Impact of Ambient Temperature Sample Storage on the Equine Fecal Microbiota

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.

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The vaginal and fecal microbiomes are related to pregnancy status in beef heifers

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-019-0401-2 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Feilong Deng ◽

Maryanna McClure ◽

Rick Rorie ◽

Xiaofan Wang ◽

Jianmin Chai ◽

...

Keyword(s):

Illumina Miseq ◽

First Trimester ◽

Fecal Microbiota ◽

Rrna Gene ◽

Beef Heifers ◽

Microbial Composition ◽

Fecal Samples ◽

Fecal Microbiome ◽

Bovine Reproduction ◽

Pregnancy Status

Abstract Background The greatest impact on profitability of a commercial beef operation is reproduction. However, in beef heifers, little is known about the vaginal and fecal microbiota with respect to their relationship with fertility. To this end, we followed heifers through gestation to examine the dynamics of vaginal and fecal microbial composition throughout pregnancy. Results Heifers were exposed to an estrus synchronization protocol, observed over a 12-day period, artificially inseminated 12 h to 18 h after observed estrus, and subsequently exposed to bulls for a 50-day breeding season. Vaginal samples were taken at pre-breeding (n = 72), during the first (n = 72), and second trimester (n = 72) for all individuals, and third trimester for individuals with confirmed pregnancies (n = 56). Fecal samples were taken at pre-breeding (n = 32) and during the first trimester (n = 32), including bred and open individuals. Next generation sequencing of the V4 region of the16S rRNA gene via the Illumina MiSeq platform was applied to all samples. Shannon indices and the number of observed bacterial features were the same in fecal samples. However, significant differences in vaginal microbiome diversity between gestation stages were observed. No differences in beta-diversity were detected in vaginal or fecal samples regarding pregnancy status, but such differences were seen with fecal microbiome over time. Random Forest was developed to identify predictors of pregnancy status in vaginal (e.g., Histophilus, Clostridiaceae, Campylobacter) and fecal (e.g., Bacteroidales, Dorea) samples. Conclusions Our study shows that bovine vaginal and fecal microbiome could be used as biomarkers of bovine reproduction. Further experiments are needed to validate these biomarkers and to examine their roles in a female’s ability to establish pregnancy.

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Analysis of the developing gut microbiota in young dairy calves—impact of colostrum microbiota and gut disturbances

Tropical Animal Health and Production ◽

10.1007/s11250-020-02535-9 ◽

2020 ◽

Vol 53 (1) ◽

Author(s):

Bui Phan Thu Hang ◽

Ewa Wredle ◽

Johan Dicksved

Keyword(s):

Average Daily Gain ◽

Amplicon Sequencing ◽

Fecal Microbiota ◽

Dairy Calves ◽

Rrna Gene ◽

Microbiota Composition ◽

Microbial Composition ◽

Fecal Samples ◽

Calf Health ◽

Significant Difference

AbstractThe aim of this study was to characterize the colostrum and fecal microbiota in calves and to investigate whether fecal microbiota composition was related to colostrum microbiota or factors associated with calf health. Colostrum samples were collected in buckets after hand milking of 76 calving cows from 38 smallholder dairy farms. Fecal samples were taken directly from the rectum of 76 calves at birth and at 14 days age. The bacterial community structure in colostrum and feces was analyzed by terminal restriction fragment length polymorphism for all samples, and the microbial composition was determined by 16S rRNA gene amplicon sequencing for a subset of the samples (8 colostrum, 40 fecal samples). There was a significant difference in fecal microbiota composition between day 0 and day 14 samples, but no associations between the microbiota and average daily gain, birth weight, or transfer of passive immunity. At 14 days of age, Faecalibacterium and Butyricicoccus were prevalent in higher relative abundances in the gut of healthy calves compared to calves with diarrhea that had been treated with antimicrobials. Colostrum showed great variation in composition of microbiota but no association to fecal microbiota. This study provides the first insights into the composition of colostrum and fecal microbiota of young dairy calves in southern Vietnam and can form the basis for future more detailed studies.

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

mSystems ◽

10.1128/msystems.01329-20 ◽

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.

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Fecal microbiota dynamics during disease activity and remission in newly diagnosed and established ulcerative colitis

Scientific Reports ◽

10.1038/s41598-021-87973-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lena Öhman ◽

Anders Lasson ◽

Anna Strömbeck ◽

Stefan Isaksson ◽

Marcus Hesselmar ◽

...

Keyword(s):

Ulcerative Colitis ◽

Gut Microbiota ◽

Disease Activity ◽

Temporal Dynamics ◽

Fecal Microbiota ◽

Disease Stage ◽

Dietary Interventions ◽

Microbial Composition ◽

Fecal Samples ◽

Specific Species

AbstractPatients with ulcerative colitis (UC) have an altered gut microbiota composition, but the microbial relationship to disease activity needs to be further elucidated. Therefore, temporal dynamics of the fecal microbial community during remission and flare was determined. Fecal samples were collected at 2–6 time-points from UC patients during established disease (cohort EST) and at diagnosis (cohort NEW). Sampling range for cohort EST was 3–10 months and for cohort NEW 36 months. Relapses were monitored for an additional three years for cohort EST. Microbial composition was assessed by Genetic Analysis GA-map Dysbiosis Test, targeting ≥ 300 bacteria. Eighteen patients in cohort EST (8 with maintained remission and 10 experiencing a flare), provided 71 fecal samples. In cohort NEW, 13 patients provided 49 fecal samples. The microbial composition showed no clustering related to disease activity in any cohort. Microbial dissimilarity was higher between than within patients for both cohorts, irrespective of presence of a flare. Microbial stability within patients was constant over time with no major shift in overall composition nor modification in the abundance of any specific species. Microbial composition was not affected by intensified medical treatment or linked to future disease course. Thus in UC, the gut microbiota is highly stable irrespective of disease stage, disease activity or treatment escalation. This suggests that prolonged dietary interventions or repeated fecal transplantations are needed to be able to induce permanent alterations of the gut microbiota.

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Bioconductor workflow for microbiome data analysis: from raw reads to community analyses

F1000Research ◽

10.12688/f1000research.8986.1 ◽

2016 ◽

Vol 5 ◽

pp. 1492 ◽

Cited By ~ 148

Author(s):

Ben J. Callahan ◽

Kris Sankaran ◽

Julia A. Fukuyama ◽

Paul J. McMurdie ◽

Susan P. Holmes

Keyword(s):

High Throughput Sequencing ◽

Linear Models ◽

Reference Database ◽

Rrna Gene ◽

Microbial Composition ◽

Nonparametric Testing ◽

Abundance Estimates ◽

Taxonomic Markers ◽

Microbiome Data ◽

Microbiome Data Analysis

High-throughput sequencing of PCR-amplified taxonomic markers (like the 16S rRNA gene) has enabled a new level of analysis of complex bacterial communities known as microbiomes. Many tools exist to quantify and compare abundance levels or microbial composition of communities in different conditions. The sequencing reads have to be denoised and assigned to the closest taxa from a reference database. Common approaches use a notion of 97% similarity and normalize the data by subsampling to equalize library sizes. In this paper, we show that statistical models allow more accurate abundance estimates. By providing a complete workflow in R, we enable the user to do sophisticated downstream statistical analyses, including both parameteric and nonparametric methods. We provide examples of using the R packages dada2, phyloseq, DESeq2, ggplot2 and vegan to filter, visualize and test microbiome data. We also provide examples of supervised analyses using random forests, partial least squares and linear models as well as nonparametric testing using community networks and the ggnetwork package.

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Probiotic Supplementation in a Clostridium difficile-Infected Gastrointestinal Model Is Associated with Restoring Metabolic Function of Microbiota

Microorganisms ◽

10.3390/microorganisms8010060 ◽

2019 ◽

Vol 8 (1) ◽

pp. 60

Author(s):

Mohd Baasir Gaisawat ◽

Chad W. MacPherson ◽

Julien Tremblay ◽

Amanda Piano ◽

Michèle M. Iskandar ◽

...

Keyword(s):

Gut Microbiota ◽

Alpha Diversity ◽

Gastrointestinal Disorder ◽

Short Chain Fatty Acids ◽

Rrna Gene ◽

Metabolic Function ◽

Microbial Composition ◽

Single Strain ◽

Fecal Samples ◽

Metabolic Functions

Clostridium (C.) difficile-infection (CDI), a nosocomial gastrointestinal disorder, is of growing concern due to its rapid rise in recent years. Antibiotic therapy of CDI is associated with disrupted metabolic function and altered gut microbiota. The use of probiotics as an adjunct is being studied extensively due to their potential to modulate metabolic functions and the gut microbiota. In the present study, we assessed the ability of several single strain probiotics and a probiotic mixture to change the metabolic functions of normal and C. difficile-infected fecal samples. The production of short-chain fatty acids (SCFAs), hydrogen sulfide (H2S), and ammonia was measured, and changes in microbial composition were assessed by 16S rRNA gene amplicon sequencing. The C. difficile-infection in fecal samples resulted in a significant decrease (p < 0.05) in SCFA and H2S production, with a lower microbial alpha diversity. All probiotic treatments were associated with significantly increased (p < 0.05) levels of SCFAs and restored H2S levels. Probiotics showed no effect on microbial composition of either normal or C. difficile-infected fecal samples. These findings indicate that probiotics may be useful to improve the metabolic dysregulation associated with C. difficile infection.

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Effect of Selected Stilbenoids on Human Fecal Microbiota

Molecules ◽

10.3390/molecules24040744 ◽

2019 ◽

Vol 24 (4) ◽

pp. 744 ◽

Cited By ~ 6

Author(s):

Jose Jaimes ◽

Veronika Jarosova ◽

Ondrej Vesely ◽

Chahrazed Mekadim ◽

Jakub Mrazek ◽

...

Keyword(s):

Gut Microbiota ◽

Relative Abundance ◽

Statistical Tests ◽

16S Rrna Gene Sequencing ◽

Fecal Microbiota ◽

Rrna Gene ◽

Microbial Composition ◽

Genus Clostridium ◽

The Family ◽

Rrna Gene Sequencing

Dietary phenolics or polyphenols are mostly metabolized by the human gut microbiota. These metabolites appear to confer the beneficial health effects attributed to phenolics. Microbial composition affects the type of metabolites produced. Reciprocally, phenolics modulate microbial composition. Understanding this relationship could be used to positively impact health by phenolic supplementation and thus create favorable colonic conditions. This study explored the effect of six stilbenoids (batatasin III, oxyresveratrol, piceatannol, pinostilbene, resveratrol, thunalbene) on the gut microbiota composition. Stilbenoids were anaerobically fermented with fecal bacteria from four donors, samples were collected at 0 and 24 h, and effects on the microbiota were assessed by 16S rRNA gene sequencing. Statistical tests identified affected microbes at three taxonomic levels. Observed microbial composition modulation by stilbenoids included a decrease in the Firmicutes to Bacteroidetes ratio, a decrease in the relative abundance of strains from the genus Clostridium, and effects on the family Lachnospiraceae. A frequently observed effect was a further decrease of the relative abundance when compared to the control. An opposite effect to the control was observed for Faecalibacterium prausnitzii, whose relative abundance increased. Observed effects were more frequently attributed to resveratrol and piceatannol, followed by thunalbene and batatasin III.

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Comparison of Methods To Collect Fecal Samples for Microbiome Studies Using Whole-Genome Shotgun Metagenomic Sequencing

mSphere ◽

10.1128/msphere.00827-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 2

Author(s):

Doratha A. Byrd ◽

Rashmi Sinha ◽

Kristi L. Hoffman ◽

Jun Chen ◽

Xing Hua ◽

...

Keyword(s):

Ambient Temperature ◽

Fecal Sample ◽

Gold Standard ◽

Beta Diversity ◽

Whole Genome ◽

Metagenomic Sequencing ◽

Sample Collection ◽

Fecal Samples ◽

Shotgun Metagenomic Sequencing ◽

Collection Methods

ABSTRACT Few previous studies have assessed stability and “gold-standard” concordance of fecal sample collection methods for whole-genome shotgun metagenomic sequencing (WGSS), an increasingly popular method for studying the gut microbiome. We used WGSS data to investigate ambient temperature stability and putative gold-standard concordance of microbial profiles in fecal samples collected and stored using fecal occult blood test (FOBT) cards, fecal immunochemical test (FIT) tubes, 95% ethanol, or RNAlater. Among 15 Mayo Clinic employees, for each collection method, we calculated intraclass correlation coefficients (ICCs) to estimate stability of fecal microbial profiles after storage for 4 days at ambient temperature and concordance with immediately frozen, no-solution samples (i.e., the putative gold standard). ICCs were estimated for multiple metrics, including relative abundances of select phyla, species, KEGG k-genes (representing any coding sequence that had >70% identity and >70% query coverage with respect to a known KEGG ortholog), KEGG modules, and KEGG pathways; species and k-gene alpha diversity; and Bray-Curtis and Jaccard species beta diversity. ICCs for microbial profile stability were excellent (≥90%) for fecal samples collected via most of the collection methods, except those preserved in 95% ethanol. Concordance with the immediately frozen, no-solution samples varied for all collection methods, but the number of observed species and the beta diversity metrics tended to have higher concordance than other metrics. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT cards, FIT tubes, and RNAlater are acceptable choices for fecal sample collection methods in future WGSS studies. IMPORTANCE A major direction for future microbiome research is implementation of fecal sample collections in large-scale, prospective epidemiologic studies. Studying microbiome-disease associations likely requires microbial data to be pooled from multiple studies. Our findings suggest collection methods that are most optimal to be used standardly across future WGSS microbiome studies.

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Maternal Gut Microbiome Biodiversity in Pregnancy

American Journal of Perinatology ◽

10.1055/s-0037-1604412 ◽

2017 ◽

Vol 35 (01) ◽

pp. 024-030 ◽

Cited By ~ 16

Author(s):

Nitasha Ricks ◽

Alexis Panzer ◽

Amber Mccoy ◽

M. Azcarate-Peril ◽

Temitope Keku ◽

...

Keyword(s):

Gut Microbiome ◽

High Throughput Sequencing ◽

White Women ◽

Phase 1 ◽

Rrna Gene ◽

Fecal Samples ◽

Gestational Weight ◽

Biodiversity Changes ◽

In Pregnancy

Objective To measure maternal gut microbiome biodiversity in pregnancy. Materials and Methods In phase 1, maternal fecal samples were collected by rectal swab in 20 healthy pregnant women (14–28 weeks gestation) to measure bacterial abundance. In phase 2, fecal samples were collected from 31 women at enrollment (<20 weeks gestation, baseline) and at 36 to 39 weeks of gestation (follow-up). We assessed cluster analysis to assess bacterial community profiles at the phylum level longitudinally through pregnancy. DNA was extracted from swabs, followed by PCR of the bacterial 16s rRNA gene and multiplex high-throughput sequencing (Ion Torrent). Results In phase 1, 16 of 20 samples yielded usable data. White women (n = 10) had greater abundance of Firmicutes (23 ± 0.15 vs. 16% ± 0.75, p = 0.007) and Bacteroidetes (24 ± 0.14 vs. 19% ± 0.68, p = 0.015) compared with non-White women (n = 6). In the 11 paired specimens, Bacteroidetes increased in abundance from baseline to follow-up. Compared with women who gained weight below the median gestational weight gain (GWG, <15.4 kg), those who gained above the median GWG had increased abundance of Bacteroidetes (p = 0.02) and other phyla (p = 0.04). Conclusion Maternal microbiome biodiversity changes as pregnancy progresses and correlates with GWG.

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Microbial Composition of Human Appendices from Patients following Appendectomy

mBio ◽

10.1128/mbio.00366-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 52

Author(s):

Caitriona M. Guinane ◽

Amany Tadrous ◽

Fiona Fouhy ◽

C. Anthony Ryan ◽

Eugene M. Dempsey ◽

...

Keyword(s):

Human Health ◽

High Throughput ◽

High Throughput Sequencing ◽

Evolutionary Development ◽

Rrna Gene ◽

Limited Data ◽

Microbial Composition ◽

Content Type ◽

Study Results ◽

Human Appendix

ABSTRACT The human appendix has historically been considered a vestige of evolutionary development with an unknown function. While limited data are available on the microbial composition of the appendix, it has been postulated that this organ could serve as a microbial reservoir for repopulating the gastrointestinal tract in times of necessity. We aimed to explore the microbial composition of the human appendix, using high-throughput sequencing of the 16S rRNA gene V4 region. Seven patients, 5 to 25 years of age, presenting with symptoms of acute appendicitis were included in this study. Results showed considerable diversity and interindividual variability among the microbial composition of the appendix samples. In general, however, Firmicutes was the dominant phylum, with the majority of additional sequences being assigned at various levels to Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Despite the large diversity in the microbiota found within the appendix, however, a few major families and genera were found to comprise the majority of the sequences present. Interestingly, also, certain taxa not generally associated with the human intestine, including the oral pathogens Gemella, Parvimonas, and Fusobacterium, were identified among the appendix samples. The prevalence of genera such as Fusobacterium could also be linked to the severity of inflammation of the organ. We conclude that the human appendix contains a robust and varied microbiota distinct from the microbiotas in other niches within the human microbiome. The microbial composition of the human appendix is subject to extreme variability and comprises a diversity of biota that may play an important, as-yet-unknown role in human health. IMPORTANCE There are currently limited data available on the microbial composition of the human appendix. It has been suggested, however, that it may serve as a “safe house” for commensal bacteria that can reinoculate the gut at need. The present study is the first comprehensive view of the microbial composition of the appendix as determined by high-throughput sequencing. We have determined that the human appendix contains a wealth of microbes, including members of 15 phyla. Important information regarding the associated bacterial diversity of the appendix which will help determine the role, if any, the appendix microbiota has in human health is presented.

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