title: Whole-Genome Enrichment Provides Deep Insights into Vibrio cholerae Metagenome from an African River.
abstract: The detection and typing of Vibrio cholerae in natural aquatic environments encounter major methodological challenges related to the fact that the bacterium is often present in environmental matrices at very low abundance in nonculturable state. This study applied, for the first time to our knowledge, a whole-genome enrichment (WGE) and next-generation sequencing (NGS) approach for direct genotyping and metagenomic analysis of low abundant V. cholerae DNA (<50 genome unit/L) from natural water collected in the Morogoro river (Tanzania). The protocol is based on the use of biotinylated RNA baits for target enrichment of V. cholerae metagenomic DNA via hybridization. An enriched V. cholerae metagenome library was generated and sequenced on an Illumina MiSeq platform. Up to 1.8 × 107bp (4.5× mean read depth) were found to map against V. cholerae reference genome sequences representing an increase of about 2500 times in target DNA coverage compared to theoretical calculations of performance for shotgun metagenomics. Analysis of metagenomic data revealed the presence of several V. cholerae virulence and virulence associated genes in river water including major virulence regions (e.g. CTX prophage and Vibrio pathogenicity island-1) and genetic markers of epidemic strains (e.g. O1-antigen biosynthesis gene cluster) that were not detectable by standard culture and molecular techniques. Overall, besides providing a powerful tool for direct genotyping of V. cholerae in complex environmental matrices, this study provides a 'proof of concept' on the methodological gap that might currently preclude a more comprehensive understanding of toxigenic V. cholerae emergence from natural aquatic environments.
authors.affiliation: Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy. luigi.vezzulli@unige.it.
authors.forename: L
authors.identifier: 0000-0002-4368-1454
authors.initials: L
authors.lastname: Vezzulli
authors.affiliation: Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Grande
authors.affiliation: Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Tassistro
authors.affiliation: Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Brettar
authors.affiliation: Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: M G
authors.initials: MG
authors.lastname: Höfle
authors.affiliation: Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: R P A
authors.initials: RP
authors.lastname: Pereira
authors.affiliation: Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Mushi
authors.affiliation: Department of Life Sciences, University of Trieste, Trieste, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Pallavicini
authors.affiliation: Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Vassallo
authors.affiliation: Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genoa, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Pruzzo
mesh_terms: D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D056186:Metagenomics / Q000379:methods*; D045483:Rivers / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013636:Tanzania; D014734:Vibrio cholerae / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial
keywords: Africa; Next-generation sequencing; Vibrio cholerae; Whole-genome enrichment