Sae Ashikawa1, Norihito Tarumoto2,3, Kazuo Imai2,3, Jun Sakai2,3, Masahiro Kodana4, Toru Kawamura4, Kenji Ikebuchi4, Takashi Murakami3,5, Kotaro Mitsutake3,6, Shigefumi Maesaki2,3, Takuya Maeda3,5. 1. 1School of Medical Technology, Faculty of Health and Medical Care, Saitama Medical University, Saitama, Japan. 2. 2Department of Infectious Disease and Infection Control, Saitama Medical University, Saitama, Japan. 3. 3Center for Clinical Infectious Diseases and Research, Saitama Medical University, Saitama, Japan. 4. 4Clinical Laboratory Medicine, Saitama Medical University Hospital, Saitama, Japan. 5. 5Department of Microbiology, Saitama Medical University, Saitama, Japan. 6. 6Department of Infectious Diseases and Infection Control, International Medical Center, Saitama Medical University, Saitama, Japan.
Abstract
PURPOSE: Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles. METHODOLOGY: 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION. RESULTS: The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The 'pass' reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing. CONCLUSION: We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.
PURPOSE: Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study, we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles. METHODOLOGY: 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains, including five types of Candida species. Barcoded amplicons were mixed, and multiplex sequencing with the MinION sequencer was performed. In addition, barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION. RESULTS: The bacterial and Candida spp. identified by MinION sequencing, based on the highest homology of reference sequences from the NCBI gene databases, agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results, excepting the closely related species Streptococcusand Escherichia coli. The 'pass' reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus, frequent sequence errors did not affect species identification based on amplicon sequencing. CONCLUSION: We have established a rapid, portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme, which replaces traditional Sanger sequencing.
Authors: Lauren M Petersen; Isabella W Martin; Wayne E Moschetti; Colleen M Kershaw; Gregory J Tsongalis Journal: J Clin Microbiol Date: 2019-12-23 Impact factor: 5.948
Authors: Amir Arastehfar; Brian L Wickes; Macit Ilkit; David H Pincus; Farnaz Daneshnia; Weihua Pan; Wenjie Fang; Teun Boekhout Journal: J Fungi (Basel) Date: 2019-09-29
Authors: Srinivas Reddy Pallerla; Do Van Dong; Le Huu Song; Thirumalaisamy P Velavan; Le Thi Kieu Linh; Trinh Van Son; Dao Thanh Quyen; Phan Quoc Hoan; Ngo Tat Trung; Nguyen Trong The; Jule Rüter; Sébastien Boutin; Dennis Nurjadi; Bui Tien Sy; Peter G Kremsner; Christian G Meyer Journal: Ann Clin Microbiol Antimicrob Date: 2022-09-05 Impact factor: 6.781