Whole-Genome Enrichment Provides Deep Insights into Vibrio cholerae Metagenome from an African River

• Published in: Microbial ecology Vol. 73; no. 3; pp. 734 - 738
• Main Authors: Vezzulli, L., Grande, C., Tassistro, G., Brettar, I., Höfle, M. G., Pereira, R. P. A., Mushi, D., Pallavicini, A., Vassallo, P., Pruzzo, C.
• Format: Journal Article
• Language: English
• Published: New York Springer Science + Business Media 01.04.2017; Springer US; Springer Nature B.V
• Subjects: Aquatic environment; Baits; Base Sequence; Biomedical and Life Sciences; Biosynthesis; Deoxyribonucleic acid; DNA; DNA, Bacterial - genetics; Ecology; Genetic markers; Genotyping Techniques; Geoecology/Natural Processes; High-Throughput Nucleotide Sequencing; Life Sciences; Metagenomics - methods; Microbial Ecology; Microbiology; Natural environment; Nature Conservation; NOTES AND SHORT COMMUNCATIONS; Notes and Short Communications; Pathogens; Rivers; Rivers - microbiology; Sequence Analysis, DNA; Tanzania; Vibrio cholerae - genetics; Vibrio cholerae - isolation & purification; Water Quality/Water Pollution; Whole-genome enrichment; Next-generation sequencing; Africa; Vibrio cholerae
• ISSN: 0095-3628; 1432-184X
• DOI: 10.1007/s00248-016-0902-x

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 × 10⁷ bp (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.