Fernbach Jonas, Baggenstos Jasmin, Svorjova Ellen-Aleksandra, Riedo Jeannine, McCallin Shawna, Loessner Martin J, Kilcher Samuel
Department of Health Science and Technology, ETH Zürich, Zürich, Switzerland.
Department of Neuro-Urology, Balgrist University Hospital, University of Zürich, Zürich, Switzerland.
Appl Environ Microbiol. 2025 Sep 17;91(9):e0201424. doi: 10.1128/aem.02014-24. Epub 2025 Aug 4.
is a major opportunistic pathogen, increasingly difficult to treat due to rising resistance to methicillin, vancomycin, and other antimicrobials. Bacteriophages offer a promising alternative, particularly when conventional therapies fail and their efficacy can be enhanced through genetic engineering. Among phages, the strictly lytic, broad-host-range members of the subfamily are among the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized K as a model to develop an efficient phage engineering platform, leveraging homologous recombination and CRISPR-Cas9-assisted counterselection. As proof of principle, this platform was utilized to construct a nanoluciferase ()-encoding reporter phage (K::) and tested as a bioluminescence-based approach for identifying viable cells. Independent of their phage-resistance profile, 100% of tested clinical isolates emitted bioluminescence upon K:: challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic phages to combat drug-resistant strains.IMPORTANCEPhage engineering, the process of modifying bacteriophages to enhance or customize their properties, offers significant potential for advancing precision antimicrobial therapies and diagnostics. While methods for engineering small phage genomes are well-established, larger phages have historically been challenging to modify. In this study, we present a novel method that enables the engineering of , a subfamily of phages known for their broad host range and strictly lytic lifestyle, making them highly relevant for diagnostic and therapeutic applications. Using this method, we successfully developed a phage-based diagnostic tool capable of rapid and sensitive detection of cells across various matrices. This approach has the potential to extend beyond diagnostics, enabling applications such as phage-mediated delivery of antimicrobial effector proteins in the future.
是一种主要的机会性病原体，由于对甲氧西林、万古霉素和其他抗菌药物的耐药性不断上升，治疗难度越来越大。噬菌体提供了一种有前景的替代方案，特别是在传统疗法失败时，并且其疗效可以通过基因工程得到增强。在噬菌体中，该亚科严格裂解、宿主范围广泛的成员是最有前景的治疗候选者之一。然而，它们庞大的基因组大小使其在工程改造方面 notoriously 困难。在本研究中，我们利用 K 作为模型开发了一个高效的噬菌体工程平台，利用同源重组和 CRISPR-Cas9 辅助的反选择。作为原理验证，该平台被用于构建一个编码纳米荧光素酶（）的报告噬菌体（K::），并作为一种基于生物发光的方法来鉴定活的 细胞进行测试。无论其噬菌体抗性谱如何，100%的测试临床 分离株在受到 K::攻击时都会发出生物发光。这种诊断测定法进一步适用于复杂基质，如人全血和牛原奶，模拟菌血症和牛乳腺炎中的 检测场景。除了基于报告噬菌体的诊断外，我们的工程技术为设计和工程改造治疗性 噬菌体以对抗耐药 菌株开辟了途径。重要性噬菌体工程，即修饰噬菌体以增强或定制其特性的过程，在推进精准抗菌疗法和诊断方面具有巨大潜力。虽然工程改造小型噬菌体基因组的方法已经很成熟，但历史上对大型噬菌体进行改造一直具有挑战性。在本研究中，我们提出了一种新方法，能够对 噬菌体亚科进行工程改造，该亚科以其广泛的宿主范围和严格的裂解生活方式而闻名，使其在诊断和治疗应用中具有高度相关性。使用这种方法，我们成功开发了一种基于噬菌体的诊断工具，能够在各种基质中快速、灵敏地检测 细胞。这种方法有可能超越诊断，在未来实现诸如噬菌体介导的抗菌效应蛋白递送等应用。
