Real-time diagnostic analysis of MinION™-based metagenomic sequencing in clinical microbiology evaluation: a case report

A 68-year-old man with a past history of schizophrenia underwent laparoscopic right hemicolectomy due to carcinoma. At postoperative day 4, he was diagnosed with aspiration pneumonia as a consequence of vomiting due to intestinal obstruction and septic shock. He was transferred to the intensive care unit for further observation and treatment (see Additional file 2). At day 46, we performed microbial laboratory evaluation based on both bacterial culture and 16S rRNA gene amplicon sequencing analysis following the pipeline established by us previously [4, 5] (see Additional file 3). MinION™-based sequencing analysis just 2 h after sample collection revealed the presence of Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Rhodococcus kyotonensis, Pseudoruegeria sabulilitoris, Corynebacterium simulans, and other microorganisms (see Additional file 4) (Fig. 1). Culture-based testing reported at 2 days thereafter detected Stenotrophomonas maltophilia and Pseudomonas aeruginosa.

Taxonomic assignment of the 16S rRNA genes in sputum samples. The samples were subjected to 16S rRNA amplicon sequencing using MinION™ (Oxford Nanopore Technologies, Oxford, UK), and the percentage of reads (with the abundance over 2%) belonging to the identified bacterial species is shown. Sequencing for 5 min generated 470,231 reads. A total of 41,136 reads were aligned with one of the bacterial 16S rRNA gene sequences, and 65.3% reads were aligned with Stenotrophomonas maltophilia and 15.7% reads were aligned with Pseudomonas aeruginosa

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We demonstrated the feasibility of the tentative point-of-care diagnosis by 16S rRNA amplicon sequencing via the MinION™ sequencer and subsequent confirmation of the results by standard culture methods. To facilitate the process of sample preparation, we amplified 16S rRNA genes directly from the specimen without DNA purification and after mechanical disruption treatment by bead-beating [6]. With the MinION™ sequencer, generated reads can be analyzed in real time, which makes this approach all the more promising [4]. In the present case, the identification process took only 2 h, including PCR amplification of 16S rRNA genes, sequencing on the MinION™, and bioinformatics analyses. The sequencing-based diagnostic approach is more sensitive than conventional culture-based tests. This feature can be useful for identifying unculturable bacteria or detecting bacteria in specimens after exposure to antimicrobial treatments.

Another advantage of the MinION™ sequencer is the long length of the read [7]. Conventional next-generation sequencing (NGS) generates relatively short reads with limited sequence information that do not allow distinguishing clearly between different bacterial species. The nanopore sequencing enables us to cover the entire region of the 16S rRNA gene, so detection on the species level has become possible (see Additional file 5) [5].

Further studies with more cases are needed to establish reliable diagnostic criteria based on the relative abundance of respiratory pathogen reads compared to those of the commensal bacteria. Clinical sequencing aided by this kind of analysis pipeline may open a way to precision medicine in the field of critical care medicine.

ARDS:

Acute respiratory distress syndrome

NGS:

Next-generation sequencing

rRNA:

Ribosomal RNA

We would like to Editage (www.editage.jp) for English language editing.

Funding

This work was supported by grants from the Japanese Society of Anesthesiologists (JSA) Pitch Contest 2017 to SK, JSPS KAKENHI grant number JP16K10975 to YM, MEXT-Supported Program for the Strategic Research Foundation at Private Universities to SN, and Japan Agency for Medical Research and Development (AMED, JP17fm0108023) to TIm.

Availability of data and materials

Sequence data from this study have been deposited in the DDBJ DRA database (accession number DRR160902).

All data generated or analyzed during this study are included in this published article.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Affiliations

1. Department of Human Stress Response Science, Institute of Biomedical Science, Kansai Medical University, Hirakata, Japan

   • Hiromasa Tanaka
   • , Yoshiyuki Matsuo
   • , Kenichiro Nishi
   • , Akihisa Okamoto
   • , Teppei Iwai
   • , Yoshiteru Tabata
   •  & Kiichi Hirota

2. Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan

   • So Nakagawa
   • , Kirill Kryukov
   •  & Tadashi Imanishi

3. Department of Anesthesiology, Osaka Red Cross Hospital, Osaka, Japan

   • Kenichiro Nishi
   •  & Akihisa Okamoto

4. Department of Anesthesia, Kyoto University Hospital, Kyoto, Japan

   • Shinichi Kai

5. Meisei Hospital, Osaka, Japan

   • Takeshi Tajima
   • , Yuji Komatsu
   •  & Motohiko Satoh

Contributions

HT, YM, and KS conducted the experiments. KN and AO collected the sample from the patient. SK, YM, SN, TI, SN, YT, TT, YK, MS, KK, TI, and KH analyzed and interpreted the data. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yoshiyuki Matsuo or Kiichi Hirota.

Ethics approval and consent to participate

The current study was approved by the Institutional Research Ethics Board.

Consent for publication

A written informed consent was obtained from the patient’s family for the publication of this case report and any accompanying images.

Competing interests

The authors declare that they have no competing interests.

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