Department of Microbiology, Division of Laboratory Medicine, Oslo University Hospital, PB 4956, Nydalen, 0424, Oslo, Norway.
Department of Biotechnology, Inland Norway University of Applied Sciences, Holsetgata 22, 2317, Hamar, Norway.
Sci Rep. 2020 May 6;10(1):7622. doi: 10.1038/s41598-020-64616-x.
Bloodstream infections (BSI) and sepsis are major causes of morbidity and mortality worldwide. Blood culture-based diagnostics usually requires 1-2 days for identification of bacterial agent and an additional 2-3 days for phenotypic determination of antibiotic susceptibility pattern. With the escalating burden of antimicrobial resistance (AMR) rapid diagnostics becomes increasingly important to secure adequate antibiotic therapy. Real-time whole genome sequencing represents a genotypic diagnostic approach with the ability to rapidly identify pathogens and AMR-encoding genes. Here we have used nanopore sequencing of bacterial DNA extracted from positive blood cultures for identification of pathogens, detection of plasmids and AMR-encoding genes. To our knowledge, this is the first study to gather the above-mentioned information from nanopore sequencing and conduct a comprehensive analysis for diagnostic purposes in real-time. Identification of pathogens was possible after 10 minutes of sequencing and all predefined AMR-encoding genes and plasmids from monoculture experiments were detected within one hour using raw nanopore sequencing data. Furthermore, we demonstrate the correct identification of plasmids and bla subtypes using de novo assembled nanopore contigs. Results from this study hold great promise for future applications in clinical microbiology and for health care surveillance purposes.
血流感染(BSI)和败血症是全球发病率和死亡率的主要原因。基于血液培养的诊断通常需要 1-2 天才能确定细菌病原体,另外还需要 2-3 天才能确定抗生素药敏模式的表型。随着抗生素耐药性(AMR)负担的不断增加,快速诊断对于确保适当的抗生素治疗变得越来越重要。实时全基因组测序是一种基因型诊断方法,能够快速识别病原体和编码 AMR 的基因。在这里,我们使用从阳性血培养物中提取的细菌 DNA 的纳米孔测序来鉴定病原体、检测质粒和编码 AMR 的基因。据我们所知,这是第一项从纳米孔测序中收集上述信息并实时进行综合分析以进行诊断的研究。在测序 10 分钟后即可鉴定出病原体,并且在一个小时内使用原始纳米孔测序数据即可检测到来自单培养实验的所有预设的编码 AMR 的基因和质粒。此外,我们还证明了使用从头组装的纳米孔连续体正确鉴定了质粒和 bla 亚型。本研究的结果为临床微生物学的未来应用和医疗保健监测目的提供了巨大的希望。