Cargando…

Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies

The current routine laboratory practices to investigate food samples in case of foodborne outbreaks still rely on attempts to isolate the pathogen in order to characterize it. We present in this study a proof of concept using Shiga toxin-producing Escherichia coli spiked food samples for a strain-le...

Descripción completa

Detalles Bibliográficos
Autores principales: Buytaers, Florence E., Saltykova, Assia, Denayer, Sarah, Verhaegen, Bavo, Vanneste, Kevin, Roosens, Nancy H. C., Piérard, Denis, Marchal, Kathleen, De Keersmaecker, Sigrid C. J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8602914/
https://www.ncbi.nlm.nih.gov/pubmed/34803953
http://dx.doi.org/10.3389/fmicb.2021.738284
_version_ 1784601664622166016
author Buytaers, Florence E.
Saltykova, Assia
Denayer, Sarah
Verhaegen, Bavo
Vanneste, Kevin
Roosens, Nancy H. C.
Piérard, Denis
Marchal, Kathleen
De Keersmaecker, Sigrid C. J.
author_facet Buytaers, Florence E.
Saltykova, Assia
Denayer, Sarah
Verhaegen, Bavo
Vanneste, Kevin
Roosens, Nancy H. C.
Piérard, Denis
Marchal, Kathleen
De Keersmaecker, Sigrid C. J.
author_sort Buytaers, Florence E.
collection PubMed
description The current routine laboratory practices to investigate food samples in case of foodborne outbreaks still rely on attempts to isolate the pathogen in order to characterize it. We present in this study a proof of concept using Shiga toxin-producing Escherichia coli spiked food samples for a strain-level metagenomics foodborne outbreak investigation method using the MinION and Flongle flow cells from Oxford Nanopore Technologies, and we compared this to Illumina short-read-based metagenomics. After 12 h of MinION sequencing, strain-level characterization could be achieved, linking the food containing a pathogen to the related human isolate of the affected patient, by means of a single-nucleotide polymorphism (SNP)-based phylogeny. The inferred strain harbored the same virulence genes as the spiked isolate and could be serotyped. This was achieved by applying a bioinformatics method on the long reads using reference-based classification. The same result could be obtained after 24-h sequencing on the more recent lower output Flongle flow cell, on an extract treated with eukaryotic host DNA removal. Moreover, an alternative approach based on in silico DNA walking allowed to obtain rapid confirmation of the presence of a putative pathogen in the food sample. The DNA fragment harboring characteristic virulence genes could be matched to the E. coli genus after sequencing only 1 h with the MinION, 1 h with the Flongle if using a host DNA removal extraction, or 5 h with the Flongle with a classical DNA extraction. This paves the way towards the use of metagenomics as a rapid, simple, one-step method for foodborne pathogen detection and for fast outbreak investigation that can be implemented in routine laboratories on samples prepared with the current standard practices.
format Online
Article
Text
id pubmed-8602914
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-86029142021-11-20 Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies Buytaers, Florence E. Saltykova, Assia Denayer, Sarah Verhaegen, Bavo Vanneste, Kevin Roosens, Nancy H. C. Piérard, Denis Marchal, Kathleen De Keersmaecker, Sigrid C. J. Front Microbiol Microbiology The current routine laboratory practices to investigate food samples in case of foodborne outbreaks still rely on attempts to isolate the pathogen in order to characterize it. We present in this study a proof of concept using Shiga toxin-producing Escherichia coli spiked food samples for a strain-level metagenomics foodborne outbreak investigation method using the MinION and Flongle flow cells from Oxford Nanopore Technologies, and we compared this to Illumina short-read-based metagenomics. After 12 h of MinION sequencing, strain-level characterization could be achieved, linking the food containing a pathogen to the related human isolate of the affected patient, by means of a single-nucleotide polymorphism (SNP)-based phylogeny. The inferred strain harbored the same virulence genes as the spiked isolate and could be serotyped. This was achieved by applying a bioinformatics method on the long reads using reference-based classification. The same result could be obtained after 24-h sequencing on the more recent lower output Flongle flow cell, on an extract treated with eukaryotic host DNA removal. Moreover, an alternative approach based on in silico DNA walking allowed to obtain rapid confirmation of the presence of a putative pathogen in the food sample. The DNA fragment harboring characteristic virulence genes could be matched to the E. coli genus after sequencing only 1 h with the MinION, 1 h with the Flongle if using a host DNA removal extraction, or 5 h with the Flongle with a classical DNA extraction. This paves the way towards the use of metagenomics as a rapid, simple, one-step method for foodborne pathogen detection and for fast outbreak investigation that can be implemented in routine laboratories on samples prepared with the current standard practices. Frontiers Media S.A. 2021-11-05 /pmc/articles/PMC8602914/ /pubmed/34803953 http://dx.doi.org/10.3389/fmicb.2021.738284 Text en Copyright © 2021 Buytaers, Saltykova, Denayer, Verhaegen, Vanneste, Roosens, Piérard, Marchal and De Keersmaecker. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Buytaers, Florence E.
Saltykova, Assia
Denayer, Sarah
Verhaegen, Bavo
Vanneste, Kevin
Roosens, Nancy H. C.
Piérard, Denis
Marchal, Kathleen
De Keersmaecker, Sigrid C. J.
Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies
title Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies
title_full Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies
title_fullStr Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies
title_full_unstemmed Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies
title_short Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies
title_sort towards real-time and affordable strain-level metagenomics-based foodborne outbreak investigations using oxford nanopore sequencing technologies
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8602914/
https://www.ncbi.nlm.nih.gov/pubmed/34803953
http://dx.doi.org/10.3389/fmicb.2021.738284
work_keys_str_mv AT buytaersflorencee towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT saltykovaassia towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT denayersarah towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT verhaegenbavo towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT vannestekevin towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT roosensnancyhc towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT pierarddenis towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT marchalkathleen towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies
AT dekeersmaeckersigridcj towardsrealtimeandaffordablestrainlevelmetagenomicsbasedfoodborneoutbreakinvestigationsusingoxfordnanoporesequencingtechnologies