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中最大的挑战之一。目前的策略依赖于血液培养，需要24-48小时，并且鉴定耐药性需要进一步的表型测试，从而延迟靶向治疗并导致经验性抗生素的过度使用。由于微生物负荷低，替代的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氏菌。裂解性噬菌体（Lytic bacteriophage，简称LBT）是一种能够感染细菌细胞的病毒，通常具有单一物种特异性，每个感染细胞迅速产生10-1000个子代。这种复制导致宿主细胞的裂解和新的细菌的释放，然后新的细菌可以感染其他细菌。我们建议检测这种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）。我们已经表明，在复制过程中噬菌体数量的增加可以使用分子诊断检测，使敏感的检测细菌感染。然而，细菌可以通过包括细胞表面受体突变、表面多糖修饰、CRISPR和毒素-抗毒素系统在内的免疫机制进化为对捕食具有抗性。这种耐药性仍然是噬菌体作为诊断工具的实施障碍。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，后代率，循环速度），并通过下一代测序对它们进行测序。使用长期进化实验和遗传操作技术的组合，您将产生更适合的宿主范围和增强的防御系统逃逸。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细菌和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来检测这些细菌作为血液中细菌的替代物，并在实验室中使用加标血液样本对检测方法进行评估，然后对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.