Engineered bacteriophages as biosensors for the rapid diagnosis of bacterial infection

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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.