Perinatal factors impact gut microbiota development in early life, however, little is known on the effects of these factors on microbes in later life. Here we sequence DNA from faecal samples of ...children over the first four years and reveal a perpetual evolution of the gut microbiota during this period. The significant impact of gestational age at birth and delivery mode on gut microbiota progression is evident in the first four years of life, while no measurable effects of antibiotics are found in the first year. Microbiota profiles are also characteristic in children dependant on gestational age and maturity. Full term delivery is characterised by Bacteroides (year one), Parabacteroides (year two) and Christensenellaceae (year four). Preterm delivery is characterised by Lactobacillus (year one), Streptococcus (year two) and Carnobacterium (year four). This study reveals that the gut retains distinct microbial profiles of perinatal factors up to four years of age.
Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial ...component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA.
The use of different primer sequences (V4-V5, V1-V2 and V1-V2 degenerate primers) resulted in differences in the genera and species detected. The V4-V5 primers gave the most comparable results across platforms. The three Ion PGM primer sets detected more of the 20 mock community species than the equivalent MiSeq primer sets. Data generated from DNA extracted using the 2 extraction methods were very similar.
Microbiota compositional data differed depending on the primers and sequencing platform that were used. The results demonstrate the risks in comparing data generated using different sequencing approaches and highlight the merits of choosing a standardised approach for sequencing in situations where a comparison across multiple sequencing runs is required.
•Children’s stress was cross-sectionally related to gut microbial composition.•New was the use of hair cortisol, heart rate variability and positive emotions.•Mainly positive emotions and ...parasympathetic activity distinguished microbiota.•A general shift towards lower Firmicutes but many associations were OTU specific.•Differential results in preadolescents versus adolescents.
Chronic stress increases disease vulnerability factors including inflammation, a pathological characteristic potentially regulated by the gut microbiota. We checked the association between the gut microbiome and psychosocial stress in children/adolescents and investigated which stress parameter (negative versus positive emotion, self-report versus parental report, events versus emotions, biomarker cortisol versus parasympathetic activity) is the most relevant indicator herein.
Gut microbiome sequencing was completed in fecal samples from 93 Belgian 8-16y olds. Stress measures included negative events, negative emotions, emotional problems reported by parents, happiness, hair cortisol and heart rate variability (pnn50 parameter reflecting parasympathetic activity). Alpha diversity, beta diversity and linear discriminant analysis were the unadjusted analyses. Age, sex, socio-economic status, diet, physical activity, sleep and weight status were adjusted for via a redundancy analysis and differential abundance via zero-inflated negative binomial regression.
High stress as reflected by low pnn50 and more negative events were associated with a lower alpha diversity as indicated by the Simpson index. Happiness and pnn50 showed significant differences between high and low stress groups based on weighted UniFrac distance, and this remained significant after confounder adjustment. Adjusted and unadjusted taxonomic differences were also most pronounced for happiness and pnn50 being associated respectively with 24 OTU (=11.8% of bacterial counts) and 31 OTU (=13.0%). As a general pattern, high stress was associated with lower Firmicutes at the phylum level and higher Bacteroides, Parabacteroides, Rhodococcus, Methanobrevibacter and Roseburia but lower Phascolarctobacterium at genus level. Several genera gave conflicting results between different stress measures e.g. Ruminococcaceae UCG014, Tenericutes, Eubacterium coprostanoligenes, Prevotella 9 and Christensenellaceae R7. Differential results in preadolescents versus adolescents were also evident.
Even in this young healthy population, stress parameters were cross-sectionally associated with gut microbial composition but this relationship was instrument specific. Positive emotions and parasympathetic activity appeared the strongest parameters and should be integrated in future microbiota projects amongst other stress measures.
High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon ...appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.
Seven faecal samples were collected from healthy adults. Samples were separated into fresh (DNA extracted immediately), snap frozen on dry ice and frozen for 7 days at -80°C prior to DNA extraction or samples frozen at -80°C for 7 days before DNA extraction. Sequencing was completed on the Illumina MiSeq platform. Culturing of total aerobes, anaerobes and bifidobacteria was also completed.
No significant differences at phylum or family levels between the treatment groups occurred. At genus level only Faecalibacterium and Leuconostoc were significantly different in the fresh samples compared to the snap frozen group (p = 0.0298; p = 0.0330 respectively). Diversity analysis indicated that samples clustered based on the individual donor, rather than by storage group. No significant differences occurred in the culture-based analysis between the fresh, snap or -80°C frozen samples.
Using the MiSeq platform coupled with culture-based analysis, this study highlighted that limited significant changes in microbiota occur following rapid freezing of faecal samples prior to DNA extraction. Thus, rapid freezing of samples prior to DNA extraction and culturing, preserves the integrity of the microbiota.
Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive ...technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance in microbiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced with MiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10 million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.
Vancomycin, metronidazole, and the bacteriocin lacticin 3147 are active against a wide range of bacterial species, including Clostridium difficile. We demonstrate that, in a human distal colon model, ...the addition of each of the three antimicrobials resulted in a significant decrease in numbers of C. difficile. However, their therapeutic use in the gastrointestinal tract may be compromised by their broad spectrum of activity, which would be expected to significantly impact on other members of the human gut microbiota. We used highthroughput pyrosequencing to compare the effect of each antimicrobial on the composition of the microbiota. All three treatments resulted in a decrease in the proportion of sequences assigned to the phyla Firmicutes and Bacteroidetes, with a corresponding increase in those assigned to members of the Proteobacteria. One possible means of avoiding such "collateral damage" would involve the application of a narrow-spectrum antimicrobial with specific anti-C. difficile activity. We tested this hypothesis using thuricin CD, a narrowspectrum bacteriocin produced by Bacillus thuringiensis, which is active against C. difficile. The results demonstrated that this bacteriocin was equally effective at killing C. difficile in the distal colon model but had no significant impact on the composition of the microbiota. This offers the possibility of developing a targeted approach to eliminating C. difficile in the colon, without collateral damage.
Male middle age is a transitional period where many physiological and psychological changes occur leading to cognitive and behavioural alterations, and a deterioration of brain function. However, the ...mechanisms underpinning such changes are unclear. The gut microbiome has been implicated as a key mediator in the communication between the gut and the brain, and in the regulation of brain homeostasis, including brain immune cell function. Thus, we tested whether targeting the gut microbiome by prebiotic supplementation may alter microglia activation and brain function in ageing. Male young adult (8 weeks) and middle-aged (10 months) C57BL/6 mice received diet enriched with a prebiotic (10% oligofructose-enriched inulin) or control chow for 14 weeks. Prebiotic supplementation differentially altered the gut microbiota profile in young and middle-aged mice with changes correlating with faecal metabolites. Functionally, this translated into a reversal of stress-induced immune priming in middle-aged mice. In addition, a reduction in ageing-induced infiltration of Ly-6C
monocytes into the brain coupled with a reversal in ageing-related increases in a subset of activated microglia (Ly-6C
) was observed. Taken together, these data highlight a potential pathway by which targeting the gut microbiome with prebiotics can modulate the peripheral immune response and alter neuroinflammation in middle age. Our data highlight a novel strategy for the amelioration of age-related neuroinflammatory pathologies and brain function.
Rationale
The impact of the microbiota on the gut-brain axis is increasingly appreciated. A growing body of literature demonstrates that use of dietary fibre and prebiotics can manipulate the ...microbiota and affect host health. However, the influence on cognition and acute stress response is less well understood.
Objectives
The objective of this study was to investigate the efficacy of a dietary fibre, polydextrose (PDX), in improving cognitive performance and acute stress responses through manipulation of the gut microbiota in a healthy population.
Methods
In this double-blind, randomised, placebo-controlled, crossover design study, 18 healthy female participants received 12.5 g Litesse®Ultra (> 90% PDX polymer) or maltodextrin for 4 weeks. Cognitive performance, mood, acute stress responses, microbiota composition, and inflammatory markers were assessed pre- and post-intervention.
Results
PDX improved cognitive flexibility as evidenced by the decrease in the number of errors made in the Intra-Extra Dimensional Set Shift (IED) task. A better performance in sustained attention was observed through higher number of correct responses and rejections in the Rapid Visual Information Processing (RVP) task. Although there was no change in microbial diversity, abundance of
Ruminiclostridium
5 significantly increased after PDX supplementation compared with placebo. PDX supplementation attenuated the increase of adhesion receptor CD62L on classical monocytes observed in the placebo group.
Conclusions
Supplementation with the PDX resulted in a modest improvement in cognitive performance. The results indicate that PDX could benefit gut-to-brain communication and modulate behavioural responses.
•The gut microbiome is sensitive the effects of chronic stress.•We examined the stress-induced temporal effects on the composition of the microbiome, which we define as volatility.•Volatility is ...correlated with several central and peripheral readouts of the stress response.•The volatility findings were replicated in an independent cohort of mice under the same conditions and a cohort of students undergoing academic stress.
The microbiome-gut-brain-axis is a complex phenomenon spanning several dynamic systems in the body which can be parsed at a molecular, cellular, physiological and ecological level. A growing body of evidence indicates that this axis is particularly sensitive to the effects of stress and that it may be relevant to stress resilience and susceptibility. Although stress-induced changes in the composition of the microbiome have been reported, the degree of compositional change over time, which we define as volatility, has not been the subject of in-depth scrutiny. Using a chronic psychosocial stress paradigm in male mice, we report that the volatility of the microbiome significantly correlated with several readouts of the stress response, including behaviour and corticosterone response. We then validated these findings in a second independent group of stressed mice. Additionally, we assessed the relationship between volatility and stress parameters in a cohort of health volunteers who were undergoing academic exams and report similar observations. Finally, we found inter-species similarities in the microbiome stress response on a functional level. Our research highlights the effects of stress on the dynamic microbiome and underscores the informative value of volatility as a parameter that should be considered in all future analyses of the microbiome.
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