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.

Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing.

Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured 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.

High-Quality Draft Single-Cell Genome Sequences of Two Gammaproteobacteria Strains Sampled from Soil in a Strawberry Farm.

Here, we present high-quality draft single-cell genome sequences of Gammaproteobacteria strains BBSC-SA01 and BBSC-SA02, obtained from uncultivated cells of soil in a strawberry farm using the single-cell sequencing platform bit-MAP. These draft genomes putatively represent novel species within Gammaproteobacteria and allow further investigation into the soil microbiome.

Cobalt-Carbon Bond Formation Reaction via Ligand Reduction of Porphycene-Cobalt(II) Complex and Its Noninnocent Reactivity.

The interesting redox properties and reactivity of metalloporphycene have been studied for decades; however, the detailed experimental investigation on the reactivity and reaction mechanism under inert condition combined with theoretical calculations had not been performed so far. In this study, the novel reactivity of the reduced form of the cobalt porphycene with alkyl halides to form cobalt-carbon (Co-C) bonds was revealed. Under electrochemical reductive conditions, not the central cobalt, but the ligand was reduced and reacted with alkyl halides to afford the cobalt-alkyl complexes under N2 atmosphere in a glovebox. The reaction mechanism was clarified by the combination of experimental and theoretical studies that the porphycene ligand works as a noninnocent ligand and allows the SN2-type Co-C bond formation. This result provides us the possibility of the reaction triggered by the reduction of ligand with macrocyclic π-conjugated system, not by the reduction of metal.

Colony fingerprint for discrimination of microbial species based on lensless imaging of microcolonies.

Detection and identification of microbial species are crucial in a wide range of industries, including production of beverages, foods, cosmetics, and pharmaceuticals. Traditionally, colony formation and its morphological analysis (e.g., size, shape, and color) with a naked eye have been employed for this purpose. However, such a conventional method is time consuming, labor intensive, and not very reproducible. To overcome these problems, we propose a novel method that detects microcolonies (diameter 10-500 µm) using a lensless imaging system. When comparing colony images of five microorganisms from different genera (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans), the images showed obvious different features. Being closely related species, St. aureus and St. epidermidis resembled each other, but the imaging analysis could extract substantial information (colony fingerprints) including the morphological and physiological features, and linear discriminant analysis of the colony fingerprints distinguished these two species with 100% of accuracy. Because this system may offer many advantages such as high-throughput testing, lower costs, more compact equipment, and ease of automation, it holds promise for microbial detection and identification in various academic and industrial areas.

Rapid imaging and detection of circulating tumor cells using a wide-field fluorescence imaging system.

Circulating tumor cells (CTCs) provide potentially accessible in vivo sources of metastatic cancer cells. As such, considerable focus has been placed on analyzing the genetics of single-CTCs. Prior to these analyses, however, CTCs must first be detected within the blood samples of cancer patients. Current methods for detection of CTCs by fluorescence microscopy require the analysis of hundreds of images per blood sample, making this a time-consuming process that creates a bottleneck in CTC analysis. In this study, we therefore developed a wide-field fluorescence imaging system for rapid CTC detection. For these analyses, CTCs were first isolated using the microcavity array (MCA), a micro-sized filter for CTC recovery that separates cells based on differences in cell size and deformability. Notably, the proposed imaging system enabled rapid (∼10 s) visualization of all stained cells within the 6 mm × 6 mm MCA field via one-shot imaging. Furthermore, the morphology of the cells in the resulting images accurately reflected that observed by conventional microscopy. In total, isolation and detection of CTCs using the MCA combined with our novel wide-field fluorescence imaging system was achieved within 1 h. Thus, our proposed system will provide rapid CTC detection system.

Simple and rapid CD4 testing based on large-field imaging system composed of microcavity array and two-dimensional photosensor.

This study presents a novel method for CD4 testing based on one-shot large-field imaging. The large-field imaging system was fabricated by a microcavity array and a two-dimensional (2D) photosensor within the desk-top-sized instrument. The microcavity array was employed to separate leukocytes from whole blood based on differences in the size of leukocytes and other blood cells. The large-field imaging system with lower side irradiation enabled acquisition of cell signatures with high signal-to-noise ratio, because the metallic substrate of the microcavity array obstructed excessive excitation light. In this setting, dual-color imaging of CD4(+) and CD8(+) T cells was achieved within the entire image area (64 mm(2)) in 2s. The practical performance of the large-field imaging system was demonstrated by determining the CD4/CD8 ratio in a few microliter of control whole blood as small as those obtained by a finger prick. The CD4/CD8 ratios measured using the large-field imaging system correlated well with those measured by microscopic analysis. These results indicate that our proposed system provides a simple and rapid CD4 testing for the application of HIV/AIDS treatment.

Digital cell counting device integrated with a single-cell array.

In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm(2) in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r(2)  = 0.99). This platform could be used at extremely low cell concentrations, i.e., 25-15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.

High-content analysis of single cells directly assembled on CMOS sensor based on color imaging.

A complementary metal oxide semiconductor (CMOS) image sensor was applied to high-content analysis of single cells which were assembled closely or directly onto the CMOS sensor surface. The direct assembling of cell groups on CMOS sensor surface allows large-field (6.66 mm×5.32 mm in entire active area of CMOS sensor) imaging within a second. Trypan blue-stained and non-stained cells in the same field area on the CMOS sensor were successfully distinguished as white- and blue-colored images under white LED light irradiation. Furthermore, the chemiluminescent signals of each cell were successfully visualized as blue-colored images on CMOS sensor only when HeLa cells were placed directly on the micro-lens array of the CMOS sensor. Our proposed approach will be a promising technique for real-time and high-content analysis of single cells in a large-field area based on color imaging.

Femtosecond laser direct writing of metallic microstructures by photoreduction of silver nitrate in a polymer matrix.

Continuous silver microstructures were produced by three-dimensional (3-D) direct laser writing using a femtosecond-pulsed laser beam with polyvinylpyrrolidone (PVP) films containing silver ions. The lines drawn by scanning a tightly focused laser beam ranged from 200 nm to 1.7 microm. Using a sample solution of high density of silver nitrate, a continuous silver line with a resistivity of 3.48 x 10(-7) ohms m was produced. Not only 3-D microstructures such as pyramidal models but also hybrid microstructures comprising polymer and silver lines were demonstrated. The 3-D direct laser writing of metallic microstructures has potential for application to 3-D electrical wiring of electronic devices and MEMS devices.