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 × 10 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.

Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next‐generation sequencing strategy

“…The protocol is based on the use of biotinylated RNA baits (on average >100-mer) for selective capturing of 884 phylogenetic and virulence markers targeting the Vibrio community and other potential pathogenic microorganisms in oyster tissues via hybridization (Table 2). This approach is estimated to increase target DNA coverage by about three orders of magnitude compared with current approaches such as shotgun metagenomics (Vezzulli et al, 2017).…”

“…Target enrichment next-generation sequencing protocol for the analysis of the bivalve Pathobiota A target enrichment next-generation sequencing protocol for the analysis of bivalve 'pathobiota' was applied following the approach previously developed for the target sequencing of Vibrio cholerae DNA in complex environmental samples (Vezzulli et al, 2017). To this aim, 884 Vibrio phylogenetic and virulence markers, as well as other bivalve and marine invertebrates microbial pathogen markers (Rosenberg et al, 2007;Travers et al, 2015) with average length of 400 nt, were identified and used to produce 100-mer biotinylated RNA baits for selective capturing of target DNA markers via hybridization (Table 2).…”

“…To this aim, 884 Vibrio phylogenetic and virulence markers, as well as other bivalve and marine invertebrates microbial pathogen markers (Rosenberg et al, 2007;Travers et al, 2015) with average length of 400 nt, were identified and used to produce 100-mer biotinylated RNA baits for selective capturing of target DNA markers via hybridization (Table 2). Five hundred nanogram of total RNA baits was produced using the MYcroarray target enrichment proprietary technology (MYcroarray, Ann Arbor, MI) and used for a capture (it is estimated that a single capture is capable of enriching single-copy nuclear loci, i.e., >99.5% of the capture target region) (Vezzulli et al, 2017).…”

“…To avoid laboratory contamination of treated samples all the analyses including DNA extraction, DNA amplification and NGS library preparations were carried out in a separate laboratory (non-aquatic/nonmicrobiological laboratory) using a dedicated set of pipettes, reagents and consumables (Vezzulli et al, 2017). Sequence reads data were archived at NCBI SRA with accession: PRJNA542081.…”

A combination of metagenomic and cultivation approaches reveals hypermutator phenotypes within Vibrio cholerae infected patients

“…In this study, only 48% of the samples from patients with acute symptoms, known to harbor a high fraction of vibrios in their stool (10 10 -10 12 vibrios per liter of stool), contained enough reads to reconstruct Vibrio cholerae MAGs and to quantify withinpatient diversity. Asymptomatic patients typically shed even less V. cholerae in their stool (Nelson et al 2009), making it even more challenging to assemble their genomes using metagenomics without depletion of host DNA or targeted sequence capture techniques (Bachmann et al 2018;Vezzulli et al 2017).…”