Björnberg Amelia, Nestor David, Peker Nilay, Sinha Bhanu, Couto Natacha, Rossen John, Sundqvist Martin, Mölling Paula
Department of Laboratory Medicine, Clinical Microbiology Örebro University Hospital and Faculty of Medicine and Health at Örebro University, Örebro, Sweden.
Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
APMIS. 2025 Jan;133(1):e13511. doi: 10.1111/apm.13511.
Shotgun metagenomics offers a broad detection of pathogens for rapid blood stream infection of pathogens but struggles with often low numbers of pathogens combined with high levels of human background DNA in clinical samples. This study aimed to develop a shotgun metagenomics protocol using blood spiked with various bacteria and to assess bacterial DNA extraction efficiency with human DNA depletion. The Blood Pathogen Kit (Molzym) was used to extract DNA from EDTA-whole blood (WB) and plasma samples, using contrived blood specimens spiked with bacteria for shotgun metagenomics diagnostics via Oxford Nanopore sequencing and PCR-based library preparation. Results showed that bacterial reads were higher in WB than plasma. Differences for Staphylococcus aureus and Streptococcus pneumoniae were more pronounced compared to Escherichia coli. Plasma samples exhibited better method reproducibility, with more consistent droplet digital PCR results for human DNA. The study found that extraction was more efficient for Gram-positive bacteria than Gram-negative, suggesting that the human DNA depletion exerts a negative effect on Gram-negative bacteria. Overall, shotgun metagenomics needs further optimisation to improve bacterial DNA recovery and enhance pathogen detection sensitivity. This study highlights some critical steps in the methodology of shotgun metagenomic-based diagnosis of blood stream infections using Nanopore sequencing.
鸟枪法宏基因组学能够广泛检测病原体，用于快速诊断血流感染中的病原体，但在临床样本中，病原体数量往往较少，同时人类背景DNA水平较高，这给检测带来了困难。本研究旨在开发一种鸟枪法宏基因组学方案，使用添加了各种细菌的血液样本，并评估经过人类DNA去除后的细菌DNA提取效率。使用血液病原体试剂盒（Molzym）从乙二胺四乙酸全血（WB）和血浆样本中提取DNA，通过牛津纳米孔测序和基于聚合酶链反应的文库制备，利用添加了细菌的人工血液标本进行鸟枪法宏基因组学诊断。结果显示，全血中的细菌读数高于血浆。与大肠杆菌相比，金黄色葡萄球菌和肺炎链球菌的差异更为明显。血浆样本表现出更好的方法重复性，人类DNA的液滴数字PCR结果更一致。研究发现，革兰氏阳性菌的提取效率高于革兰氏阴性菌，这表明去除人类DNA对革兰氏阴性菌有负面影响。总体而言，鸟枪法宏基因组学需要进一步优化，以提高细菌DNA回收率并增强病原体检测灵敏度。本研究强调了使用纳米孔测序基于鸟枪法宏基因组学诊断血流感染方法中的一些关键步骤。
