Engineered bacteriophages as biosensors for the rapid diagnosis of bacterial infection

直接从血液中快速检测细菌病原体仍然是诊断微生物学的最大挑战之一。目前的策略依赖于血液培养，需要24-48小时，并且确定耐药性需要进一步的表型检测，延迟靶向治疗并导致过度使用经验性抗生素。由于生物负荷低，其他分子技术通常不够灵敏。主要病原体包括革兰氏阴性菌，如大肠杆菌和肺炎克雷伯菌。溶解性噬菌体（噬菌体）是一种能够感染细菌细胞的多种病毒，通常具有单一种特异性，每个被感染细胞迅速产生10-1000个后代。这种复制导致宿主细胞的裂解和新的噬菌体的释放，这些噬菌体可以感染更多的细菌。我们建议检测噬菌体数量的增加作为检测细菌的代理（噬菌体只在活细菌中繁殖）。我们已经证明，在复制过程中噬菌体数量的增加可以使用分子诊断来检测，从而能够敏感地检测细菌感染。然而，细菌可以通过细胞表面受体突变、表面多糖修饰、CRISPR和毒素-抗毒素系统等机制进化为抵抗噬菌体的捕食。这种耐药性仍然是将噬菌体用作诊断工具的障碍。在这个项目中，你将分离新的噬菌体并筛选它们的诊断潜力（宿主范围，子代率，循环速度），并通过下一代测序对它们进行表征。通过长期进化实验和基因操作技术的结合，你将生产出更适合的噬菌体，它们具有更好的宿主范围和增强的防御系统逃逸能力。测序和分子生物学技术将用于确定致病突变及其机制，这将是理解感染期间噬菌体与细菌防御之间相互作用的关键。然后将设计分子诊断检测方法，以检测这些噬菌体作为血液中细菌的替代品，并且将在实验室使用加了尖刺的血液样本对这些检测方法进行评估，然后再对来自疑似菌血症患者的临床样本进行检测。

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.