A Culture-Independent Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of Shiga-Toxigenic Escherichia coli O104:H4
A Culture-Independent Sequence-Based Metagenomics Approach to the Investigation of an Outbreak of Shiga-Toxigenic Escherichia coli O104:H4
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DOI：
10.1001/jama.2013.3231
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发表时间：
2013-04-10
影响因子：
120.7
通讯作者：
Pallen, Mark J.
中科院分区：
文献类型：
--
作者：
Loman, Nicholas J.;Constantinidou, Chrystala;Pallen, Mark J.
确定引起暴发的细菌的重要性有助于疾病管理。然而，传统的基于培养的诊断可能很困难，特别是在没有针对暴发菌株的特定诊断测试的情况下。目的探索元基因组学(从微生物复杂样本中提取的DNA直接测序)作为开放式临床发现平台的潜力，该平台能够在没有实验室培养的情况下识别和表征暴发疫情中的细菌菌株。设计、背景和患者在一项回顾性调查中，从2011年德国产志贺毒素大肠杆菌(STEC)O104：H4暴发期间腹泻患者的粪便样本中选择45份样本。对样品进行高通量测序(2012年8月至9月)，然后进行三阶段分析(2012年11月至2013年2月)。在第一阶段，开发了从头组装的方法来获得暴发毒株的基因组草稿。在第二阶段，在每个样本中确定暴发毒株基因组的覆盖深度。在第三阶段，将每个样本的序列与已知细菌的序列进行比较，以识别暴发菌株以外的病原体。主要结果和衡量基因组序列数据的恢复，以便从粪便样本中鉴定和鉴定暴发菌株和其他病原体。结果在第一阶段，获得了STEC暴发菌株的基因组草图。在第二阶段，从超过10倍覆盖率的10个样本和超过1倍覆盖率的26个样本中恢复了暴发毒株基因组。在40份STEC阳性样本中，有27份(67%)检测到志贺毒素基因序列。在阶段3中，回收了艰难梭菌、空肠弯曲杆菌、简缩弯曲杆菌和肠沙门氏菌的序列。结论和相关性这些结果表明，元基因组学作为一种独立于培养的方法在腹泻疾病暴发期间鉴定细菌病原体是有潜力的。挑战包括提高诊断灵敏度、加快和简化工作流程以及降低成本。贾玛2013；309(14)：1502-1510 www.jama.com
Importance Identification of the bacterium responsible for an outbreak can aid in disease management. However, traditional culture-based diagnosis can be difficult, particularly if no specific diagnostic test is available for an outbreak strain.Objective To explore the potential of metagenomics, which is the direct sequencing of DNA extracted from microbiologically complex samples, as an open-ended clinical discovery platform capable of identifying and characterizing bacterial strains from an outbreak without laboratory culture.Design, Setting, and Patients In a retrospective investigation, 45 samples were selected from fecal specimens obtained from patients with diarrhea during the 2011 outbreak of Shiga-toxigenic Escherichia coli (STEC) O104:H4 in Germany. Samples were subjected to high-throughput sequencing (August-September 2012), followed by a 3-phase analysis (November 2012-February 2013). In phase 1, a de novo assembly approach was developed to obtain a draft genome of the outbreak strain. In phase 2, the depth of coverage of the outbreak strain genome was determined in each sample. In phase 3, sequences from each sample were compared with sequences from known bacteria to identify pathogens other than the outbreak strain.Main Outcomes and Measures The recovery of genome sequence data for the purposes of identification and characterization of the outbreak strain and other pathogens from fecal samples.Results During phase 1, a draft genome of the STEC outbreak strain was obtained. During phase 2, the outbreak strain genome was recovered from 10 samples at greater than 10-fold coverage and from 26 samples at greater than 1-fold coverage. Sequences from the Shiga-toxin genes were detected in 27 of 40 STEC-positive samples (67%). In phase 3, sequences from Clostridium difficile, Campylobacter jejuni, Campylobacter concisus, and Salmonella enterica were recovered.Conclusions and Relevance These results suggest the potential of metagenomics as a culture-independent approach for the identification of bacterial pathogens during an outbreak of diarrheal disease. Challenges include improving diagnostic sensitivity, speeding up and simplifying workflows, and reducing costs. JAMA. 2013;309(14):1502-1510 www.jama.com