Target enrichment of uncultured human oral bacteria with phage-derived molecules found by single-cell genomics.

Advances in culture-independent microbial analysis, such as metagenomics and single-cell genomics, have significantly increased our understanding of microbial lineages. While these methods have uncovered a large number of novel microbial taxa, many remain uncultured, and their function and mode of existence in the environment are still unknown. This study aims to explore the use of bacteriophage-derived molecules as probes for detecting and isolating uncultured bacteria. Here, we proposed multiplex single-cell sequencing to obtain massive uncultured oral bacterial genomes and searched prophage sequences from over 450 obtained human oral bacterial single-amplified genomes (SAGs). The focus was on the cell wall binding domain (CBD) in phage endolysin, and fluorescent protein-fused CBDs were generated based on several CBD gene sequences predicted from Streptococcus SAGs. The ability of the Streptococcus prophage-derived CBDs to detect and enrich specific Streptococcus species from human saliva while maintaining cell viability was confirmed by magnetic separation and flow cytometry. The approach to phage-derived molecule generation based on uncultured bacterial SAG is expected to improve the process of designing molecules that selectively capture or detect specific bacteria, notably from uncultured gram-positive bacteria, and will have applications in isolation and in situ detection of beneficial or pathogenic bacteria.

Exploring strain diversity of dominant human skin bacterial species using single-cell genome sequencing.

To understand the role of the skin commensal bacterial community in skin health and the spread of pathogens, it is crucial to identify genetic differences in the bacterial strains corresponding to human individuals. A culture-independent genomics approach is an effective tool for obtaining massive high-quality bacterial genomes. Here we present a single-cell genome sequencing to obtain comprehensive whole-genome sequences of uncultured skin bacteria from skin swabs. We recovered 281 high-quality (HQ) and 244 medium-quality single-amplified genomes (SAGs) of multiple skin bacterial species from eight individuals, including cohabiting group. Single-cell sequencing outperformed in the genome recovery from the same skin swabs, showing 10-fold non-redundant strain genomes compared to the shotgun metagenomic sequencing and binning approach. We then focused on the abundant skin bacteria and identified intra-species diversity, especially in 47 Moraxella osloensis derived HQ SAGs, characterizing the strain-level heterogeneity at mobile genetic element profiles, including plasmids and prophages. Even between the cohabiting individual hosts, they have unique skin bacterial strains in the same species, which shows microdiversity in each host. Genetic and functional differences between skin bacterial strains are predictive of in vivo competition to adapt bacterial genome to utilize the sparse nutrients available on the skin or produce molecules that inhibit the colonization of other microbes or alter their behavior. Thus, single-cell sequencing provides a large number of genomes of higher resolution and quality than conventional metagenomic analysis and helps explore the skin commensal bacteria at the strain level, linking taxonomic and functional information.

Strain-level profiling of viable microbial community by selective single-cell genome sequencing.

Culture-independent analysis with high-throughput sequencing has been widely used to characterize bacterial communities. However, signals derived from non-viable bacteria and non-cell DNA may inhibit its characterization. Here, we present a method for viable bacteria-targeted single-cell genome sequencing, called PMA-SAG-gel, to obtain comprehensive whole-genome sequences of surviving uncultured bacteria from microbial communities. PMA-SAG-gel uses gel matrixes that enable sequential enzymatic reactions for cell lysis and genome amplification of viable single cells from the microbial communities. PMA-SAG-gel removed the single-amplified genomes (SAGs) derived from dead bacteria and enabled selective sequencing of viable bacteria in the model samples of Escherichia coli and Bacillus subtilis. Next, we demonstrated the recovery of near-complete SAGs of eight oxygen-tolerant bacteria, including Bacteroides spp. and Phocaeicola spp., from 1331 human feces SAGs. We found the presence of two different strains in each species and identified their specific genes to investigate the metabolic functions. The survival profile of an entire population at the strain level will provide the information for understanding the characteristics of the surviving bacteria under the specific environments or sample processing and insights for quality assessment of live bacterial products or fecal microbiota transplantation and for understanding the effect of antimicrobial treatments.

Recovery of strain-resolved genomes from human microbiome through an integration framework of single-cell genomics and metagenomics.

BACKGROUND: Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once. RESULTS: Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis). CONCLUSIONS: SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker . Video abstract.