The current anti-microbial resistance (AMR) crisis is well documented and the need for a very rapid antibiotic susceptibility test is key to preventing mis-diagnosis and subsequent mis-prescribing. Recent advances in the understanding of microbial lag phases and the use of flow cytometry and fluorescent dyes has bought this possibility to the forefront of AMR research, with prediciton of AMR within 20 miniutes being demonstrated (1,2).
The development of optimised dyes is imperative in order for this to be achieved across multiple pathogens, antibiotics and matrices. Historically fluorescent dyes have suffered from poor aqueous solubility, poor tissue permeability, sub-optimal photophysics and off target effects. This project aims to address these issues through the synthesis and subsequent assessment of novel dyes that fluoresce in the Red/NIR region, which is necessary to avoid overlap with the fluorescence of intrinsic cellular material.
A number of strategies will be undertaken and potentially combined in order to achieve this:
• Synthesis of Diketopyrrolopyrrole (DPP) analogues – DPPs are of significant interest due to their impressive photo and thermal stability, and high fluorescence efficiencies in the red/NIR region. In addition, DPPs are easily modified, which is critical to improving their spectroscopic characteristics, increasing solubility in biological media, ability to penetrate gram –ve bacteria (in particular) and functionalization with targeting moieties.
• Incorporation of fluoromodules – Complexes which form upon noncovalent binding of a fluorogenic dye to its biomolecular partner to enhance the quantum yield of the dye. This approach has been successfully applied to blue fluorescent dyes 3 and should be amenable to red/NIR fluorescent dyes.
• Expansion to other red/NIR fluorescent chemotypes. There are numerous potential alternative chemotypes that could be investigated. In silico assessment of these structures for predicted aqueous solubility and penetration into gram –ve bacteria 4 will be undertaken to select the most promising chemotypes for onward investigation.
Following successful synthesis the compounds will be assessed through high-throughput flow cytometry, both in wild types and strains from our laboratory collection as well as in the Keio collection of gene knockouts (to establish which transporters are being used to take up the dyes).
It is envisaged that the PhD student would undertake both the chemical synthesis of the dyes and their biological assessment, thereby maximising multidisciplinary training.
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website: http://www.dimen.org.uk/
1. Jindal S, Thampy H, J. DP, Kell DB: Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of bacterial (re)growth Microbiology 2019; 165:439-454.
2. Jindal S, Yang L, Day PJ, Kell DB: Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli. BMC Microbiol 2019; 19:195; also bioRxiv 603688v603681.
3. Zanotti KJ, Silva GL, Creeger Y, Robertson KL, Waggoner AS, Berget PB, Armitage BA: Blue fluorescent dye-protein complexes based on fluorogenic cyanine dyes and single chain antibody fragments. Org Biomol Chem 2011; 9:1012-1020.