Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis

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.

快速鉴定致病病原菌对于脓毒症、肺炎等感染性疾病患者的救治至关重要。然而，当前基于培养的检测方法存在诸多局限，仅能鉴定可培养的细菌物种。为规避上述问题，本研究尝试利用下一代DNA测序（next-generation DNA sequencing, NGS）技术检测血液中的细菌DNA。本研究使用Ion个人基因组测序仪，对细菌人工模拟阳性血液中提取的DNA开展宏基因组测序与16S核糖体RNA（16S rRNA）基因扩增子测序。研究通过自主开发的分析流程Genome Search Toolkit及数据库GenomeSync对NGS数据进行分析。宏基因组测序分析成功检测出人工添加至血液中的3株革兰氏阳性菌与3株革兰氏阴性菌，但即便在DNA提取前通过低速离心分离了人体血细胞，仍存在大量非细菌序列读段。针对16S rRNA基因扩增子的7个可变区开展测序分析，同样成功检测出血液中添加的全部6株细菌。本研究使用6名脓毒症患者与4名健康志愿者的血液DNA对16S rRNA基因扩增子分析方法进行了验证，实现了血液中潜在病原菌的物种级鉴定。上述研究结果表明，本研究建立的检测体系有望成为未来临床实用诊断的潜在平台。