Culture-and amplification free nanopore sequencing for rapid detection of pathogens and antimicrobial resistance genes from urine. Culture-and amplification free nanopore sequencing for rapid detection of pathogens and antimicrobial resistance genes from urine

Urinary Tract Infections (UTIs) are among the most prevalent infections globally. Every year, approximately 150 million people are diagnosed with UTIs worldwide. The current state-of-the-art routine method diagnostic methods are culture-based and have a turnaround time of 2-4 days for pathogen identification and susceptibility testing. This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Urine samples were spiked at clinically significant concentrations of 103-105 CFU/ml. Three DNA extraction kits (BioStic, PowerFood, and Blood and Tissue) were investigated based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. Results: The Blood and Tissue kit outperformed the other kits based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. Using nanopore sequencing, we demonstrated that identifying some pathogens and antibiotic-resistance genes (ARG) was possible at as low as 103 colony formation units (CFU/mL). However, all the pathogens and corresponding genes were only identified at a spike concentration of 105 CFU/ml, achieved only after 10 min and 3 hours of sequencing, respectively. The overall turnaround time, from sample preparation to the sequencing information about pathogen ID and antimicrobial resistance (AMR) genes, was five hours. This study shows great promise in reducing the time required for informed antibiotic administration from approximately 48 hours to five hours, thereby reducing the number of empirical doses and saving lives.

尿路感染（Urinary Tract Infections, UTIs）是全球范围内最为高发的感染性病症之一。全球每年约有1.5亿人被确诊为尿路感染患者。当前主流的常规诊断方法均基于培养法，病原体鉴定与药敏试验的周转时长长达2~4天。本研究首先建立了一种基于光密度的培养法，向健康尿液样本中接种6种最为常见的尿路致病菌。所接种的尿液样本浓度均设置为临床相关阈值：10³~10⁵菌落形成单位每毫升（Colony Formation Unit, CFU/mL）。本研究针对3种DNA提取试剂盒（BioStic、PowerFood及Blood and Tissue）开展对比评估，评估维度涵盖DNA得率、平均处理时长、洗脱体积以及单次提取的平均成本。实验结果显示：Blood and Tissue试剂盒在上述所有评估维度上均优于其余两款试剂盒。借助纳米孔测序（Nanopore Sequencing）技术，本研究证实：在低至10³ CFU/mL的样本浓度下，即可实现部分致病菌与抗生素耐药基因（Antibiotic-Resistance Genes, ARGs）的鉴定。但仅当接种浓度达到10⁵ CFU/mL时，方可实现所有致病菌及其对应耐药基因的精准鉴定，此时分别仅需10分钟与3小时的测序时长。从样本制备到获取致病菌鉴定与抗菌药物耐药（Antimicrobial Resistance, AMR）基因相关测序结果的整体周转时长仅为5小时。本研究展现出巨大的应用潜力：可将针对性抗生素给药所需的决策时长从约48小时缩短至5小时，进而减少经验性给药剂量，挽救更多生命。