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...

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Autores principales:	Florence E. Buytaers, Assia Saltykova, Sarah Denayer, Bavo Verhaegen, Kevin Vanneste, Nancy H. C. Roosens, Denis Piérard, Kathleen Marchal, Sigrid C. J. De Keersmaecker
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	metagenomics nanopore Flongle strain-level outbreak food surveillance Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/22e9e819288c42708a17b0f8043a6160
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spelling	oai:doaj.org-article:22e9e819288c42708a17b0f8043a61602021-11-05T14:48:52ZTowards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies1664-302X10.3389/fmicb.2021.738284https://doaj.org/article/22e9e819288c42708a17b0f8043a61602021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.738284/fullhttps://doaj.org/toc/1664-302XThe 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.Florence E. BuytaersFlorence E. BuytaersAssia SaltykovaAssia SaltykovaSarah DenayerBavo VerhaegenKevin VannesteNancy H. C. RoosensDenis PiérardKathleen MarchalKathleen MarchalSigrid C. J. De KeersmaeckerFrontiers Media S.A.articlemetagenomicsnanoporeFlonglestrain-leveloutbreakfood surveillanceMicrobiologyQR1-502ENFrontiers in Microbiology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	metagenomics nanopore Flongle strain-level outbreak food surveillance Microbiology QR1-502
spellingShingle	metagenomics nanopore Flongle strain-level outbreak food surveillance Microbiology QR1-502 Florence E. Buytaers Florence E. Buytaers Assia Saltykova Assia Saltykova Sarah Denayer Bavo Verhaegen Kevin Vanneste Nancy H. C. Roosens Denis Piérard Kathleen Marchal Kathleen Marchal Sigrid C. J. De Keersmaecker Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies
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	article
author	Florence E. Buytaers Florence E. Buytaers Assia Saltykova Assia Saltykova Sarah Denayer Bavo Verhaegen Kevin Vanneste Nancy H. C. Roosens Denis Piérard Kathleen Marchal Kathleen Marchal Sigrid C. J. De Keersmaecker
author_facet	Florence E. Buytaers Florence E. Buytaers Assia Saltykova Assia Saltykova Sarah Denayer Bavo Verhaegen Kevin Vanneste Nancy H. C. Roosens Denis Piérard Kathleen Marchal Kathleen Marchal Sigrid C. J. De Keersmaecker
author_sort	Florence E. Buytaers
title	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_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_sort	towards real-time and affordable strain-level metagenomics-based foodborne outbreak investigations using oxford nanopore sequencing technologies
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/22e9e819288c42708a17b0f8043a6160
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Towards Real-Time and Affordable Strain-Level Metagenomics-Based Foodborne Outbreak Investigations Using Oxford Nanopore Sequencing Technologies

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