scholarly journals A technical pipeline for screening microbial communities as a function of substrate specificity through single cell fluorescent imaging

2021 ◽  
Author(s):  
Shaun A. Leivers ◽  
Leidy Lagos ◽  
Sabina Leanti La Rosa ◽  
Bjørge Westereng
Keyword(s):  
Substrate Specificity ◽  
Microbial Communities ◽  
Single Cell ◽  
Monosaccharide Composition ◽  
Fluorescent Imaging ◽  
Fluorescent Label ◽  
Human Gut ◽  
Single Strain ◽  
Multifaceted Approach ◽  
The Common

The study of specific glycan uptake and metabolism has been shown to be an effective tool in aiding with the continued unravelling of the complexities in the human gut microbiome. To this aim fluorescent labelling of glycans may provide a powerful route towards target. In this study, we successfully used the fluorescent label 2-aminobenzamide (2-AB), most commonly employed for enhancing the detection of protein anchored glycans, to monitor and study microbial degradation of labelled glycans. Both single strain and co-cultured fermentations of microbes from the common human-gut derived Bacteroides genus, were able to grow when supplemented with 2-AB labelled glycans of different monosaccharide composition, degrees of acetylation and polymerization. Utilizing a multifaceted approach that combines chromatography, mass spectrometry, microscopy and flow cytometry techniques, it was possible to comprehensively track the metabolism of the labelled glycans in both supernatants and at a single cell level. We envisage this combination of complimentary techniques will help further the understanding of substrate specificity and the role it plays within microbial communities.

scholarly journals Microbe-seq: high-throughput, single-microbe genomics with strain resolution, applied to a human gut microbiome

2020 ◽  
Author(s):  
Wenshan Zheng ◽  
Shijie Zhao ◽  
Yehang Yin ◽  
Huidan Zhang ◽  
David M. Needham ◽  
...  
Keyword(s):  
Microbial Communities ◽  
High Throughput ◽  
Gut Microbiome ◽  
Bacterial Species ◽  
Microbial Interactions ◽  
Single Individual ◽  
Human Gut ◽  
Single Strain ◽  
Human Gut Microbiome

AbstractWe present Microbe-seq, a high-throughput single-microbe method that yields strain-resolved genomes from complex microbial communities. We encapsulate individual microbes into droplets with microfluidics and liberate their DNA, which we amplify, tag with droplet-specific barcodes, and sequence. We use Microbe-seq to explore the human gut microbiome; we collect stool samples from a single individual, sequence over 20,000 microbes, and reconstruct nearly-complete genomes of almost 100 bacterial species, including several with multiple subspecies strains. We use these genomes to probe genomic signatures of microbial interactions: we reconstruct the horizontal gene transfer (HGT) network within the individual and observe far greater exchange within the same bacterial phylum than between different phyla. We probe bacteria-virus interactions; unexpectedly, we identify a significant in vivo association between crAssphage, an abundant bacteriophage, and a single strain of Bacteroides vulgatus. Microbe-seq contributes high-throughput culture-free capabilities to investigate genomic blueprints of complex microbial communities with single-microbe resolution.

scholarly journals Influence of pig farming on human Gut Microbiota: role of airborne microbial communities

Gut Microbes ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 1-13
Author(s):  
Julia Moor ◽  
Tsering Wüthrich ◽  
Suzanne Aebi ◽  
Nadezda Mostacci ◽  
Gudrun Overesch ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Microbial Communities ◽  
Human Gut ◽  
Human Gut Microbiota ◽  
Pig Farming

scholarly journals Discovery of novel community-relevant small proteins in a simplified human intestinal microbiome

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Hannes Petruschke ◽  
Christian Schori ◽  
Sebastian Canzler ◽  
Sarah Riesbeck ◽  
Anja Poehlein ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Intestinal Microbiota ◽  
De Novo ◽  
Bacterial Species ◽  
Intestinal Microbiome ◽  
Single Strain ◽  
Small Proteins ◽  
Human Intestinal Microbiota ◽  
Wide Range

Abstract Background The intestinal microbiota plays a crucial role in protecting the host from pathogenic microbes, modulating immunity and regulating metabolic processes. We studied the simplified human intestinal microbiota (SIHUMIx) consisting of eight bacterial species with a particular focus on the discovery of novel small proteins with less than 100 amino acids (= sProteins), some of which may contribute to shape the simplified human intestinal microbiota. Although sProteins carry out a wide range of important functions, they are still often missed in genome annotations, and little is known about their structure and function in individual microbes and especially in microbial communities. Results We created a multi-species integrated proteogenomics search database (iPtgxDB) to enable a comprehensive identification of novel sProteins. Six of the eight SIHUMIx species, for which no complete genomes were available, were sequenced and de novo assembled. Several proteomics approaches including two earlier optimized sProtein enrichment strategies were applied to specifically increase the chances for novel sProtein discovery. The search of tandem mass spectrometry (MS/MS) data against the multi-species iPtgxDB enabled the identification of 31 novel sProteins, of which the expression of 30 was supported by metatranscriptomics data. Using synthetic peptides, we were able to validate the expression of 25 novel sProteins. The comparison of sProtein expression in each single strain versus a multi-species community cultivation showed that six of these sProteins were only identified in the SIHUMIx community indicating a potentially important role of sProteins in the organization of microbial communities. Two of these novel sProteins have a potential antimicrobial function. Metabolic modelling revealed that a third sProtein is located in a genomic region encoding several enzymes relevant for the community metabolism within SIHUMIx. Conclusions We outline an integrated experimental and bioinformatics workflow for the discovery of novel sProteins in a simplified intestinal model system that can be generically applied to other microbial communities. The further analysis of novel sProteins uniquely expressed in the SIHUMIx multi-species community is expected to enable new insights into the role of sProteins on the functionality of bacterial communities such as those of the human intestinal tract.

Effects of Community Versus Single Strain Inoculants on the Biocontrol of Salmonella and Microbial Community Dynamics in Alfalfa Sprouts†

2005 ◽  
Vol 68 (1) ◽  
pp. 40-48 ◽  
Author(s):  
ANABELLE MATOS ◽  
JAY L. GARLAND
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Community Dynamics ◽  
Community Level ◽  
Microbial Consortia ◽  
Single Strain ◽  
Acridine Orange Staining ◽  
Physiological Profiles ◽  
Alfalfa Sprouts ◽  
Alfalfa Seeds

Potential biological control inoculants, Pseudomonas fluorescens 2-79 and microbial communities derived from market sprouts or laboratory-grown alfalfa sprouts, were introduced into alfalfa seeds with and without a Salmonella inoculum. We examined their ability to inhibit the growth of this foodborne pathogen and assess the relative effects of the inoculants on the alfalfa microbial community structure and function. Alfalfa seeds contaminated with a Salmonella cocktail were soaked for 2 h in bacterial suspensions from each inoculant tested. Inoculated alfalfa seeds were grown for 7 days and sampled during days 1, 3, and 7. At each sampling, alfalfa sprouts were sonicated for 7 min to recover microflora from the surface, and the resulting suspensions were diluted and plated on selective and nonselective media. Total bacterial counts were obtained using acridine orange staining, and the percentage culturability was calculated. Phenotypic potential of sprout-associated microbial communities inoculated with biocontrol treatments was assessed using community-level physiological profiles based on patterns of use of 95 separate carbon sources in Biolog plates. Community-level physiological profiles were also determined using oxygen-sensitive fluorophore in BD microtiter plates to examine functional patterns in these communities. No significant differences in total and mesophilic aerobe microbial cell density or microbial richness resulting from the introduction of inoculants on alfalfa seeds with and without Salmonella were observed. P. fluorescens 2-79 exhibited the greatest reduction in the growth of Salmonella early during alfalfa growth (4.22 log at day 1), while the market sprout inoculum had the reverse effect, resulting in a maximum log reduction (5.48) of Salmonella on day 7. Community-level physiological profiles analyses revealed that market sprout communities peaked higher and faster compared with the other inoculants tested. These results suggest that different modes of actions of single versus microbial consortia biocontrol treatments may be involved.

scholarly journals EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

2021 ◽  
Vol 9 (8) ◽  
pp. 1649
Author(s):  
Véronica L. Roman ◽  
Christophe Merlin ◽  
Marko P. J. Virta ◽  
Xavier Bellanger
Keyword(s):  
Microbial Communities ◽  
Single Cell ◽  
Operational Taxonomic Unit ◽  
Rrna Gene ◽  
Environmental Matrices ◽  
Taxonomic Assignment ◽  
Integrative And Conjugative Elements ◽  
Functional Sequence ◽  
Methodological Improvement ◽  
New Gene

EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.

Preparation of Single Cell Suspensions of the Intra-Epithelial Layer and Lamina Propria from Human Intestinal Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Pranay Dogra ◽  
Josh Gray ◽  
Peter A. Szabo ◽  
Daniel Caron ◽  
...  
Keyword(s):  
Single Cell ◽  
Lamina Propria ◽  
Intestinal Tract ◽  
Distal Colon ◽  
Cell Suspensions ◽  
Epithelial Layer ◽  
Dilution Factor ◽  
Human Gut ◽  
Intestinal Tissue ◽  
Defined Media

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from the epithelial layer and the lamina propria of human gut sections of about one gram of tissue. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples. This protocol can be used for any section of the intestinal tract from duodenum to distal colon.

scholarly journals Integrated single-cell analysis unveils diverging immune features of COVID-19, influenza, and other community-acquired pneumonia

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alex R Schuurman ◽  
Tom DY Reijnders ◽  
Anno Saris ◽  
Ivan Ramirez Moral ◽  
Michiel Schinkel ◽  
...  
Keyword(s):  
Single Cell ◽  
Influenza A ◽  
Mononuclear Cells ◽  
Single Cell Analysis ◽  
Community Acquired Pneumonia ◽  
Type I ◽  
Cell Analysis ◽  
Peripheral Blood Mononuclear ◽  
The Common ◽  
Peripheral Immune Response

The exact immunopathophysiology of community-acquired pneumonia (CAP) caused by SARS-CoV-2 (COVID-19) remains clouded by a general lack of relevant disease controls. The scarcity of single-cell investigations in the broader population of patients with CAP renders it difficult to distinguish immune features unique to COVID-19 from the common characteristics of a dysregulated host response to pneumonia. We performed integrated single-cell transcriptomic and proteomic analyses in peripheral blood mononuclear cells from a matched cohort of eight patients with COVID-19, eight patients with CAP caused by Influenza A or other pathogens, and four non-infectious control subjects. Using this balanced, multi-omics approach, we describe shared and diverging transcriptional and phenotypic patterns—including increased levels of type I interferon-stimulated natural killer cells in COVID-19, cytotoxic CD8 T EMRA cells in both COVID-19 and influenza, and distinctive monocyte compositions between all groups—and thereby expand our understanding of the peripheral immune response in different etiologies of pneumonia.

scholarly journals Microbial Eukaryotes: a Missing Link in Gut Microbiome Studies

mSystems ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Isabelle Laforest-Lapointe ◽  
Marie-Claire Arrieta
Keyword(s):  
Microbial Communities ◽  
Microbial Ecology ◽  
High Throughput Sequencing ◽  
Sequencing Technology ◽  
Human Gut ◽  
Microbial Eukaryotes ◽  
Host Health ◽  
High Level ◽  
Eukaryotic Organisms ◽  
New Evidence

ABSTRACTHuman-associated microbial communities include prokaryotic and eukaryotic organisms across high-level clades of the tree of life. While advances in high-throughput sequencing technology allow for the study of diverse lineages, the vast majority of studies are limited to bacteria, and very little is known on how eukaryote microbes fit in the overall microbial ecology of the human gut. As recent studies consider eukaryotes in their surveys, it is becoming increasingly clear that eukaryotes play important ecological roles in the microbiome as well as in host health. In this perspective, we discuss new evidence on eukaryotes as fundamental species of the human gut and emphasize that future microbiome studies should characterize the multitrophic interactions between microeukaryotes, other microorganisms, and the host.

scholarly journals Outlier Detection for Minor Compositional Variations in Taxonomic Abundance Data

2019 ◽  
Vol 9 (7) ◽  
pp. 1355
Author(s):  
Koji Ishiya ◽  
Sachiyo Aburatani
Keyword(s):  
Microbial Communities ◽  
Natural Environments ◽  
Human Gut ◽  
Short Term ◽  
Computational Framework ◽  
Dietary Changes ◽  
Compositional Changes ◽  
Compositional Variations ◽  
Living Organisms ◽  
Shed Light

To understand the activities of complex microbial communities in various natural environments and living organisms, we need to capture the compositional changes in their taxonomic abundance. Here, we propose a new computational framework to detect compositional changes in microorganisms, including minor bacteria. This framework is designed to statistically assess relative variations in taxonomic abundance. By using this approach, we detected compositional changes in the human gut microbiome that might be associated with short-term human dietary changes. Our approach can shed light on the compositional changes of minor microorganisms that are easily overlooked.

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