scholarly journals Evaluation of the Effect of Storage Methods on Fecal, Saliva, and Skin Microbiome Composition

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Clarisse Marotz ◽  
Kellen J. Cavagnero ◽  
Se Jin Song ◽  
Daniel McDonald ◽  
Stephen Wandro ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Room Temperature ◽  
Cost Effective ◽  
Rrna Gene ◽  
Sequencing Analysis ◽  
Skin Microbiome ◽  
Metagenomic Sequencing ◽  
Sample Storage ◽  
Microbial Composition ◽  
Optimal Ratio

ABSTRACT As the number of human microbiome studies expand, it is increasingly important to identify cost-effective, practical preservatives that allow for room temperature sample storage. Here, we reanalyzed 16S rRNA gene amplicon sequencing data from a large sample storage study published in 2016 and performed shotgun metagenomic sequencing on remnant DNA from this experiment. Both results support the initial findings that 95% ethanol, a nontoxic, cost-effective preservative, is effective at preserving samples at room temperature for weeks. We expanded on this analysis by collecting a new set of fecal, saliva, and skin samples to determine the optimal ratio of 95% ethanol to sample. We identified optimal collection protocols for fecal samples (storing a fecal swab in 95% ethanol) and saliva samples (storing unstimulated saliva in 95% ethanol at a ratio of 1:2). Storing skin swabs in 95% ethanol reduced microbial biomass and disrupted community composition, highlighting the difficulties of low biomass sample preservation. The results from this study identify practical solutions for large-scale analyses of fecal and oral microbial communities. IMPORTANCE Expanding our knowledge of microbial communities across diverse environments includes collecting samples in places far from the laboratory. Identifying cost-effective preservatives that will enable room temperature storage of microbial communities for sequencing analysis is crucial to enabling microbiome analyses across diverse populations. Here, we validate findings that 95% ethanol efficiently preserves microbial composition at room temperature for weeks. We also identified the optimal ratio of 95% ethanol to sample for stool and saliva to preserve both microbial load and composition. These results provide rationale for an accessible, nontoxic, cost-effective solution that will enable crowdsourcing microbiome studies, such as The Microsetta Initiative, and lower the barrier for collecting diverse samples.

scholarly journals Comparative analysis of amplicon and metagenomic sequencing methods reveals key features in the evolution of animal metaorganisms

2019 ◽  
Author(s):  
Philipp Rausch ◽  
Malte Rühlemann ◽  
Britt Hermes ◽  
Shauni Doms ◽  
Tal Dagan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Microbial Composition ◽  
Evolutionary Patterns ◽  
Wide Range ◽  
Taxonomic Groups ◽  
And Function

AbstractBackgroundThe interplay between hosts and their associated microbiome is now recognized as a fundamental basis of the ecology, evolution and development of both players. These interdependencies inspired a new view of multicellular organisms as “metaorganisms”. The goal of the Collaborative Research Center “Origin and Function of Metaorganisms” is to understand why and how microbial communities form long-term associations with hosts from diverse taxonomic groups, ranging from sponges to humans in addition to plants.MethodsIn order to optimize the choice of analysis procedures, which may differ according to the host organism and question at hand, we systematically compared the two main technical approaches for profiling microbial communities, 16S rRNA gene amplicon- and metagenomic shotgun sequencing across our panel of ten host taxa. This includes two commonly used 16S rRNA gene regions and two amplification procedures, thus totaling five different microbial profiles per host sample.ConclusionWhile 16S rRNA gene-based analyses are subject to much skepticism, we demonstrate that many aspects of bacterial community characterization are consistent across methods and that metagenomic shotgun results are largely dependent on the employed pipeline. The resulting insight facilitates the selection of appropriate methods across a wide range of host taxa. Finally, by contrasting taxonomic and functional profiles and performing phylogenetic analysis, we provide important and novel insight into broad evolutionary patterns among metaorganisms, whereby the transition of animals from an aquatic to a terrestrial habitat marks a major event in the evolution of host-associated microbial composition.

scholarly journals Impact of Ambient Temperature Sample Storage on the Equine Fecal Microbiota

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 819
Author(s):  
Michelle Martin de Bustamante ◽  
Caryn Plummer ◽  
Jennifer MacNicol ◽  
Diego Gomez
Keyword(s):  
Ambient Temperature ◽  
Room Temperature ◽  
High Throughput Sequencing ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Sample Storage ◽  
Sample Collection ◽  
Microbial Composition ◽  
Fecal Samples ◽  
Community Membership

Sample storage conditions are an important factor in fecal microbiota analyses in general. The objective of this study was to investigate the effect of sample storage at room temperature on the equine fecal microbiota composition. Fecal samples were collected from 11 healthy horses. Each sample was divided into 7 sealed aliquots. One aliquot was immediately frozen at −80 °C; the remaining aliquots were stored at room temperature (21 to 22 °C) with one transferred to the freezer at each of the following time points: 6, 12, 24, 48, 72 and 96 h. The Illumina MiSeq sequencer was used for high-throughput sequencing of the V4 region of the 16S rRNA gene. Fibrobacteraceae (Fibrobacter) and Ruminococcaceae (Ruminococcus) were enriched in samples from 0 h and 6 h, whereas taxa from the families Bacillaceae, Planococcaceae, Enterobacteriaceae and Moraxellaceae were enriched in samples stored at room temperature for 24 h or greater. Samples frozen within the first 12 h after collection shared similar community membership. The community structure was similar for samples collected at 0 h and 6 h, but it was significantly different between samples frozen at 0 h and 12 h or greater. In conclusion, storage of equine fecal samples at ambient temperature for up to 6 h before freezing following sample collection had minimal effect on the microbial composition. Longer-term storage at ambient temperature resulted in alterations in alpha-diversity, community membership and structure and the enrichment of different taxa when compared to fecal samples immediately frozen at −80 °C.

scholarly journals Comparative analysis of amplicon and metagenomic sequencing methods reveals key features in the evolution of animal metaorganisms

Microbiome ◽  
2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Philipp Rausch ◽  
Malte Rühlemann ◽  
Britt M. Hermes ◽  
Shauni Doms ◽  
Tal Dagan ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Microbial Communities ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Microbial Composition ◽  
Evolutionary Patterns ◽  
Trade Offs ◽  
Wide Range ◽  
Taxonomic Groups

Abstract Background The interplay between hosts and their associated microbiome is now recognized as a fundamental basis of the ecology, evolution, and development of both players. These interdependencies inspired a new view of multicellular organisms as “metaorganisms.” The goal of the Collaborative Research Center “Origin and Function of Metaorganisms” is to understand why and how microbial communities form long-term associations with hosts from diverse taxonomic groups, ranging from sponges to humans in addition to plants. Methods In order to optimize the choice of analysis procedures, which may differ according to the host organism and question at hand, we systematically compared the two main technical approaches for profiling microbial communities, 16S rRNA gene amplicon and metagenomic shotgun sequencing across our panel of ten host taxa. This includes two commonly used 16S rRNA gene regions and two amplification procedures, thus totaling five different microbial profiles per host sample. Conclusion While 16S rRNA gene-based analyses are subject to much skepticism, we demonstrate that many aspects of bacterial community characterization are consistent across methods. The resulting insight facilitates the selection of appropriate methods across a wide range of host taxa. Overall, we recommend single- over multi-step amplification procedures, and although exceptions and trade-offs exist, the V3 V4 over the V1 V2 region of the 16S rRNA gene. Finally, by contrasting taxonomic and functional profiles and performing phylogenetic analysis, we provide important and novel insight into broad evolutionary patterns among metaorganisms, whereby the transition of animals from an aquatic to a terrestrial habitat marks a major event in the evolution of host-associated microbial composition.

scholarly journals Dissimilarity of microbial diversity of pond water, shrimp intestine and sediment in Aquamimicry system

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Shenzheng Zeng ◽  
Sukontorn Khoruamkid ◽  
Warinphorn Kongpakdee ◽  
Dongdong Wei ◽  
Lingfei Yu ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Well Being ◽  
Pacific White Shrimp ◽  
Shrimp Farming ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Shrimp Culture ◽  
Water And Sediment ◽  
Surrounding Environment ◽  
Shrimp Production

Abstract The Pacific white shrimp, with the largest production in shrimp industry, has suffered from multiple severe viral and bacterial diseases, which calls for a more reliable and environmentally friendly system to promote shrimp culture. The “Aquamimicry system”, mimicking the nature of aquatic ecosystems for the well-being of aquatic animals, has effectively increased shrimp production and been adapted in many countries. However, the microbial communities in the shrimp intestine and surrounding environment that act as an essential component in Aquamimicry remain largely unknown. In this study, the microbial composition and diversity alteration in shrimp intestine, surrounding water and sediment at different culture stages were investigated by high throughput sequencing of 16S rRNA gene, obtaining 13,562 operational taxonomic units (OTUs). Results showed that the microbial communities in shrimp intestine and surrounding environment were significantly distinct from each other, and 23 distinguished taxa for each habitat were further characterized. The microbial communities differed significantly at different culture stages, confirmed by a great number of OTUs dramatically altered during the culture period. A small part of these altered OTUs were shared between shrimp intestine and surrounding environment, suggesting that the microbial alteration of intestine was not consistent with that of water and sediment. Regarding the high production of Aquamimicry farm used as a case in this study, the dissimilarity between intestinal and surrounding microbiota might be considered as a potential indicator for healthy status of shrimp farming, which provided hints on the appropriate culture practices to improve shrimp production.

scholarly journals A Metagenomics Investigation of Intergenerational Effects of Non-nutritive Sweeteners on Gut Microbiome

2022 ◽  
Vol 8 ◽  
Author(s):  
Weilan Wang ◽  
Jodi E. Nettleton ◽  
Michael G. Gänzle ◽  
Raylene A. Reimer
Keyword(s):  
Body Weight ◽  
Body Fat ◽  
Body Weight Gain ◽  
Lactate Production ◽  
16S Rrna Gene Sequencing ◽  
Liver Weight ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Microbial Composition ◽  
Maternal Consumption

To identify possible mechanisms by which maternal consumption of non-nutritive sweeteners increases obesity risk in offspring, we reconstructed the major alterations in the cecal microbiome of 3-week-old offspring of obese dams consuming high fat/sucrose (HFS) diet with or without aspartame (5–7 mg/kg/day) or stevia (2–3 mg/kg/day) by shotgun metagenomic sequencing (n = 36). High throughput 16S rRNA gene sequencing (n = 105) was performed for dams, 3- and 18-week-old offspring. Maternal consumption of sweeteners altered cecal microbial composition and metabolism of propionate/lactate in their offspring. Offspring daily body weight gain, liver weight and body fat were positively correlated to the relative abundance of key microbes and enzymes involved in succinate/propionate production while negatively correlated to that of lactose degradation and lactate production. The altered propionate/lactate production in the cecum of weanlings from aspartame and stevia consuming dams implicates an altered ratio of dietary carbohydrate digestion, mainly lactose, in the small intestine vs. microbial fermentation in the large intestine. The reconstructed microbiome alterations could explain increased offspring body weight and body fat. This study demonstrates that intense sweet tastants have a lasting and intergenerational effect on gut microbiota, microbial metabolites and host health.

scholarly journals Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

2021 ◽  
Author(s):  
Seppo Virtanen ◽  
Schahzad Saqib ◽  
Tinja Kanerva ◽  
Pekka Nieminen ◽  
Ilkka Kalliala ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Cost Effective ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Extensive Literature ◽  
Metagenomic Sequencing ◽  
Text Extraction ◽  
Healthy Women ◽  
Bacteria And Fungi

Abstract Background: Amplicon sequencing of kingdom-specific tags such as 16S rRNA gene for bacteria and internal transcribed spacer (ITS) region for fungi are widely used for investigating microbial populations. So far most human studies have focused on bacteria while studies on host-associated fungi in health and disease have only recently started to accumulate. To enable cost-effective parallel analysis of bacterial and fungal communities in human and environmental samples, we developed a method where 16S rRNA gene and ITS-1 amplicons were pooled together for a single Illumina MiSeq or HiSeq run and analysed after primer-based segregation. Taxonomic assignments were performed with Blast in combination with an iterative text-extraction based filtration approach, which uses extensive literature records from public databases to select the most probable hits that were further validated by shotgun metagenomic sequencing. Results: Using 50 vaginal samples, we show that the combined run provides comparable results on bacterial composition and diversity to conventional 16S rRNA gene amplicon sequencing. The text-extraction-based taxonomic assignment guided tool provided ecosystem specific annotations that were confirmed by Metagenomic Phylogenetic Analysis (MetaPhlAn). The metagenome analysis revealed distinct functional differences between the bacterial community types while fungi were undetected, despite being identified in all samples based on ITS amplicons. Co-abundance analysis of bacteria and fungi did not show strong between-kingdom correlations within the vaginal ecosystem of healthy women.Conclusion: Combined amplicon sequencing for bacteria and fungi provides a simple and cost-effective method for simultaneous analysis of microbiota and mycobiota within the same samples. Text extraction-based annotation tool facilitates the characterization and interpretation of defined microbial communities from rapidly accumulating sequencing and metadata readily available through public databases.

scholarly journals Human gut microbial communities dictate efficacy of anti-PD-1 therapy in a humanized microbiome mouse model of glioma

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Kory J Dees ◽  
Hyunmin Koo ◽  
J Fraser Humphreys ◽  
Joseph A Hakim ◽  
David K Crossman ◽  
...  
Keyword(s):  
Mouse Model ◽  
Microbial Communities ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Microbial Composition ◽  
Healthy Human ◽  
Fecal Transplantation ◽  
Host Interaction ◽  
Shotgun Metagenomic Sequencing ◽  
Mouse Lines

Abstract Background Although immunotherapy works well in glioblastoma (GBM) preclinical mouse models, the therapy has not demonstrated efficacy in humans. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a preclinical mouse model of GBM. Methods We used 5 healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate 5 independent humanized mouse lines (HuM1-HuM5). Results Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. All HuM mouse lines were susceptible to GBM transplantation, and exhibited similar median survival ranging from 19 to 26 days. Interestingly, we found that HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM1, HuM4, and HuM5 mice are nonresponders to anti-PD-1, while HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls. Bray-Curtis cluster analysis of the 5 HuM gut microbial communities revealed that responders HuM2 and HuM3 were closely related, and detailed taxonomic comparison analysis revealed that Bacteroides cellulosilyticus was commonly found in HuM2 and HuM3 with high abundances. Conclusions The results of our study establish the utility of humanized microbiome mice as avatars to delineate features of the host interaction with gut microbial communities needed for effective immunotherapy against GBM.

IMMU-09. HUMAN MICROBIOTA INFLUENCE THE EFFICACY OF IMMUNOTHERAPY IN A MOUSE MODEL OF GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi93-vi94
Author(s):  
Kory Dees ◽  
Hyunmin Koo ◽  
James Humphreys ◽  
Joseph Hakim ◽  
David Crossman ◽  
...  
Keyword(s):  
Mouse Model ◽  
Microbial Communities ◽  
Mouse Models ◽  
Gut Microbiome ◽  
Positive Response ◽  
Checkpoint Inhibitors ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Microbial Composition ◽  
Healthy Human

Abstract Although immunotherapy works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has unfortunately not demonstrated efficacy in humans. In melanoma and other cancers, the composition of the gut microbiome has been shown to determine responsiveness or resistance to immune checkpoint inhibitors (anti-PD-1). Most pre-clinical cancer studies have been done in mouse models using mouse gut microbiomes, but there are significant differences between mouse and human microbial gut compositions. To address this inconsistency, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a pre-clinical mouse model of GBM. We used five healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate five independent humanized mouse lines (HuM1-HuM5). Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. Interestingly, we found that the HuM lines responded differently to anti-PD-1. Specifically, we demonstrate that HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls, while HuM1, HuM4, and HuM5 mice are resistant to anti-PD-1. These mice are genetically identical, and only differ in the composition of the gut microbiome. In a correlative experiment, we found that disrupting the responder HuM2 microbiome with antibiotics abrogated the positive response to anti-PD-1, indicating that HuM2 microbiota must be present in the mice to elicit the positive response to anti-PD-1 in the GBM model. The question remains of whether the “responsive” microbial communities in HuM2 and HuM3 can be therapeutically exploited and applicable in other tumor models, or if the “resistant” microbial communities in HuM1, HuM4, and HuM5 can be depleted and/or replaced. Future studies will assess responder microbial transplants as a method of enhancing immunotherapy.

scholarly journals Energy-Efficient Single-Stage Nitrite Shunt Denitrification with Saline Sewage through Concise Dissolved Oxygen (DO) Supply: Process Performance and Microbial Communities

2020 ◽  
Vol 8 (6) ◽  
pp. 919 ◽  
Author(s):  
Huichuan Zhuang ◽  
Zhuoying Wu ◽  
Linji Xu ◽  
Shao-Yuan Leu ◽  
Po-Heng Lee
Keyword(s):  
Dissolved Oxygen ◽  
Microbial Communities ◽  
Energy Efficient ◽  
Oxygen Demand ◽  
Operating Conditions ◽  
Single Stage ◽  
Rrna Gene ◽  
Unexpected Finding ◽  
Sequencing Analysis ◽  
Ratio Requirement

Single-stage nitrite shunt denitrification (through nitrite rather than nitrate) with low dissolved oxygen (DO) supply is a better alternative in terms of energy-efficiency, short-footprint, and low C/N-ratio requirement. This study investigates the optimal DO level with temperature effect, with saline sewage at the fixed hydraulic and solids retention times of 8 h and 8 d, respectively. Moreover, 16S rRNA gene sequencing analysis corresponding with total nitrogen (TN) and chemical oxygen demand (COD) removals in each operating condition were performed. Results showed that DO of 0.3 mg/L at 20 °C achieved over 60.7% and over 97.9% of TN and COD removal, respectively, suggesting that such condition achieved effective nitrite-oxidizing bacteria inhibition and efficient denitrification. An unexpected finding was that sulfur-reducing Haematobacter and nitrogen-fixing Geofilum and Shinella were highly abundant with the copredominance of ammonia-oxidizing Comamonas and Nitrosomonas, nitrite-oxidizing Limnohabitans, and denitrifying Simplicispira, Castellaniella, and Nitratireductor. Further, canonical correspondence analysis (CCA) with respect to the operating conditions associated with phenotype prediction via R-based tool Tax4Fun was performed for a preliminary diagnosis of microbial functionality. The effects of DO, temperature, nitrite, and nitrate in various extents toward each predominant microbe were discussed. Collectively, DO is likely pivotal in single-stage nitrite shunt denitrification, as well as microbial communities, for energy-efficient saline sewage treatment.

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