Microbiota-based prediction of chronic infections is promising yet not well established. Early childhood caries (ECC) is the most common infection in children. Here we simultaneously tracked ...microbiota development at plaque and saliva in 50 4-year-old preschoolers for 2 years; children either stayed healthy, transitioned into cariogenesis, or experienced caries exacerbation. Caries onset delayed microbiota development, which is otherwise correlated with aging in healthy children. Both plaque and saliva microbiota are more correlated with changes in ECC severity (dmfs) during onset than progression. By distinguishing between aging- and disease-associated taxa and exploiting the distinct microbiota dynamics between onset and progression, we developed a model, Microbial Indicators of Caries, to diagnose ECC from healthy samples with 70% accuracy and predict, with 81% accuracy, future ECC onsets for samples clinically perceived as healthy. Thus, caries onset in apparently healthy teeth can be predicted using microbiota, when appropriately de-trended for age.
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•Oral microbiota in 50 four-year-old children were tracked for 2 years•Age-dependent microbiota development is perturbed by early childhood caries (ECC) onset•Shifts in microbiota precede manifestation of clinical symptoms of ECC•Microbial Indicators of Caries, when de-trended for age, can predict ECC onset
Teng et al. tracked plaque and saliva microbiota of 50 4-year-old children for 2 years. By distinguishing between aging- and disease-associated taxa and exploiting the distinct microbiota dynamics between disease onset and progression, a predictive model, Microbial Indicators of Caries, is proposed as a method to predict future caries onset.
High-throughput sequencing of 16S ribosomal RNA gene amplicons has facilitated understanding of complex microbial communities, but the inherent noise in PCR and DNA sequencing limits differentiation ...of closely related bacteria. Although many scientific questions can be addressed with broad taxonomic profiles, clinical, food safety, and some ecological applications require higher specificity. Here we introduce a novel sub-operational-taxonomic-unit (sOTU) approach, Deblur, that uses error profiles to obtain putative error-free sequences from Illumina MiSeq and HiSeq sequencing platforms. Deblur substantially reduces computational demands relative to similar sOTU methods and does so with similar or better sensitivity and specificity. Using simulations, mock mixtures, and real data sets, we detected closely related bacterial sequences with single nucleotide differences while removing false positives and maintaining stability in detection, suggesting that Deblur is limited only by read length and diversity within the amplicon sequences. Because Deblur operates on a per-sample level, it scales to modern data sets and meta-analyses. To highlight Deblur's ability to integrate data sets, we include an interactive exploration of its application to multiple distinct sequencing rounds of the American Gut Project. Deblur is open source under the Berkeley Software Distribution (BSD) license, easily installable, and downloadable from https://github.com/biocore/deblur.
Deblur provides a rapid and sensitive means to assess ecological patterns driven by differentiation of closely related taxa. This algorithm provides a solution to the problem of identifying real ecological differences between taxa whose amplicons differ by a single base pair, is applicable in an automated fashion to large-scale sequencing data sets, and can integrate sequencing runs collected over time.
The vitamin D receptor is highly expressed in the gastrointestinal tract where it transacts gene expression. With current limited understanding of the interactions between the gut microbiome and ...vitamin D, we conduct a cross-sectional analysis of 567 older men quantifying serum vitamin D metabolites using LC-MSMS and defining stool sub-Operational Taxonomic Units from16S ribosomal RNA gene sequencing data. Faith's Phylogenetic Diversity and non-redundant covariate analyses reveal that the serum 1,25(OH)
D level explains 5% of variance in α-diversity. In β-diversity analyses using unweighted UniFrac, 1,25(OH)
D is the strongest factor assessed, explaining 2% of variance. Random forest analyses identify 12 taxa, 11 in the phylum Firmicutes, eight of which are positively associated with either 1,25(OH)
D and/or the hormone-to-prohormone 1,25(OH)
D/25(OH)D "activation ratio." Men with higher levels of 1,25(OH)
D and higher activation ratios, but not 25(OH)D itself, are more likely to possess butyrate producing bacteria that are associated with better gut microbial health.
Our understanding of the interaction between the gut microbiota and host health has recently improved dramatically. However, the effects of toxic metal exposure on the gut microbiota remain poorly ...characterized. As this microbiota creates a critical interface between the external environment and the host's cells, it may play an important role in host outcomes during exposure. We therefore used 16S ribosomal RNA (rRNA) gene sequencing to track changes in the gut microbiota composition of rats exposed to heavy metals. Rats were exposed daily for five days to arsenic, cadmium, cobalt, chromium, nickel, or a vehicle control. Significant changes to microbiota composition were observed in response to high doses of chromium and cobalt, and significant dose-dependent changes were observed in response to arsenic, cadmium and nickel. Many of these perturbations were not uniform across metals. However, bacteria with higher numbers of iron-importing gene orthologs were overly represented after exposure to arsenic and nickel, suggesting some possibility of a shared response. These findings support the utility of the microbiota as a pre-clinical tool for identifying exposures to specific heavy metals. It is also clear that characterizing changes to the functional capabilities of microbiota is critical to understanding responses to metal exposure.
Recent algorithmic advances in amplicon-based microbiome studies enable the inference of exact amplicon sequence fragments. These new methods enable the investigation of sub-operational taxonomic ...units (sOTU) by removing erroneous sequences. However, short (e.g., 150-nucleotide nt) DNA sequence fragments do not contain sufficient phylogenetic signal to reproduce a reasonable tree, introducing a barrier in the utilization of critical phylogenetically aware metrics such as Faith's PD or UniFrac. Although fragment insertion methods do exist, those methods have not been tested for sOTUs from high-throughput amplicon studies in insertions against a broad reference phylogeny. We benchmarked the SATé-enabled phylogenetic placement (SEPP) technique explicitly against 16S V4 sequence fragments and showed that it outperforms the conceptually problematic but often-used practice of reconstructing
phylogenies. In addition, we provide a BSD-licensed QIIME2 plugin (https://github.com/biocore/q2-fragment-insertion) for SEPP and integration into the microbial study management platform QIITA.
The move from OTU-based to sOTU-based analysis, while providing additional resolution, also introduces computational challenges. We demonstrate that one popular method of dealing with sOTUs (building a
tree from the short sequences) can provide incorrect results in human gut metagenomic studies and show that phylogenetic placement of the new sequences with SEPP resolves this problem while also yielding other benefits over existing methods.
Recent studies suggest that gut microbiomes of urban-industrialized societies are different from those of traditional peoples. Here we examine the relationship between lifeways and gut microbiota ...through taxonomic and functional potential characterization of faecal samples from hunter-gatherer and traditional agriculturalist communities in Peru and an urban-industrialized community from the US. We find that in addition to taxonomic and metabolic differences between urban and traditional lifestyles, hunter-gatherers form a distinct sub-group among traditional peoples. As observed in previous studies, we find that Treponema are characteristic of traditional gut microbiomes. Moreover, through genome reconstruction (2.2-2.5 MB, coverage depth × 26-513) and functional potential characterization, we discover these Treponema are diverse, fall outside of pathogenic clades and are similar to Treponema succinifaciens, a known carbohydrate metabolizer in swine. Gut Treponema are found in non-human primates and all traditional peoples studied to date, suggesting they are symbionts lost in urban-industrialized societies.
Abstract
Background
Alteration of the gut microbiota may contribute to the development of inflammatory bowel disease (IBD). Epigallocatechin-3-gallate (EGCG), a major bioactive constituent of green ...tea, is known to be beneficial in IBD alleviation. However, it is unclear whether the gut microbiota exerts an effect when EGCG attenuates IBD.
Results
We first explored the effect of oral or rectal EGCG delivery on the DSS-induced murine colitis. Our results revealed that anti-inflammatory effect and colonic barrier integrity were enhanced by oral, but not rectal, EGCG. We observed a distinct EGCG-mediated alteration in the gut microbiome by increasing
Akkermansia
abundance and butyrate production. Next, we demonstrated that the EGCG pre-supplementation induced similar beneficial outcomes to oral EGCG administration. Prophylactic EGCG attenuated colitis and significantly enriched short-chain fatty acids (SCFAs)-producing bacteria such as
Akkermansia
and SCFAs production in DSS-induced mice. To validate these discoveries, we performed fecal microbiota transplantation (FMT) and sterile fecal filtrate (SFF) to inoculate DSS-treated mice. Microbiota from EGCG-dosed mice alleviated the colitis over microbiota from control mice and SFF shown by superiorly anti-inflammatory effect and colonic barrier integrity, and also enriched bacteria such as
Akkermansia
and SCFAs. Collectively, the attenuation of colitis by oral EGCG suggests an intimate involvement of SCFAs-producing bacteria
Akkermansia
, and SCFAs, which was further demonstrated by prophylaxis and FMT.
Conclusions
This study provides the first data indicating that oral EGCG ameliorated the colonic inflammation in a gut microbiota-dependent manner. Our findings provide novel insights into EGCG-mediated remission of IBD and EGCG as a potential modulator for gut microbiota to prevent and treat IBD.
Many factors affect the microbiomes of humans, mice, and other mammals, but substantial challenges remain in determining which of these factors are of practical importance. Considering the relative ...effect sizes of both biological and technical covariates can help improve study design and the quality of biological conclusions. Care must be taken to avoid technical bias that can lead to incorrect biological conclusions. The presentation of quantitative effect sizes in addition to P values will improve our ability to perform meta-analysis and to evaluate potentially relevant biological effects. A better consideration of effect size and statistical power will lead to more robust biological conclusions in microbiome studies.
Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife ...conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment.
Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.
Data from 16S ribosomal RNA (rRNA) amplicon sequencing present challenges to ecological and statistical interpretation. In particular, library sizes often vary over several ranges of magnitude, and ...the data contains many zeros. Although we are typically interested in comparing relative abundance of taxa in the ecosystem of two or more groups, we can only measure the taxon relative abundance in specimens obtained from the ecosystems. Because the comparison of taxon relative abundance in the specimen is not equivalent to the comparison of taxon relative abundance in the ecosystems, this presents a special challenge. Second, because the relative abundance of taxa in the specimen (as well as in the ecosystem) sum to 1, these are compositional data. Because the compositional data are constrained by the simplex (sum to 1) and are not unconstrained in the Euclidean space, many standard methods of analysis are not applicable. Here, we evaluate how these challenges impact the performance of existing normalization methods and differential abundance analyses.
Effects on normalization: Most normalization methods enable successful clustering of samples according to biological origin when the groups differ substantially in their overall microbial composition. Rarefying more clearly clusters samples according to biological origin than other normalization techniques do for ordination metrics based on presence or absence. Alternate normalization measures are potentially vulnerable to artifacts due to library size. Effects on differential abundance testing: We build on a previous work to evaluate seven proposed statistical methods using rarefied as well as raw data. Our simulation studies suggest that the false discovery rates of many differential abundance-testing methods are not increased by rarefying itself, although of course rarefying results in a loss of sensitivity due to elimination of a portion of available data. For groups with large (~10×) differences in the average library size, rarefying lowers the false discovery rate. DESeq2, without addition of a constant, increased sensitivity on smaller datasets (<20 samples per group) but tends towards a higher false discovery rate with more samples, very uneven (~10×) library sizes, and/or compositional effects. For drawing inferences regarding taxon abundance in the ecosystem, analysis of composition of microbiomes (ANCOM) is not only very sensitive (for >20 samples per group) but also critically the only method tested that has a good control of false discovery rate.
These findings guide which normalization and differential abundance techniques to use based on the data characteristics of a given study.
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