Engineered bacteriophages as biosensors for the rapid diagnosis of bacterial infection

Rapid detection of bacterial pathogens directly from blood remains one of the greatest challenges in diagnostic microbiology. Current strategies rely on blood culture, which takes 24-48 hours, and identifying drug resistance requires further phenotypic tests, delaying targeted therapy and the causing the overuse of empirical antibiotics. Alternative molecular techniques are usually insufficiently sensitive, due to the low organism load. Key pathogens include Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae.

Lytic bacteriophages (phages) are a diverse family of viruses capable of infecting bacterial cells, often with single species specificity, rapidly generating 10-1000 progeny per infected cell. This replication results in the lysis of the host cell and the release of new phages, which can then infect further bacteria. We propose to detect this increase in phage numbers as a proxy for detecting bacteria (phages only multiply in live bacteria). We have shown that the increase in phage numbers during replication can be detected using molecular diagnostics, enabling sensitive detection of a bacterial infection. However, bacteria can evolve to be resistant to predation by phages via mechanisms including mutations in cell surface receptors, modifications to surface polysaccharides, CRISPR, and toxin-anti-toxin systems. This resistance remains a barrier to the implementation of bacteriophages as diagnostic tools.

In this project you will Isolate novel bacteriophage and screen them for their diagnostic potential (host range, progeny rate, cycle speed), and characterise them via next generation sequencing. Using a combination of long-term evolutionary experiments, and genetic manipulation techniques you will produce fitter phages with improved host ranges and enhanced defence system escape. Sequencing and molecular biology techniques will be used to determine causative mutations, and their mechanisms, which will be key in understanding the interactions between the phages and the bacterial defences during infection.

Molecular diagnostic assays will then be designed to detect these phages as a proxy for bacteria in blood, and the assays will be evaluated using spiked blood samples in the laboratory, before being tested on clinical samples from patients suspected of bacteraemia. 

Where does the project lie on the Translational Pathway?

T1 – Basic Research

Expected Outputs

The project will be expected to produce several high quality (3/4*) publications, in the fields of phage engineering, bacteria/phage interactions, and phage as biosensors.  Recent work from TE and EH on multidrug-resistant bacterial population dynamics has resulted in high-impact publications in Genome Biology, Nature Communications and Microbial Genomics. The data generated will support larger (3-5 years) funding applications to UK research councils and the Wellcome Trust for enhanced activity in this area. We expect the activities of the PhD to generate Intellectual property (IP) for LSTM, which will be key for realising the translational potential of this work. The development of a successful assay and associated IP could for the basis of a “spin out” or will be licensed to a collaborator for commercialisation.

 

AMR research is a priority area within the LSTM research strategy. This project will expand activity and visibility in this area, and strengthen LSTMs outputs in the T1 infectious diseases category, as aimed for in the 2017-2023 strategic plan. The development of new rapid diagnostics for identification of bacterial infections and AMR is one of the ten priority interventions recommended by the influential Wellcome Trust and the UK Department of Health Review on Antimicrobial Resistance (https://amr-review.org/) and the World Health Organisation’s Global Action Plan for AMR.

Training Opportunities

The student will receive training on a wide variety of molecular and microbiology techniques in the laboratory of TE; short and long read DNA sequencing, qPCR, DNA cloning, classical microbiology, phage isolation.  The lab of EH will provide advanced bioinformatics training, including genome assembly and annotation, comparative genomics and phylogenetics. Training on cutting edge phage engineering techniques will be provided at the lab of co-supervisor and phage biologist AS (University of Warwick), and the student will undertake placements here during the project.  We will encourage the student to identify both internal and external training opportunities that are of interest and will support their attendance.

Skills Required

Basic molecular biology, microbiology and bioinformatics would be beneficial

Key Publications associated with this project

Atienzar, AIC, Williams, C. T., Karkey, A., Dongol, S., Sulochana,  M., Rajendra, S., Hobbs, G., Evans, K.,  Musicha, P.,  Feasey, N., Cuevas, L. E.,  Adams, E. R.,  Edwards, T. (2021). A novel air-dried multiplex high resolution melt assay for the detection of extended spectrum beta-lactamase and carbapenemase genes. Journal of Global Antimicrobial resistance. 27 :123-131

Avramucz et al. (2021) Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7. Biology. 10:6

Grigonyte et al. (2021) Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7. Viruses. 12:2

Hubbard, A., Mason, J., Roberts, P., Parry, C., Corless, C., van Aartsen, J., Howard, A., Fraser, A., Adams, E., Roberts, A., Edwards, T. (2020). Piperacillin/tazobactam resistance in a clinical isolate of Escherichia coli due to IS26-mediated amplification of blaTEM-1BNature Communications. 11:4915 DOI: 10.1038/s41467-020-18668-2 

2020 Horesh G, Fino C, Harms A, Dorman MD, Parts L, Gerdes K, Heinz E*, Thomson NR*. *Shared corresponding author. Type II and Type IV Toxin-Antitoxin systems show different evolutionary patterns in the global K. pneumoniae population. Nucleic Acids Research. doi: 10.1093/nar/gkaa198.

Now Accepting Applications 

CLOSING DATE FOR APPLICATIONS: Application Portal closes: Wednesday 9th February 2022 (12:00 noon UK time)

Shortlisting complete by: End Feb/early March 2022

Interviews by: Late March/early April 2022

For more information on Eligibility, funding and how to apply please visit the MRC DTP/CASE pages