scholarly journals Harnessing Colon Chip Technology to Identify Commensal Bacteria That Promote Host Tolerance to Infection

2021 ◽  
Vol 11 ◽  
Author(s):  
Francesca S. Gazzaniga ◽  
Diogo M. Camacho ◽  
Meng Wu ◽  
Matheus F. Silva Palazzo ◽  
Alexandre L. M. Dinis ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Human Microbiome ◽  
Commensal Bacteria ◽  
Bacterial Strains ◽  
Mucus Production ◽  
16S Sequencing ◽  
Chip Technology ◽  
Host Microbe Interactions ◽  
Host Tolerance

Commensal bacteria within the gut microbiome contribute to development of host tolerance to infection, however, identifying specific microbes responsible for this response is difficult. Here we describe methods for developing microfluidic organ-on-a-chip models of small and large intestine lined with epithelial cells isolated from duodenal, jejunal, ileal, or colon organoids derived from wild type or transgenic mice. To focus on host-microbiome interactions, we carried out studies with the mouse Colon Chip and demonstrated that it can support co-culture with living gut microbiome and enable assessment of effects on epithelial adhesion, tight junctions, barrier function, mucus production, and cytokine release. Moreover, infection of the Colon Chips with the pathogenic bacterium, Salmonella typhimurium, resulted in epithelial detachment, decreased tight junction staining, and increased release of chemokines (CXCL1, CXCL2, and CCL20) that closely mimicked changes previously seen in mice. Symbiosis between microbiome bacteria and the intestinal epithelium was also recapitulated by populating Colon Chips with complex living mouse or human microbiome. By taking advantage of differences in the composition between complex microbiome samples cultured on each chip using 16s sequencing, we were able to identify Enterococcus faecium as a positive contributor to host tolerance, confirming past findings obtained in mouse experiments. Thus, mouse Intestine Chips may represent new experimental in vitro platforms for identifying particular bacterial strains that modulate host response to pathogens, as well as for investigating the cellular and molecular basis of host-microbe interactions.

scholarly journals Harnessing Colon Chip technology to identify commensal bacteria that promote host tolerance to infection

2020 ◽  
Author(s):  
Francesca S. Gazzaniga ◽  
Diogo M. Camacho ◽  
Meng Wu ◽  
Matheus Palazzo ◽  
Alexandre Dinis ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Human Microbiome ◽  
Commensal Bacteria ◽  
Bacterial Strains ◽  
Mucus Production ◽  
16S Sequencing ◽  
Chip Technology ◽  
Host Microbe Interactions ◽  
Host Tolerance

ABSTRACTCommensal bacteria within the gut microbiome contribute to development of host tolerance to infection, however, identifying specific microbes responsible for this response is difficult. Here we describe methods for developing microfluidic organ-on-a-chip models of small and large intestine lined with epithelial cells isolated from duodenal, jejunal, ileal, or colon organoids derived from wild type or transgenic mice. To focus on host-microbiome interactions, we carried out studies with the mouse Colon Chip and demonstrated that it can support co-culture with living gut microbiome and enable assessment of effects on epithelial adhesion, tight junctions, barrier function, mucus production, and cytokine release. Moreover, infection of the Colon Chips with the pathogenic bacterium, Salmonella typhimurium, resulted in epithelial detachment, decreased tight junction staining, and increased release of cytokines (CXCL1, CXCL2, and CCL20) that closely mimicked changes previously seen in mice. Symbiosis between microbiome bacteria and the intestinal epithelium was also recapitulated by populating Colon Chips with complex living mouse or human microbiome. By taking advantage of differences in the composition between complex microbiome samples cultured on each chip using 16s sequencing, we were able to identify Enterococcus faecium as a positive contributor to host tolerance, confirming past findings obtained in mouse experiments. Thus, mouse Intestine Chips may represent new experimental in vitro platforms for identifying particular bacterial strains that modulate host response to pathogens, as well as for investigating the cellular and molecular basis of host-microbe interactions.

scholarly journals A high-resolution pipeline for 16S-sequencing identifies bacterial strains in human microbiome

2019 ◽  
Author(s):  
Igor Segota ◽  
Tao Long
Keyword(s):  
Bacterial Species ◽  
Human Microbiome ◽  
Amplicon Sequencing ◽  
R Package ◽  
Strain Level ◽  
Sequencing Data ◽  
Bacterial Strains ◽  
16S Sequencing ◽  
16S Amplicon Sequencing ◽  
Sequencing Data Analysis

We developed a High-resolution Microbial Analysis Pipeline (HiMAP) for 16S amplicon sequencing data analysis, aiming at bacterial species or strain-level identification from human microbiome to enable experimental validation for causal effects of the associated bacterial strains on health and diseases. HiMAP achieved higher accuracy in identifying species in human microbiome mock community than other pipelines. HiMAP identified majority of the species, with strain-level resolution wherever possible, as detected by whole genome shotgun sequencing using MetaPhlAn2 and reported comparable relative abundances. HiMAP is an open-source R package available at https://github.com/taolonglab/himap.

scholarly journals Composition of the Gut Microbiome Influences Production of Sulforaphane-Nitrile and Iberin-Nitrile from Glucosinolates in Broccoli Sprouts

Nutrients ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 3013
Author(s):  
John A. Bouranis ◽  
Laura M. Beaver ◽  
Jaewoo Choi ◽  
Carmen P. Wong ◽  
Duo Jiang ◽  
...  
Keyword(s):  
Gut Microbiome ◽  
Ex Vivo ◽  
Human Subjects ◽  
Individual Variability ◽  
16S Sequencing ◽  
Microbiome Composition ◽  
Broccoli Sprouts ◽  
Broccoli Sprout

Isothiocyanates, such as sulforaphane and iberin, derived from glucosinolates (GLS) in cruciferous vegetables, are known to prevent and suppress cancer development. GLS can also be converted by bacteria to biologically inert nitriles, such as sulforaphane-nitrile (SFN-NIT) and iberin-nitrile (IBN-NIT), but the role of the gut microbiome in this process is relatively undescribed and SFN-NIT excretion in humans is unknown. An ex vivo fecal incubation model with in vitro digested broccoli sprouts and 16S sequencing was utilized to explore the role of the gut microbiome in SFN- and IBN-NIT production. SFN-NIT excretion was measured among human subjects following broccoli sprout consumption. The fecal culture model showed high inter-individual variability in nitrile production and identified two sub-populations of microbial communities among the fecal cultures, which coincided with a differing abundance of nitriles. The Clostridiaceae family was associated with high levels, while individuals with a low abundance of nitriles were more enriched with taxa from the Enterobacteriaceae family. High levels of inter-individual variation in urine SFN-NIT levels were also observed, with peak excretion of SFN-NIT at 24 h post broccoli sprout consumption. These results suggest that nitrile production from broccoli, as opposed to isothiocyanates, could be influenced by gut microbiome composition, potentially lowering efficacy of cruciferous vegetable interventions.

scholarly journals Identification of Commensal Bacterial Strains That Modulate Yersinia enterocolitica and Dextran Sodium Sulfate-Induced Inflammatory Responses: Implications for the Development of Probiotics

2007 ◽  
Vol 75 (7) ◽  
pp. 3490-3497 ◽  
Author(s):  
Julia S. Frick ◽  
Kerstin Fink ◽  
Frauke Kahl ◽  
Maria J. Niemiec ◽  
Matteo Quitadamo ◽  
...  
Keyword(s):  
Sodium Sulfate ◽  
Inflammatory Responses ◽  
Dextran Sodium Sulfate ◽  
Commensal Bacteria ◽  
Bacterial Strains ◽  
Necrosis Factor Alpha ◽  
Bifidobacterium Adolescentis ◽  
Enteric Infections

ABSTRACT An increasing body of evidence suggests that probiotic bacteria are effective in the treatment of enteric infections, although the molecular basis of this activity remains elusive. To identify putative probiotics, we tested commensal bacteria in terms of their toxicity, invasiveness, inhibition of Yersinia-induced inflammation in vitro and in vivo, and modulation of dextran sodium sulfate (DSS)-induced colitis in mice. The commensal bacteria Escherichia coli, Bifidobacterium adolescentis, Bacteroides vulgatus, Bacteroides distasonis, and Streptococcus salivarius were screened for adhesion to, invasion of, and toxicity for host epithelial cells (EC), and the strains were tested for their ability to inhibit Y. enterocolitica-induced NF-κB activation. Additionally, B. adolescentis was administered to mice orally infected with Y. enterocolitica and to mice with mucosae impaired by DSS treatment. None of the commensal bacteria tested was toxic for or invaded the EC. B. adolescentis, B. distasonis, B. vulgatus, and S. salivarius inhibited the Y. enterocolitica-induced NF-κB activation and interleukin-8 production in EC. In line with these findings, B. adolescentis-fed mice had significantly lower results for mean pathogen burden in the visceral organs, intestinal tumor necrosis factor alpha mRNA expression, and loss of body weight upon oral infection with Y. enterocolitica. In addition, the administration of B. adolescentis decelerated inflammation upon DSS treatment in mice. We suggest that our approach might help to identify new probiotics to be used for the treatment of inflammatory and infectious gastrointestinal disorders.

scholarly journals Highly reproducible 16S sequencing facilitates measurement of host genetic influences on the stickleback gut microbiome

2018 ◽  
Author(s):  
Clayton M. Small ◽  
Mark Currey ◽  
Emily A. Beck ◽  
Susan Bassham ◽  
William A. Cresko
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
Gut Microbiome ◽  
Dna Isolation ◽  
Minor Effect ◽  
Tissue Preparation ◽  
Host Genotype ◽  
16S Sequencing ◽  
Host Microbe Interactions ◽  
Microbiome Data

AbstractMulticellular organisms interact with resident microbes in important ways, and a better understanding of host-microbe interactions is aided by tools such as high-throughput 16S sequencing. However, rigorous evaluation of the veracity of these tools in a different context from which they were developed has often lagged behind. Our goal was to perform one such critical test by examining how variation in tissue preparation and DNA isolation could affect inferences about gut microbiome variation between two genetically divergent lines of threespine stickleback fish maintained in the same lab environment. Using careful experimental design and intensive sampling of individuals, we addressed technical and biological sources of variation in 16S-based estimates of microbial diversity. After employing a two-tiered bead beating approach consisting of tissue homogenization followed by microbial lysis in subsamples, we found an extremely minor effect of DNA isolation protocol relative to among-host microbial diversity differences. Individual abundance estimates for rare OTUs, however, showed much lower reproducibility. We found that the stickleback gut microbiome was highly variable, even among siblings housed together, but that an effect of host genotype (stickleback lineage) was detectable for some microbial taxa. Our findings demonstrate the importance of appropriately quantifying biological and technical variance components when attempting to understand major influences on high-throughput microbiome data.

scholarly journals Primary Human Dendritic Cells and Whole-Blood based Assays to Evaluate Immuno-Modulatory Properties of Heat-Killed Commensal Bacteria

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 225
Author(s):  
James E. Norton Norton, Jr. ◽  
Sushma Kommineni ◽  
Patricia Akrivoulis ◽  
Dario A. Gutierrez ◽  
Daria J. Hazuda ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Whole Blood ◽  
Culture System ◽  
Immune Cell ◽  
Ex Vivo ◽  
Critical Role ◽  
Commensal Bacteria ◽  
Host Immune System ◽  
Bacterial Strains

There is mounting evidence that the microbiome plays a critical role in training and maturation of the host immune system. Pre-clinical and clinical studies have shown that microbiome perturbation is correlated with sub-optimal host responses to vaccines and cancer immunotherapy. As such, identifying species of commensal bacteria capable of modulating immunological outcomes is of considerable interest. Currently, the lack of reliable primary immune cell-based assays capable of differentiating immuno-modulatory properties of various commensal bacteria is a major limitation. Here, we demonstrate that primary human monocyte-derived dendritic cells (MoDC) are capable of stratifying different strains of live and heat-killed commensal bacteria in an in vitro culture system. Specifically, heat-killed bacterial strains were able to differentially modulate co-stimulation/maturation markers CD80, CD83, and HLA-DR, as well as cytokine/chemokine signatures, such as IL-1b, MIP-1a, and TNFa in primary human MoDC. We further validated our observations using the TruCulture® (Myriad RBM, Inc., Austin, TX, USA) whole-blood ex vivo culture system. Using this ex vivo system allowed us to measure immune-altering effects of commensal bacteria in primary human whole-blood. As such, we report that both these primary in vitro and ex vivo systems are robust and enable identification, stratification, and differentiation of various commensal bacteria as potential modulators of host immunity.

scholarly journals Biomimetic Gut Model Systems for Development of Targeted Microbial Solutions for Enhancing Warfighter Health and Performance

mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Lauren M. Brinkac ◽  
Nandita Rahman ◽  
Loun-Loun Chua ◽  
Sterling Thomas
Keyword(s):  
Human Health ◽  
Gut Microbiome ◽  
Human Microbiome ◽  
Research Priorities ◽  
Performance Outcomes ◽  
Vital Role ◽  
Model Systems ◽  
Experimental Model Systems ◽  
And Performance

ABSTRACT The human gut microbiome plays a vital role in both health and disease states and as a mediator of cognitive and physical performance. Despite major advances in our understanding of the role of gut microbes in host physiology, mechanisms underlying human-microbiome dynamics have yet to be fully elucidated. This knowledge gap represents a major hurdle to the development of targeted gut microbiome solutions influencing human health and performance outcomes. The microbiome as it relates to warfighter health and performance is of interest to the Department of Defense (DoD) with the development of interventions impacting gut microbiome resiliency among its top research priorities. While technological advancements are enabling the development of experimental model systems that facilitate mechanistic insights underpinning human health, disease, and performance, translatability to human outcomes is still questionable. This review discusses some of the drivers influencing the DoD’s interest in the warfighter gut microbiome and describes current in vitro gut model systems supporting direct microbial-host interactions.

scholarly journals Highly Reproducible 16S Sequencing Facilitates Measurement of Host Genetic Influences on the Stickleback Gut Microbiome

mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Clayton M. Small ◽  
Mark Currey ◽  
Emily A. Beck ◽  
Susan Bassham ◽  
William A. Cresko
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
Gut Microbiome ◽  
Dna Isolation ◽  
Minor Effect ◽  
Tissue Preparation ◽  
Host Genotype ◽  
16S Sequencing ◽  
Microbiome Composition ◽  
Host Microbe Interactions

ABSTRACT Multicellular organisms interact with resident microbes in important ways, and a better understanding of host-microbe interactions is aided by tools such as high-throughput 16S sequencing. However, rigorous evaluation of the veracity of these tools in a different context from which they were developed has often lagged behind. Our goal was to perform one such critical test by examining how variation in tissue preparation and DNA isolation could affect inferences about gut microbiome variation between two genetically divergent lines of threespine stickleback fish maintained in the same laboratory environment. Using careful experimental design and intensive sampling of individuals, we addressed technical and biological sources of variation in 16S-based estimates of microbial diversity. After employing a two-tiered bead beating approach that comprised tissue homogenization followed by microbial lysis in subsamples, we found an extremely minor effect of DNA isolation protocol relative to among-host microbial diversity differences. Abundance estimates for rare operational taxonomic units (OTUs), however, showed much lower reproducibility. Gut microbiome composition was highly variable across fish—even among cohoused siblings—relative to technical replicates, but a subtle effect of host genotype (stickleback line) was nevertheless detected for some microbial taxa. IMPORTANCE Our findings demonstrate the importance of appropriately quantifying biological and technical variance components when attempting to understand major influences on high-throughput microbiome data. Our focus was on understanding among-host (biological) variance in community metrics and its magnitude in relation to within-host (technical) variance, because meaningful comparisons among individuals are necessary in addressing major questions in host-microbe ecology and evolution, such as heritability of the microbiome. Our study design and insights should provide a useful example for others desiring to quantify microbiome variation at biological levels in the face of various technical factors in a variety of systems.

scholarly journals 2224 Determining if intestinal commensal bacteria enhance the frequency of reassortment of an enteric, segmented virus, reovirus

2018 ◽  
Vol 2 (S1) ◽  
pp. 9-9
Author(s):  
Matthew Lanahan ◽  
Andrea Erickson ◽  
Julie Pfeiffer
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Bacterial Species ◽  
Plaque Assay ◽  
Virus Evolution ◽  
Commensal Bacteria ◽  
Bacterial Strains ◽  
Rt Pcr ◽  
Sds Page

OBJECTIVES/SPECIFIC AIMS: The overall goal is to determine if intestinal commensal bacteria play a role in enteric virus evolution. We will use reovirus, an enteric segmented virus, to investigate specific goals. First, we will determine if specific bacterial species enhance the coinfection frequency of 2 separate strains of reovirus. Second, we will determine if the presence/absence of different bacterial species in the microbiota of mice results in different reovirus reassortment frequencies. Finally, we will discover if reassortant reovirus is present in human populations. METHODS/STUDY POPULATION: My first goal is to determine if specific bacterial species enhance the coinfection frequency of 2 strains of reovirus. In our lab, we have a panel of commensal intestinal bacterial strains, as well as a number of lab adapted bacterial strains. We will use this panel of bacteria to determine if reovirus binds to different species of bacteria using a binding assay involving radiolabeled virus. Additionally, we will determine if specific species of bacteria alter the coinfection frequency through a Flow cytometry based assay. This will involve mixing virus with bacteria, infecting cells in culture, and straining for reovirus proteins for flow cytometry. Our second goal is to determine if specific bacteria promote reassortment of reovirus in a mouse model of infection. To do this, we will use gnotobiotic techniques to create mice harboring different intestinal bacteria populations. Mice will be infected with 2 strains of reovirus, and then feces and organs will be collected. Progeny virus will be subjected to a plaque assay on 2 different types of cells. The first type of cells will be normal cells in culture in which all viable viruses will form plaques. The second will be a cell line that stably expresses siRNAs against specific reovirus segments in which only specific reassortants will form plaques. These 2 plaque assays will be used to quantify the total number of viruses present and the total number of reassortant viruses present. Additionally, SDS-PAGE and RT-PCR will be used to confirm reassortants. Our third goal is to determine if reassortant reovirus is present in infected humans. To do this, I will obtain feces from reovirus-infected children and isolate reovirus. One specific reovirus reassortant is known to propogate in dual-infected mice. I will use the plaque assay technique to determine if this reassortant is also present in humans. To determine if other reassortants are present, I will use RT-PCR and SDS-PAGE. RESULTS/ANTICIPATED RESULTS: Based on previous studies with other enteric viruses, we suspect that specific bacterial species bind reovirus strains with different efficiencies. It is likely that a number of bacterial species will promote coinfection. The bacterial strains that binds both reovirus strains at a high efficiency will likely enhance coinfection by the greatest amount. It is likely that mice harboring different bacterial populations will produce different reovirus reassortment frequencies. We predict that bacteria that enhance reovirus coinfection in vitro should also enhance reovirus reassortment in our mouse model. Therefore, mice specifically lacking bacteria that promote coinfection should have significantly lower amounts of reassortant reovirus. It will be important to control for the overall amount of replication within mice with different microbiotas, as this will affect the basal reassortment frequency. We suspect that reovirus reassortants are present in humans. Work done both in vitro and in mouse models indicates that reassortment happens at high frequencies. Additionally, one specific reassortant commonly propogates in mice due to an enhanced cellular attachment phenotype. Therefore, we predict that this reassortant also commonly emerges after coinfection and reassortment in humans. DISCUSSION/SIGNIFICANCE OF IMPACT: Segmented viruses, such as influenza and rotavirus, are important human pathogens. Viral reassortment poses a unique threat to humans, as it enables new viruses to emerge and cause pandemics or epidemics. However, little is known about what factors promote viral reassortment. This study will provide insight into a novel mechanism of segmented virus evolution.

scholarly journals Controlled Complexity: Optimized Systems to Study the Role of the Gut Microbiome in Host Physiology

2021 ◽  
Vol 12 ◽  
Author(s):  
Robert W. P. Glowacki ◽  
Morgan J. Engelhart ◽  
Philip P. Ahern
Keyword(s):  
Gut Microbiome ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Human Microbiome ◽  
Model Systems ◽  
Wild Mice ◽  
Host Health ◽  
Host Physiology ◽  
The Impact

The profound impact of the gut microbiome on host health has led to a revolution in biomedical research, motivating researchers from disparate fields to define the specific molecular mechanisms that mediate host-beneficial effects. The advent of genomic technologies allied to the use of model microbiomes in gnotobiotic mouse models has transformed our understanding of intestinal microbial ecology and the impact of the microbiome on the host. However, despite incredible advances, our understanding of the host-microbiome dialogue that shapes host physiology is still in its infancy. Progress has been limited by challenges associated with developing model systems that are both tractable enough to provide key mechanistic insights while also reflecting the enormous complexity of the gut ecosystem. Simplified model microbiomes have facilitated detailed interrogation of transcriptional and metabolic functions of the microbiome but do not recapitulate the interactions seen in complex communities. Conversely, intact complex communities from mice or humans provide a more physiologically relevant community type, but can limit our ability to uncover high-resolution insights into microbiome function. Moreover, complex microbiomes from lab-derived mice or humans often do not readily imprint human-like phenotypes. Therefore, improved model microbiomes that are highly defined and tractable, but that more accurately recapitulate human microbiome-induced phenotypic variation are required to improve understanding of fundamental processes governing host-microbiome mutualism. This improved understanding will enhance the translational relevance of studies that address how the microbiome promotes host health and influences disease states. Microbial exposures in wild mice, both symbiotic and infectious in nature, have recently been established to more readily recapitulate human-like phenotypes. The development of synthetic model communities from such “wild mice” therefore represents an attractive strategy to overcome the limitations of current approaches. Advances in microbial culturing approaches that allow for the generation of large and diverse libraries of isolates, coupled to ever more affordable large-scale genomic sequencing, mean that we are now ideally positioned to develop such systems. Furthermore, the development of sophisticated in vitro systems is allowing for detailed insights into host-microbiome interactions to be obtained. Here we discuss the need to leverage such approaches and highlight key challenges that remain to be addressed.

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