scholarly journals Optimisation of sample storage and DNA extraction for human gut microbiota studies​

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jekaterina Kazantseva ◽  
Esther Malv ◽  
Aleksei Kaleda ◽  
Aili Kallastu ◽  
Anne Meikas
Keyword(s):  
Gut Microbiota ◽  
Dna Extraction ◽  
Personalised Medicine ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Human Gut ◽  
Human Gut Microbiota ◽  
Sequencing Technologies ◽  
Different Temperatures ◽  
Reliable Solution

Abstract Background New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition studies. Metagenomic analysis of the gut microbiota is paramount for the characterization of human health and wellbeing. Despite the intensive research, there is a huge gap and inconsistency between different studies due to the non-standardised and biased pipeline. Methodical and systemic understanding of every stage in the process is necessary to overcome all bottlenecks and grey zones of gut microbiota studies, where all details and interactions between processes are important. Results Here we show that an inexpensive, but reliable iSeq 100 platform is an excellent tool to perform the analysis of the human gut microbiota by amplicon sequencing of the 16 S rRNA gene. Two commercial DNA extraction kits and different starting materials performed similarly regarding the taxonomic distribution of identified bacteria. DNA/RNA Shield reagent proved to be a reliable solution for stool samples collection, preservation, and storage, as the storage of faecal material in DNA/RNA Shield for three weeks at different temperatures and thawing cycles had a low impact on the bacterial distribution. Conclusions Altogether, a thoroughly elaborated pipeline with close attention to details ensures high reproducibility with significant biological but not technical variations.

scholarly journals Changes in human gut microbiota composition are linked to the energy metabolic switch during 10 d of Buchinger fasting

2019 ◽  
Vol 8 ◽  
Author(s):  
Robin Mesnage ◽  
Franziska Grundler ◽  
Andreas Schwiertz ◽  
Yvon Le Maho ◽  
Françoise Wilhelmi de Toledo
Keyword(s):  
Gut Microbiota ◽  
Inflammatory Diseases ◽  
Well Being ◽  
Amplicon Sequencing ◽  
Serum Biochemistry ◽  
Rrna Gene ◽  
Human Gut ◽  
Metabolic Switch ◽  
Human Gut Microbiota ◽  
Inflammatory Status

Abstract Fasting is increasingly popular to manage metabolic and inflammatory diseases. Despite the role that the human gut microbiota plays in health and diseases, little is known about its composition and functional capacity during prolonged fasting when the external nutrient supply is reduced or suppressed. We analysed the effects of a 10-d periodic fasting on the faecal microbiota of fifteen healthy men. Participants fasted according to the peer-reviewed Buchinger fasting guidelines, which involve a daily energy intake of about 1046 kJ (250 kcal) and an enema every 2 d. Serum biochemistry confirmed the metabolic switch from carbohydrates to fatty acids and ketones. Emotional and physical well-being were enhanced. Faecal 16S rRNA gene amplicon sequencing showed that fasting caused a decrease in the abundance of bacteria known to degrade dietary polysaccharides such as Lachnospiraceae and Ruminococcaceae. There was a concomitant increase in Bacteroidetes and Proteobacteria (Escherichia coli and Bilophila wadsworthia), known to use host-derived energy substrates. Changes in taxa abundance were associated with serum glucose and faecal branched-chain amino acids (BCAA), suggesting that fasting-induced changes in the gut microbiota are associated with host energy metabolism. These effects were reversed after 3 months. SCFA levels were unchanged at the end of the fasting. We also monitored intestinal permeability and inflammatory status. IL-6, IL-10, interferon γ and TNFα levels increased when food was reintroduced, suggesting a reactivation of the postprandial immune response. We suggest that changes in the gut microbiota are part of the physiological adaptations to a 10-d periodic fasting, potentially influencing its beneficial health effects.

scholarly journals Production of conjugated linoleic acid (CLA): effect of inulin on microbial composition and CLA concentration in a human intestinal model

2020 ◽  
Vol 79 (OCE2) ◽  
Author(s):  
Ilaria Carafa ◽  
Domenico Masuero ◽  
Urska Vrhovsek ◽  
Giovanni Bittante ◽  
Elena Franciosi ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Gut Microbiota ◽  
Relative Abundance ◽  
Illumina Miseq ◽  
Ultra Performance Liquid Chromatography ◽  
Rrna Gene ◽  
Human Gut ◽  
Microbial Composition ◽  
Human Gut Microbiota

AbstractConjugated linoleic acids (CLAs) show a number of putative health-promoting activities including anti-carcinogenic, anti-adipogenic, anti-diabetogenic, anti-inflammatory and antioxidant actions. CLAs are naturally produced by ruminal bacteria and several studies demonstrate that various lactobacilli and bifidobacteria are also able to produce CLAs in vitro from linoleic acid (LA). However, the ability of the human gut microbiota to produce CLA is less extensively studied. Our hypothesis is that the human gut microbiota is able to convert LA to CLA, and that the readily fermentable fiber inulin would positively modulate the growth of CLA-producing bacteria and, consequently increase the CLA content in the intestine.The capability of the faecal microbiota from five healthy donors to produce CLA was tested in anaerobic batch cultures for 48 hours at pH 5.5 and 6.5. Test treatments were linoleic acid (LA; 1 mg/mL) + bovine serum albumin (BSA; 0.2 mg/mL), and LA (1 mg/mL) + BSA (0.2 mg/mL) + inulin (1%, w/v) compared to a control BSA (0.2 mg/mL) fermentation. The microbial composition was analyzed 0, 24 and 48 hours after starting the fermentation by 16S rRNA gene Illumina MiSeq sequencing (V3-V4 region). CLAs were quantified by Ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) and bi-dimensional gas chromatography (GC x GC).The inclusion of LA + BSA + inulin at pH 5.5 significantly increased the relative abundance of Collinsella aerofaciens (p < 0.05), and tended to increase the relative abundance of bifidobacteria. LA + BSA + inulin at both pH 5.5 and 6.5 reduced the relative abundance of Parabacteroides, Bilophila, Clostridia and Enterobacteriaceae (p < 0.05). The concentration of CLA, in particular the isomer cis9,trans11 C18:2, was significantly higher in the LA + BSA + inulin group at pH 5.5 after 24 and 48 hours fermentation.The data show that the treatment LA + BSA + inulin at pH 5.5 induce substantial changes in microbiota composition, including bifidogenesis and CLA production in a human intestinal microbiota model. The changes of relative abundance detected are consistent with changes in gut bacteria previously linked to human health. Collinsella aerofaciens has been reported for reducing bloating, in particular in subjects suffering from irritable bowel syndrome, while Clostridia, Bilophila and Enterobacteriaceae causes human infections. In addition, the increase of bifidobacteria and LAB, which have previously been shown in vitro to produce CLA, may also be involved in CLA production under simulated cecal microbiome. These preclinical observations warrant confirmation in suitably designed animal and human mechanistic studies.

scholarly journals Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shoichiro Kameoka ◽  
Daisuke Motooka ◽  
Satoshi Watanabe ◽  
Ryuichi Kubo ◽  
Nicolas Jung ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Amplicon Sequencing ◽  
Analysis Data ◽  
Rrna Gene ◽  
Bacterial Composition ◽  
Sequencing Technologies ◽  
Primer Sets ◽  
Microbiome Data

Abstract Background 16S rRNA gene amplicon sequencing (16S analysis) is widely used to analyze microbiota with next-generation sequencing technologies. Here, we compared fecal 16S analysis data from 192 Japanese volunteers using the modified V1–V2 (V12) and the standard V3–V4 primer (V34) sets to optimize the gut microbiota analysis protocol. Results QIIME1 and QIIME2 analysis revealed a higher number of unclassified representative sequences in the V34 data than in the V12 data. The comparison of bacterial composition demonstrated that at the phylum level, Actinobacteria and Verrucomicrobia were detected at higher levels with V34 than with V12. Among these phyla, we observed higher relative compositions of Bifidobacterium and Akkermansia with V34. To estimate the actual abundance, we performed quantitative real-time polymerase chain reaction (qPCR) assays for Akkermansia and Bifidobacterium. We found that the abundance of Akkermansia as detected by qPCR was close to that in V12 data, but was markedly lower than that in V34 data. The abundance of Bifidobacterium detected by qPCR was higher than that in V12 and V34 data. Conclusions These results indicate that the bacterial composition derived from the V34 region might differ from the actual abundance for specific gut bacteria. We conclude that the use of the modified V12 primer set is more desirable in the 16S analysis of the Japanese gut microbiota.

scholarly journals An extended reconstruction of human gut microbiota metabolism of dietary compounds

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Telmo Blasco ◽  
Sergio Pérez-Burillo ◽  
Francesco Balzerani ◽  
Daniel Hinojosa-Nogueira ◽  
Alberto Lerma-Aguilera ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Human Health ◽  
Metabolic Networks ◽  
16S Rrna Gene Sequencing ◽  
Complex Problem ◽  
Rrna Gene ◽  
Limited Information ◽  
Sequencing Data ◽  
Human Gut ◽  
Human Gut Microbiota

AbstractUnderstanding how diet and gut microbiota interact in the context of human health is a key question in personalized nutrition. Genome-scale metabolic networks and constraint-based modeling approaches are promising to systematically address this complex problem. However, when applied to nutritional questions, a major issue in existing reconstructions is the limited information about compounds in the diet that are metabolized by the gut microbiota. Here, we present AGREDA, an extended reconstruction of diet metabolism in the human gut microbiota. AGREDA adds the degradation pathways of 209 compounds present in the human diet, mainly phenolic compounds, a family of metabolites highly relevant for human health and nutrition. We show that AGREDA outperforms existing reconstructions in predicting diet-specific output metabolites from the gut microbiota. Using 16S rRNA gene sequencing data of faecal samples from Spanish children representing different clinical conditions, we illustrate the potential of AGREDA to establish relevant metabolic interactions between diet and gut microbiota.

scholarly journals Ramadan Fasting Leads to Shifts in Human Gut Microbiota Structured by Dietary Composition

2021 ◽  
Vol 12 ◽  
Author(s):  
Ikram Ali ◽  
Ke Liu ◽  
Danfeng Long ◽  
Shah Faisal ◽  
Mian Gul Hilal ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Ethnic Groups ◽  
Alpha Diversity ◽  
Rrna Gene ◽  
Dietary Composition ◽  
Human Gut ◽  
Ramadan Fasting ◽  
Human Gut Microbiota ◽  
Before And After ◽  
Chinese Group

The structure and diversity of human gut microbiota are directly related to diet, though less is known about the influences of ethnicity and diet-related behaviors, such as fasting (intermittent caloric restriction). In this study, we investigated whether fasting for Ramadan altered the microbiota in Chinese and Pakistani individuals. Using high-throughput 16S rRNA gene sequencing and self-reported dietary intake surveys, we determined that both the microbiota and dietary composition were significantly different with little overlap between ethnic groups. Principal Coordinate Analyses (PCoA) comparison of samples collected from both groups before and after fasting showed partial separation of microbiota related to fasting in the Pakistani group, but not in the Chinese group. Measurement of alpha diversity showed that Ramadan fasting significantly altered the coverage and ACE indices among Chinese subjects, but otherwise incurred no changes among either group. Specifically, Prevotella and Faecalibacterium drove predominance of Bacteroidetes and Firmicutes in the Pakistani group, while Bacteroides (phylum Bacteroidetes) were the most prevalent among Chinese participants both before and after fasting. We observed significant enrichment of some specific taxa and depletion of others in individuals of both populations, suggesting that fasting could affect beta diversity. Notably, Dorea, Klebsiella, and Faecalibacterium were more abundant in the Chinese group after fasting, while Sutterella, Parabacteroides, and Alistipes were significantly enriched after fasting in the Pakistani group. Evaluation of the combined groups showed that genera Coprococcus, Clostridium_XlV, and Lachnospiracea were all significantly decreased after fasting. Analysis of food intake and macronutrient energy sources showed that fat-derived energy was positively associated with Oscillibacter and Prevotella, but negatively associated with Bacteroides. In addition, the consumption of sweets was significantly positively correlated with the prevalence of Akkermansia. Our study indicated that diet was the most significant influence on microbiota, and correlated with ethnic groups, while fasting led to enrichment of specific bacterial taxa in some individuals. Given the dearth of understanding about the impacts of fasting on microbiota, our results provide valuable inroads for future study aimed at novel, personalized, behavior-based treatments targeting specific gut microbes for prevention or treatment of digestive disorders.

scholarly journals Gilbert’s Syndrome and the Gut Microbiota – Insights From the Case-Control BILIHEALTH Study

2021 ◽  
Vol 11 ◽  
Author(s):  
Patrick A. Zöhrer ◽  
Claudia A. Hana ◽  
Nazlisadat Seyed Khoei ◽  
Christine Mölzer ◽  
Marlies Hörmann-Wallner ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Bacterial Species ◽  
Elevated Serum ◽  
Study Groups ◽  
Rrna Gene ◽  
Human Gut ◽  
Gilbert’S Syndrome ◽  
Alpha And Beta Diversity ◽  
Human Gut Microbiota ◽  
Gilbert's Syndrome

The heme catabolite bilirubin has anti-inflammatory, anti-oxidative and anti-mutagenic effects and its relation to colorectal cancer (CRC) risk is currently under evaluation. Although the main metabolic steps of bilirubin metabolism, including the formation of stercobilin and urobilin, take place in the human gastrointestinal tract, potential interactions with the human gut microbiota are unexplored. This study investigated, whether gut microbiota composition is altered in Gilbert’s Syndrome (GS), a mild form of chronically elevated serum unconjugated bilirubin (UCB) compared to matched controls. Potential differences in the incidence of CRC-associated bacterial species in GS were also assessed. To this end, a secondary investigation of the BILIHEALTH study was performed, assessing 45 adults with elevated UCB levels (GS) against 45 age- and sex-matched controls (C). Fecal microbiota analysis was performed using 16S rRNA gene sequencing. No association between mildly increased UCB and the composition of the gut microbiota in this healthy cohort was found. The alpha and beta diversity did not differ between C and GS and both groups showed a typical representation of the known dominant phyla. Furthermore, no difference in abundance of Firmicutes and Proteobacteria, which have been associated with the mucosa of CRC patients were observed between the groups. A sequence related to the Christensenella minuta strain YIT 12065 was identified with a weak association value of 0.521 as an indicator species in the GS group. This strain has been previously associated with a lower body mass index, which is typical for the GS phenotype. Overall, sex was the only driver for an identifiable difference in the study groups, as demonstrated by a greater bacterial diversity in women. After adjusting for confounding factors and multiple testing, we can conclude that the GS phenotype does not affect the composition of the human gut microbiota in this generally healthy study group.

scholarly journals The Impact of a Dynamic Working Environment on Human Gut Microbiota

2021 ◽  
Author(s):  
Lu Ling ◽  
Jun Zhou ◽  
Qianlong Meng ◽  
Ziran Zhang ◽  
Wenkun Li ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Clinical Characteristics ◽  
Dietary Habits ◽  
Living Environment ◽  
Working Environment ◽  
Rrna Gene ◽  
Human Gut ◽  
Human Gut Microbiota ◽  
Train Drivers ◽  
The Impact

Gut microbiota dysbiosis is associated with a variety of diseases, such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), metabolic diseases, allergic diseases, neurodevelopmental disorders and cancer. The human gut microbiota can be influenced by a variety of factors, including geography, dietary habits, living environment, age and altered lifestyle etc. This study was conducted to explore the gut microbiota compositions in officials who are in a stable working environment and train drivers who are in a dynamic working environment. Microbiota communities in the feces of 80 officials and 88 train drivers were analyzed using Illumina MiSeq sequencing targeting the V3-V4 region of 16S ribosomal RNA (rRNA) gene and ITS1 region of fungi. There were significant differences between the two groups in diversity and richness of gut microbiota, while the microbial community compositions of the two groups were similar. The relationship between gut microbiota and clinical characteristics was investigated. We found that more bacteria and fungi were positively correlated with clinical characteristics. Functional prediction analysis of the gut microbiota between the two groups by PICRUSt2 revealed significant differences between the official group and the train driver group. Elucidating these differences of the microbiome between the two groups will provide a foundation understanding of the impact of a dynamic environment on gut microbiota.

scholarly journals Can Physical Activity Influence Human Gut Microbiota Composition Independently of Diet? A Systematic Review

Nutrients ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1890
Author(s):  
Barbara Dorelli ◽  
Francesca Gallè ◽  
Corrado De Vito ◽  
Guglielmo Duranti ◽  
Matteo Iachini ◽  
...  
Keyword(s):  
Physical Activity ◽  
Gut Microbiota ◽  
Amplicon Sequencing ◽  
Microbiota Composition ◽  
Human Gut ◽  
Exercise Interventions ◽  
Human Gut Microbiota ◽  
16S Rrna Amplicon Sequencing ◽  
New Perspective ◽  
Gut Microbiota Composition

Evidence suggests that physical activity (PA) influences the human gut microbiota composition, but its role is unclear because of dietary interference. The aim of this review is to clarify this issue from this new perspective in healthy individuals. Articles analyzing intestinal microbiota from fecal samples by 16S rRNA amplicon sequencing were selected by searching the electronic databases PubMed, Scopus, and Web of Science until December 2020. For each study, methodological quality was assessed, and results about microbiota biodiversity indices, phylum and genus composition, and information on PA and diet were considered. From 997 potentially relevant articles, 10 met the inclusion criteria and were analyzed. Five studies involved athletes, three were performed on active people classified on the basis of habitual PA level, and two among sedentary subjects undergoing exercise interventions. The majority of the studies reported higher variability and prevalence of the phylum Firmicutes (genera Ruminococcaceae or Fecalibacteria) in active compared to inactive individuals, especially in athletes. The assessment of diet as a possible confounder of PA/exercise effects was completed only in four studies. They reported a similar abundance of Lachnospiraceae, Paraprevotellaceae, Ruminococcaceae, and Veillonellaceae, which are involved in metabolic, protective, structural, and histological functions. Further studies are needed to confirm these findings.

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