scholarly journals Guanidine thiocyanate solution facilitates sample collection for plant rhizosphere microbiome analysis

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6440
Author(s):  
Xiaoxiao Sun ◽  
Meiling Wang ◽  
Lin Guo ◽  
Changlong Shu ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Large Scale ◽  
Room Temperature ◽  
Quantitative Polymerase Chain Reaction ◽  
Alpha Diversity ◽  
Operational Taxonomic Unit ◽  
Rrna Gene ◽  
Guanidine Thiocyanate ◽  
Sample Collection ◽  
Thiocyanate Solution ◽  
Health And Development

The interactions between rhizosphere microorganisms and plants are important for the health and development of crops. Analysis of plant rhizosphere bacterial compositions, particularly of those with resistance to biotic/abiotic stresses, may improve their applications in sustainable agriculture. Large-scale rhizosphere samplings in the field are usually required; however, such samples, cannot be immediately frozen. We found that the storage of samples at room temperature for 2 days leads to a considerable reduction in the operational taxonomic unit (OTU) number and the indices of bacterial alpha-diversity of rhizosphere communities. In this study, in order to overcome these problems, we established a method using guanidine thiocyanate (GTC) solution for the preservation of rhizosphere samples after their collection. This method allowed the maintenance of the samples for at least 1 day at room temperature prior to their cryopreservation and was shown to be compatible with conventional DNA isolation protocols. Illumina sequencing of V3 and V4 hypervariable regions of the 16S rRNA gene was used to assess the feasibility and reliability of this method, and no significant differences were observed in the number of OTUs and in the Chao and Shannon indices between samples stored at −70 °C and those stored in GTC solution. Moreover, the representation of Pseudomonas spp. in samples stored in GTC solution was not significantly different from that in samples stored at −70 °C, as determined by real-time quantitative polymerase chain reaction (p > 0.05). Both types of samples were shown to cluster together according to principal coordinate analysis. Furthermore, GTC solution did not affect the bacterial taxon profiles at different storage periods compared with those observed when storing the samples below −70 °C. Even incubation of thawed samples (frozen at −70 °C) for 15 min at room temperature induced minor changes in the bacterial composition. Taken together, our results demonstrated that GTC solution may provide a reliable alternative for the preservation of rhizosphere samples in the field.

scholarly journals The colorectal cancer-associated faecal microbiome of developing countries resembles that of developed countries

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Caroline Young ◽  
Henry M. Wood ◽  
Ramakrishnan Ayloor Seshadri ◽  
Pham Van Nang ◽  
Carlos Vaccaro ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Developing Countries ◽  
Large Scale ◽  
Room Temperature ◽  
16S Rrna Gene Sequencing ◽  
Occult Blood ◽  
Developed Countries ◽  
Rrna Gene ◽  
Sample Collection ◽  
Faecal Microbiome

Abstract Background The incidence of colorectal cancer (CRC) is increasing in developing countries, yet limited research on the CRC- associated microbiota has been conducted in these areas, in part due to scarce resources, facilities, and the difficulty of fresh or frozen stool storage/transport. Here, we aimed (1) to establish a broad representation of diverse developing countries (Argentina, Chile, India, and Vietnam); (2) to validate a ‘resource-light’ sample-collection protocol translatable in these settings using guaiac faecal occult blood test (gFOBT) cards stored and, importantly, shipped internationally at room temperature; (3) to perform initial profiling of the collective CRC-associated microbiome of these developing countries; and (4) to compare this quantitatively with established CRC biomarkers from developed countries. Methods We assessed the effect of international storage and transport at room temperature by replicating gFOBT from five UK volunteers, storing two in the UK, and sending replicates to institutes in the four countries. Next, to determine the effect of prolonged UK storage, DNA extraction replicates for a subset of samples were performed up to 252 days apart. To profile the CRC-associated microbiome of developing countries, gFOBT were collected from 41 treatment-naïve CRC patients and 40 non-CRC controls from across the four institutes, and V4 16S rRNA gene sequencing was performed. Finally, we constructed a random forest (RF) model that was trained and tested against existing datasets from developed countries. Results The microbiome was stably assayed when samples were stored/transported at room temperature and after prolonged UK storage. Large-scale microbiome structure was separated by country and continent, with a smaller effect from CRC. Importantly, the RF model performed similarly to models trained using external datasets and identified similar taxa of importance (Parvimonas, Peptostreptococcus, Fusobacterium, Alistipes, and Escherichia). Conclusions This study demonstrates that gFOBT, stored and transported at room temperature, represents a suitable method of faecal sample collection for amplicon-based microbiome biomarkers in developing countries and suggests a CRC-faecal microbiome association that is consistent between developed and developing countries.

Effect of rice bran fermented with Saccharomyces cerevisiae and Lactobacillus plantarum on gut microbiome of mice fed high-sucrose diet

2019 ◽  
Vol 10 (7) ◽  
pp. 811-821
Author(s):  
J. Shibayama ◽  
M. Goto ◽  
T. Kuda ◽  
M. Fukunaga ◽  
H. Takahashi ◽  
...  
Keyword(s):  
Bile Acid ◽  
Rice Bran ◽  
Gut Microbiome ◽  
Control Diet ◽  
Alpha Diversity ◽  
Blood Lipid ◽  
Operational Taxonomic Unit ◽  
Rrna Gene ◽  
High Sucrose

To clarify the effect of rice bran (RB) and fermented RB (FRB) in a high-sucrose and low-dietary fibre diet on the gut microbiome, the in vitro bile acid-lowering capacity and caecal microbiota of ICR mice fed with 20% RB or FRB diets for two weeks were determined. The caecal microbiome was analysed by 16S rRNA gene amplicon sequencing. The in vitro bile acid-lowering capacity was high for FRB. In mouse experiments, triacylglycerol and total cholesterol were generally lower with FRB, although the faecal frequency was highest in mice fed with RB. The Shannon-Wiener and Simpson’s indices for alpha-diversity in the microbiome of mice fed with RB and FRB, were higher than mice fed the control diet. At the phylum level in the caecal microbiome, Firmicutes and Bacteroidetes were high with FRB and RB, respectively. At the operational taxonomic unit level, some bacterial groups related to diabetes and gut toxicity, such as Lachnospiraceae and Enterorhabdus mucosicola, were high for RB but not for FRB diets. These results suggest that FRB, rather than RB, intake improve the intestinal environment and blood lipid condition.

scholarly journals Minor compositional alterations in faecal microbiota after five weeks and five months storage at room temperature on filter papers

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sebastian von Huth ◽  
Louise Bruun Thingholm ◽  
Corinna Bang ◽  
Malte C. Rühlemann ◽  
Andre Franke ◽  
...  
Keyword(s):  
Room Temperature ◽  
Alpha Diversity ◽  
16S Rrna Gene Sequencing ◽  
Occult Blood ◽  
Faecal Occult Blood Test ◽  
Rrna Gene ◽  
Long Term Storage ◽  
Fresh Frozen ◽  
Rrna Gene Sequencing ◽  
Filter Papers

AbstractThe gut microbiota is recognized as having major impact in health and disease. Sample storage is an important aspect to obtain reliable results. Mostly recommended is immediate freezing, however, this is not always feasible. Faecal occult blood test (FOBT) papers are an appealing solution in such situations, and most studies find these to be applicable, showing no major changes within 7 days storage at room temperature (RT). As fieldwork often requires RT storage for longer periods, evaluation of this is warranted. We performed 16S rRNA gene sequencing of 19 paired faecal samples immediately frozen or kept five weeks and five months at RT on FOBT papers. Alpha-diversity evaluation revealed no effect of FOBT storage, and evaluation of beta-diversity showed that host explained 65% of community variation, while storage method explained 5%. Evaluation of community dispersion and the Firmicutes/Bacteroidetes ratio revealed a larger effect of storage time for fresh-frozen samples. Single taxa evaluation (order-to-genus level) showed significant alterations of four (of 37) genera after five weeks and five genera after five months. When comparing the two timepoints, alterations were only detectable for fresh-frozen samples. Our findings reveal that long term storage on FOBT papers is an applicable approach for microbiota research.

scholarly journals High stability of faecal microbiome composition in guanidine thiocyanate solution at room temperature and robustness during colonoscopy

Gut ◽  
2016 ◽  
Vol 65 (9) ◽  
pp. 1574-1575 ◽  
Author(s):  
Yuichiro Nishimoto ◽  
Sayaka Mizutani ◽  
Takeshi Nakajima ◽  
Fumie Hosoda ◽  
Hikaru Watanabe ◽  
...  
Keyword(s):  
High Stability ◽  
Room Temperature ◽  
Guanidine Thiocyanate ◽  
Microbiome Composition ◽  
Thiocyanate Solution ◽  
Faecal Microbiome

scholarly journals Impact of inter- and intra-individual variation, sample storage and sampling fraction on human stool microbial community profiles

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6172 ◽  
Author(s):  
Yun Kit Yeoh ◽  
Zigui Chen ◽  
Mamie Hui ◽  
Martin C.S. Wong ◽  
Wendy C.S. Ho ◽  
...  
Keyword(s):  
Microbial Community ◽  
Community Composition ◽  
Individual Variation ◽  
Large Scale ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
Relative Effect ◽  
Individual Variability ◽  
Rrna Gene ◽  
Sample Collection

Stools are commonly used as proxies for studying human gut microbial communities as sample collection is straightforward, cheap and non-invasive. In large-scale human population surveys, however, sample integrity becomes an issue as it is not logistically feasible for researchers to personally collect stools from every participant. Instead, participants are usually given guidelines on sample packaging and storage, and asked to deliver their stools to a centralised facility. Here, we tested a number of delivery conditions (temperature, duration and addition of preservative medium) and assessed their effects on stool microbial community composition using 16S rRNA gene amplicon sequencing. The largest source of variability in stool community composition was attributable to inter-individual differences regardless of delivery condition. Although the relative effect of delivery condition on community composition was small compared to inter-individual variability (1.6% vs. 60.5%, permutational multivariate analysis of variance [PERMANOVA]) and temporal variation within subjects over 10 weeks (5.2%), shifts in microbial taxa associated with delivery conditions were non-systematic and subject-specific. These findings indicated that it is not possible to model or accurately predict shifts in stool community composition associated with sampling logistics. Based on our findings, we recommend delivery of fresh, preservative-free stool samples to laboratories within 2 hr either at ambient or chilled temperatures to minimise perturbations to microbial community composition. In addition, subsamples from different fractions of the same stool displayed a small (3.3% vs. 72.6% inter-individual variation, PERMANOVA) but significant effect on community composition. Collection of larger sample volumes for homogenisation is recommended.

scholarly journals Resident microbes of lactation rooms and daycares

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8168
Author(s):  
Diana H. Taft ◽  
Samir Akre ◽  
Nicolas Madrid ◽  
Andre Knoesen ◽  
David A. Mills ◽  
...  
Keyword(s):  
Beta Diversity ◽  
Alpha Diversity ◽  
Illumina Miseq ◽  
Shannon Index ◽  
Rrna Gene ◽  
Sample Collection ◽  
Unifrac Distance ◽  
Modern Development ◽  
Temperature And Humidity ◽  
The Impact

Dedicated lactation rooms are a modern development as mothers return to work while still providing breastmilk to their absent infants. This study describes the built environment microbiome of lactation rooms and daycares, and explores the influence of temperature and humidity on the microbiome of lactation rooms. Sterile swabs were used to collect samples from five different sites in lactation rooms at University of California, Davis and from five different sites in daycares located in Davis, California. DNA from the swabs was extracted and the V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Temperature and relative humidity data were collected on a subset of the lactation rooms. Sampled lactation rooms could be either dedicated lactation rooms or could also serve other functions (e.g., combined lactation room and restroom lounge). The majority of sequence reads were identified as belonging to family Moraxellaceae, with 73% of all reads included in analysis identified as an unknown species of Acinetobacter. Alpha diversity was analyzed using the Shannon index, while beta diversity was analyzed using unweighted and weighted UniFrac distance. The Jaccard distance was used to measure amount of change at sampling locations between time points for analysis of the impact of temperature and humidity on the microbiome. There were significant differences in the beta diversity of the microbiome of lactation rooms by room type. There were also significant differences in the beta diversity of the microbiome by sample collection location. There were no significant differences in either alpha or beta diversity associated with room temperature or humidity. Additional studies are needed to understand if the differences in lactation room type may result in differences in the breastmilk microbiome of milk collected in those rooms, and to what extent any such differences may influence the infant microbiome.

scholarly journals Diet induces reproducible alterations in the mouse and human gut microbiome

2019 ◽  
Author(s):  
Jordan E. Bisanz ◽  
Vaibhav Upadhyay ◽  
Jessie A. Turnbaugh ◽  
Kimberly Ly ◽  
Peter J. Turnbaugh
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Meta Analysis ◽  
Alpha Diversity ◽  
Operational Taxonomic Unit ◽  
Molecular Testing ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Dietary Interventions

SummaryThe degree to which diet reproducibly alters the human and mouse gut microbiota remains unclear. Here, we focus on the consumption of a high-fat diet (HFD), one of the most frequently studied dietary interventions in mice. We employed a subject-level meta-analysis framework for unbiased collection and analysis of publicly available 16S rRNA gene and metagenomic sequencing data from studies examining HFD in rodent models. In total, we re-analyzed 27 studies, 1101 samples, and 106 million reads mapping to 16S rRNA gene sequences. We report reproducible changes in gut microbial community structure both within and between studies, including a significant increase in the Firmicutes phylum and decrease in the Bacteroidetes phylum; however, reduced alpha diversity is not a consistent feature of HFD. Finer taxonomic analysis revealed that the strongest signal of HFD on microbiota species composition is Lactococcus spp., which we demonstrate is a common dietary contaminant through the molecular testing of dietary ingredients, culturing, microscopy, and germ-free mouse experiments. After in silico removal of Lactococcus spp., we employed machine learning to define a unique operational taxonomic unit (OTU)-based signature capable of predicting the dietary intake of mice and demonstrate that phylogenetic and gene-family transformations of this model are capable of accurately predicting human samples in controlled feeding settings (area under the receiver operator curve = 0.75 and 0.88 respectively). Together, these results demonstrate the utility of microbiome meta-analyses in identifying robust bacterial signals for mechanistic studies and creates a framework for the routine meta-analysis of microbiome studies in preclinical models.

scholarly journals Rare Taxa Maintain Microbial Diversity and Contribute to Terrestrial Community Dynamics throughout Bark Beetle Infestation

2016 ◽  
Vol 82 (23) ◽  
pp. 6912-6919 ◽  
Author(s):  
Kristin M. Mikkelson ◽  
Chelsea M. Bokman ◽  
Jonathan O. Sharp
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Bark Beetle ◽  
Large Scale ◽  
Tree Mortality ◽  
Community Dynamics ◽  
Alpha Diversity ◽  
Biogeochemical Cycling ◽  
Rrna Gene ◽  
Edaphic Parameters

ABSTRACTA global phenomenon of increasing bark beetle-induced tree mortality has heightened concerns regarding ecosystem response and biogeochemical implications. Here, we explore microbial dynamics under lodgepole pines through the analysis of bulk (16S rRNA gene) and potentially active (16S rRNA) communities to understand the terrestrial ecosystem responses that are associated with this form of large-scale tree mortality. We found that the relative abundances of bulk and potentially active taxa were correlated across taxonomic levels, but at lower levels, cladal differences became more apparent. Despite this correlation, there was a strong differentiation of community composition between bulk and potentially active taxa, with further clustering associated with the stages of tree mortality. Surprisingly, community clustering as a function of tree phase had limited correlation to soil water content and total nitrogen concentrations, which were the only two measured edaphic parameters to differ in association with tree phase. Bacterial clustering is more readily explained by the observed decrease in the abundance of active, rare microorganisms after tree death in conjunction with stable alpha diversity measurements. This enables the rare fraction of the terrestrial microbial community to maintain metabolic diversity by transitioning between metabolically active and dormant states during this ecosystem disturbance and contributes disproportionately to community dynamics and archived metabolic capabilities. These results suggest that analyzing bulk and potentially active communities after beetle infestation may be a more sensitive indicator of disruption than measuring local edaphic parameters.IMPORTANCEForests around the world are experiencing unprecedented mortality due to insect infestations that are fueled in part by a changing climate. While aboveground processes have been explored, changes at the terrestrial interface that are relevant to microbial biogeochemical cycling remain largely unknown. In this study, we investigated the changing bulk and potentially active microbial communities beneath healthy and beetle-killed trees. We found that, even though few edaphic parameters were altered from beetle infestation, the rare microbes were more likely to be active and fluctuate between dormancy and metabolic activity. This indicates that rare as opposed to abundant taxa contribute disproportionately to microbial community dynamics and presumably biogeochemical cycling within these types of perturbed ecosystems.

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