Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis.
Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis.
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DOI：
10.1371/journal.pone.0202049
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发表时间：
2018
期刊：
影响因子：
3.7
通讯作者：
Inoue S
中科院分区：
文献类型：
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作者：
Watanabe N;Kryukov K;Nakagawa S;Takeuchi JS;Takeshita M;Kirimura Y;Mitsuhashi S;Ishihara T;Aoki H;Inokuchi S;Imanishi T;Inoue S
及时鉴定致病性病原菌对于治疗患有感染性疾病（包括败血症和肺炎）的患者至关重要。然而，目前基于培养的方法具有几个缺点，包括它们对可培养细菌物种的使用的限制。为了解决这些问题，我们尝试使用下一代DNA测序（NGS）技术检测血液中的细菌DNA。我们使用Ion Personal Genome Machine对从细菌掺入血液中提取的DNA进行宏基因组和16S核糖体RNA（rRNA）基因扩增子测序。使用我们内部的管道基因组搜索工具包和数据库GenomeSync分析NGS数据。宏基因组测序分析成功地检测到血液中掺入的三种革兰氏阳性和三种革兰氏阴性细菌，这与相当大一部分非细菌读数相关，即使在DNA提取之前通过低速离心分离人类血细胞。对16S rRNA基因扩增子的七个可变区的测序分析也成功地检测到血液中掺入的所有六种细菌。使用来自6名败血症患者和4名健康志愿者血液中的DNA验证了使用16S rRNA基因扩增子分析的方法，血液中的潜在致病菌在物种水平上被鉴定。这些发现表明，我们的系统将成为未来实用诊断的潜在平台。
Prompt identification of causative pathogenic bacteria is imperative for the treatment of patients suffering from infectious diseases, including sepsis and pneumonia. However, current culture-based methodologies have several drawbacks including their limitation of use to culturable bacterial species. To circumvent these problems, we attempted to detect bacterial DNA in blood using next-generation DNA sequencing (NGS) technology. We conducted metagenomic and 16S ribosomal RNA (rRNA) gene amplicon sequencing of DNA extracted from bacteria-spiked blood using an Ion Personal Genome Machine. NGS data was analyzed using our in-house pipeline Genome Search Toolkit and database GenomeSync. The metagenomic sequencing analysis successfully detected three gram-positive and three gram-negative bacteria spiked in the blood, which was associated with a significant portion of non-bacterial reads, even though human blood cells were separated by low-speed centrifugation prior to DNA extraction. Sequencing analysis of seven variable regions of the 16S rRNA gene amplicon also successfully detected all six bacteria spiked in the blood. The methodology using 16S rRNA gene amplicon analysis was verified using DNA from the blood of six patients with sepsis and four healthy volunteers with potential pathogenic bacteria in the blood being identified at the species level. These findings suggest that our system will be a potential platform for practical diagnosis in the future.