. Their ability to expel a broad range of toxic substances out of bacteria significantly contributes to multidrug resistance against structurally and functionally diverse antimicrobial drugs. Understanding their structural dynamics is important, as these fundamental fluctuations frequently represent motions and states that are critical for protein function and drug efflux. To do this, chemical biology and advanced mass spectrometry strategies are being developed which enable membrane protein dynamics to be deciphered within complex environments, including in live cells. Using techniques such as Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS), which measures the extent and rate of exchange of protein backbone amide hydrogens for deuterium, both global and local information on protein interactions, ligand binding, and structural dynamics can be delivered. This will enable an unprecedented insight into the structure, dynamics, and function of these systems, particularly on the impact of drug and lipid interactions, and clinically relevant mutations. With the achievement of cellular structural dynamic insight offering a huge step forward in our understanding of how they shape the function of healthy and diseased cells. So far, we have explored prototypical resistance-nodulation-division (RND) multidrug efflux systems within a planktonic context. Planktonic bacteria are 'free-living' or 'free flowing' in suspension, commonly grown in flask cultures in the laboratory. They are not fixed to a community of bacteria and are designed to colonize new niches, but with a lower chance of survival. However, bacterial populations found naturally often form structured communities of cells called biofilms, which provide a more secure way for bacteria to reproduce and survive. Biofilms typically pose a great issue for implants as they provide an ideal solid support to promote growth, thus treatment of such infections is extremely difficult, normally resulting in the removal of the implant. Within this renewal we plan to expand our research in two ways: 1) the exploration of related efflux proteins and systems, away from the prototypical, to broaden our understanding of the fundamental role structural dynamics plays in the multidrug resistance phenotype, and 2) adapt our methods to interface with biofilm systems, so as to gain a 'true' context of the role these efflux systems play in bacterial infection and resistance. In conclusion there is an abundance of evidence to support the influence of RND pumps in the pathogenicity of bacteria. By understanding these modes of pathogenicity therapeutic methods can be designed to overcome them and treat infection. By utilizing different methods, focused on targeting RND function with efflux pump inhibitors (EPIs), reliance on new, more potent antibiotics can be ameliorated.

This project aims to develop an integrated mathematical model to explore the early stages of bacterial biofilm formation. The project requires the development of new mathematical models that can correctly capture details of how bacteria move in fluid environments and colonize surfaces. Furthermore, recent experiments on surface-attached bacteria have identified new movement patterns that are not currently captured in existing mathematical models. We will, therefore, be undertaking mathematical research to tackle the important societal and economic challenge of biofilms. The resulting new mathematical models and techniques will also be of relevance to many other phenomena concerning active particles that can transition between existing in the bulk fluid and being attached to a surface. Bacteria are among the most primitive forms of life. Yet, despite their relative simplicity and small size, bacteria can actively sense a remarkable diversity of different environmental signals, and use this information to direct their motility towards more favourable environments. This ability to move profoundly affects where we expect to find bacteria. It is important to study biofilms because during bacterial infection the emergence of anti-microbial resistance frequently occurs within biofilms; and combatting bacterial infections in a major health challenge. Furthermore biofilms have impact beyond health: a study by the National Biofilm Innovation Centre estimated that biofilms act on a $4 trillion global industrial base operating across many sectors, including contamination of food and water supplies, disruption of oil and gas and biofouling in marine environments, and also benefitting waste-water treatment processes, biorefining and biotechnology. Many factors affect how biofilms form. In this project we focus on the very early stages of biofilm formation where the behaviour of cells, in particular the way in which they move and compete with each other, can profoundly impact what happens in the later stages. By developing a mathematical framework, we will clarify the complexity of the problem, and be able to test biological hypothesis concerning how different bacterial species compete and colonize surfaces. The ability of bacteria to swim and move up chemical gradients (chemotaxis) has been well-known for several decades. However we still cannot fully predict where the bacteria are, and how likely they are to encounter a surface, in flow environments such as the digestive tract or circulatory system. This is the challenge we address in our first objective (shape & shear). It has recently been discovered that some surface-attached bacteria can undergo chemotaxis, and our second objective (search) aims to develop a new model to explain the mechanisms for this and develop a model which can predict where bacteria will accumulate on a surface. Our final objective (strife) will investigate how bacterial strains with different growth and motility signatures compete, either indirectly through competition for resources, or directly for example through toxin production. By developing a mathematical model of this we can investigate the early spatial patterning of bacteria on a surface, which will impact the composition of resultant biofilms.

There is an absence of qualitative, interdisciplinary research on the personal application of infection prevention (IP) measures, like hand-washing and mask-wearing, and its effectiveness beyond the healthcare setting. In this crisis, IP measures are critical to building confidence to resume leisure and economic activity out of the home. The project advances previous work by this team that identified a need for novel IP research which integrates behavioural, microbiological and aesthetic approaches to creatively demonstrate the interactions of human movement with microbial/viral transmission. The case study is the public transport bus and its diverse community of users, including BAME and other higher-risk groups. The research will: i) investigate the structural challenges in consistent application of IP in public (and private) spaces; ii) provide microbiological and sociological evidence to inform and improve effective cleaning practices for bus operators and safe travel practices for bus users; iii) generate wider public knowledge and understanding of infection risk/prevention and their geographies in shared indoor spaces. This project will build confidence by addressing unknowns about the potential viral threat of boarding the bus. The team will work quickly to undertake and integrate findings from an ethnographic research and a microbiome study to assess the effectiveness of bus cleaning routines and passenger PPE. A fluorescents mapping simulation using ultraviolet powders and sprays will mimic and demonstrate visually the mobility of 'mock' SARS-CoV-2 through contact and aerosols if IP measures are not implemented. Outputs include the creation of novel viral aesthetic materials to communicate the effectiveness of IP.

Biofilms are microbial cells embedded within a self-secreted extracellular polymeric substance (EPS) matrix which adhere to substrates. Biofilms are central to some of the most urgent global challenges across diverse fields of application, from medicine to industry to the environment and exert considerable economic and social impact. For example, catheter-associated urinary tract infections (CAUTI) in hospitals has been estimated to cause additional health-care costs of £1-2.5 billion in the United Kingdom alone (Ramstedt et al, Macromolec. Biosci. 19, 2019) and to cause over 2000 deaths per year (Feneley et al, J. Med. Eng. Technol. 39, 2015). To combat biofilm growth on surfaces, chemical-based approaches using immobilization of antimicrobial agents (i.e. antibiotics, silver particles) can trigger antimicrobial resistance (AMR), but are often not sustainable. Alternatively, bio-inspired nanostructured surfaces (e.g. cicada wing, lotus leaf) can be used, but their effects often may not last. A recent innovation in creating slippery surfaces has been inspired by the slippery surface strategy of the carnivorous Nepenthes pitcher plant. These slippery surfaces involve the impregnation of a porous or textured solid surface with a liquid lubricant locked-in to the structure. Such liquid surfaces have been shown to have promise as antifouling surfaces by inhibiting the direct access to the solid surface for biofilm attachment, adhesion and growth. However, the antibiofilm performance of these new liquid surfaces under flow conditions remains a concern due to flow-induced depletion of lubricant. Here we propose a novel anti-biofilm surface by creating permanently bound slippery liquid-like solid surfaces. Success would transform our understanding about bacteria living on surfaces and open-up new design paradigms for the development of next generation antibiofilm surfaces for a wide range of applications (e.g. biomedical devices and ship hulls). To enable the successful delivery of this project, it requires us to combine cross-disciplinary skills ranging from materials chemistry, physical and chemical characterisations of materials surfaces, nanomechanics, microbiology, biomechanics, to computational mechanics. The project objectives well align with EPSRC Healthcare Technologies Grand Challenges, addressing the topics of controlling the amount of physical intervention required, optimizing treatment, and transforming community health and care. In parallel, we shall contribute to the advancement of Cross-Cutting Research Capabilities (e.g. advanced materials, future manufacturing technologies and sustainable design of medical devices) that are essential for delivering these Grand Challenges. In particular, this research will employ nanomechanical tests to determine bacteria adhesion and microfluidics techniques for biofilm characterisation, which enables us to create novel approaches in computational engineering through the formulation and validation of sophisticated numerical models of bacteria attachment and biofilm mechanics.

AMR is a global threat needing urgent coordinated actions via a transdisciplinary approach with pooling of resources and research efforts to expedite practical solutions for new - diagnostics, therapies and vaccines - the three lines of defence against AMR. In 2016, the O'Neill report 'analysed the global problem of rising drug resistance and proposed concrete actions'. However, almost a decade later, without 'fit-for-purpose' diagnostics, the report's recommendation of diagnostics-guided antimicrobial treatments by 2020 remains unrealistic even today. In addition, to have a meaningful impact in controlling AMR, One Health approach is crucial for all AMR interventions including diagnostics - as emergence and spread of AMR is interlinked between humans, animals, plants and the environment. Animals raised for food account for 73% of global antimicrobial use, and >75% of human pathogens detected (last 3 decades) have originated in animals, highlighting the context and the need for diagnostics for domesticated animals. Plant health depends heavily on fungicides for the control of fungal and oomycete diseases. However, resistance against multiple fungicide classes has led to control problems in key diseases in wheat, barley, potatoes, and fruits. There are concerns about the impact of agricultural fungicides on antifungal resistance in human pathogens, especially Aspergillus fumigatus. Thus, diagnostics are needed for timely detection to prevent spread. Environment Chemical pollutants, heavy metals, antimicrobials, co-selectors and pathogens and pesticides - all drive selection of AMR, thus needing prompt and precise detection and control. Clinical need for appropriate diagnostics is well-documented. AMR from Bacterial pathogens are associated with ~5 million AMR deaths annually and the threat from fungal pathogens and their resistances are high too. Hence, our Network's focus is One Health diagnostics. While the UK is well-placed to meet the scientific challenges of developing such technologies and become an international leader, a step-change in our approach is needed if we are to transition the country's scientific excellence into a coordinated drive to develop practical solutions that can be implemented and adopted across these sectors. Thus, through a transdisciplinary team, ARREST-AMR will support the successful development and smooth journey of technologies from research labs to adoption and use in 'real life' through 5 objectives: Identify 'needs': Across all the sectors (i) identify areas (such as diseases, pathogens, chemical co-selectors) where the diagnostics are needed the most (ii) what types of technologies are needed (iii) where should they be placed to provide the most useful information at the right time and at the right cost. To achieve this, the Network will conduct extensive stakeholder engagements across all sectors. Innovate: Experts such as scientists, engineers, clinicians, veterinarians, crop-protection professionals, experts in One Health and biologists who work in fundamental biology of AMR - will together develop research projects to contribute to better understanding of AMR, with the knowledge-generation focussed to develop new products that address the 'needs'; and help existing UK technologies improve their diagnostic performance/economic utility and reconcile the 'needs'. Evaluate: Supporting with standardised approaches for performance, economic and utility evidence generation for each sector will engender a culture of translational-focussed research. Implement: We will support translational aspects including Regulatory and behavioural aspects, identifying facilitators and barriers for adoption. Cross-pollinate: Will help exchange of best practices, needs, regulatory aspects and product applications within and outside - their sectors and the Network.