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. 2018 Aug 15;13(8):e0202049.
doi: 10.1371/journal.pone.0202049. eCollection 2018.

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

Nobuo Watanabe  1 , Kirill Kryukov  2 , So Nakagawa  2   3 , Junko S Takeuchi  2 , Meiko Takeshita  1 , Yukiko Kirimura  1 , Satomi Mitsuhashi  2 , Toru Ishihara  4 , Hiromichi Aoki  1 , Sadaki Inokuchi  1 , Tadashi Imanishi  2 , Shigeaki Inoue  1
Affiliations

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

Nobuo Watanabe et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Bacterial composition of whole blood and its 20 × g plasma evaluated using metagenome sequencing and GSTK analysis.
DNA was extracted from control blood (A), gram-positive bacteria-spiked blood (B), gram-negative bacteria-spiked blood (C), or their respective plasma after clarification by 20 × g centrifugation. The numbers in the parenthesis to the right show the number of reads obtained from the sequencing run. The abbreviation are as follows: SA, Staphylococcus aureus; SP, Streptococcus pneumoniae; En, Enterococcus faecalis; PA, Pseudomonas aeruginosa; E, Escherichia coli; and HI, Haemophilus influenzae.
Fig 2
Fig 2. Bacterial composition identified by 16S rRNA gene amplicon sequencing followed by GSTK or Ion Reporter analysis of control and bacteria-spiked blood.
Seven variable regions of the 16S rRNA gene were amplified by PCR from either gram-positive bacteria-spiked whole blood (A) or gram-negative bacteria-spiked whole blood (B) used in Fig 1, and sequenced using Ion PGM. The sequence reads were analyzed using GSTK or Ion Reporter suite software.
Fig 3
Fig 3. Bacterial composition in whole blood from healthy volunteers and sepsis patients.
A heat map chart of possible infecting species of bacteria and possible environment-contaminated bacterial species detected in blood samples of healthy volunteers and sepsis patients. An asterisk indicates that the bacterial species was also detected by blood culture testing. Amplicons were prepared using 30 ng of input DNA for the PCR amplification. The figure was generated using a gplots module in the R software.
See this image and copyright information in PMC

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