Microbial community profiling using 16S rRNA gene sequences requires accurate taxonomy assignments. 'Universal' primers target conserved sequences and amplify sequences from many taxa, but they ...provide variable coverage of different environments, and regions of the rRNA gene differ in taxonomic informativeness--especially when high-throughput short-read sequencing technologies (for example, 454 and Illumina) are used. We introduce a new evaluation procedure that provides an improved measure of expected taxonomic precision when classifying environmental sequence reads from a given primer. Applying this measure to thousands of combinations of primers and read lengths, simulating single-ended and paired-end sequencing, reveals that these choices greatly affect taxonomic informativeness. The most informative sequence region may differ by environment, partly due to variable coverage of different environments in reference databases. Using our Rtax method of classifying paired-end reads, we found that paired-end sequencing provides substantial benefit in some environments including human gut, but not in others. Optimal primer choice for short reads totaling 96 nt provides 82-100% of the confident genus classifications available from longer reads.
To understand how different diets, the consumers’ gut microbiota, and the enteric nervous system (ENS) interact to regulate gut motility, we developed a gnotobiotic mouse model that mimics short-term ...dietary changes that happen when humans are traveling to places with different culinary traditions. Studying animals transplanted with the microbiota from humans representing diverse culinary traditions and fed a sequence of diets representing those of all donors, we found that correlations between bacterial species abundances and transit times are diet dependent. However, the levels of unconjugated bile acids—generated by bacterial bile salt hydrolases (BSH)—correlated with faster transit, including during consumption of a Bangladeshi diet. Mice harboring a consortium of sequenced cultured bacterial strains from the Bangladeshi donor’s microbiota and fed a Bangladeshi diet revealed that the commonly used cholekinetic spice, turmeric, affects gut motility through a mechanism that reflects bacterial BSH activity and Ret signaling in the ENS. These results demonstrate how a single food ingredient interacts with a functional microbiota trait to regulate host physiology.
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•Gut motility is affected by different human gut microbiota-diet combinations•Turmeric slows motility in gnotobiotic mice with a Bangladeshi microbiota/diet•Turmeric’s effect involves bile acid secretion/deconjugation and Ret signaling•Gnotobiotic mice can define key interactions between traditional foods and microbiota
A mouse model of short-term dietary changes, which happen when humans are traveling to places with different culinary traditions, reveals how a single food ingredient interacts with a functional microbiota trait to regulate host physiology.
COVID-19 (coronavirus disease 2019) patients exhibiting gastrointestinal symptoms are reported to have worse prognosis. Ace2 (angiotensin-converting enzyme 2), the gene encoding the host protein to ...which SARS-CoV-2 spike proteins bind, is expressed in the gut and therefore may be a target for preventing or reducing severity of COVID-19. Here we test the hypothesis that Ace2 expression in the gastrointestinal and respiratory tracts is modulated by the microbiome. We used quantitative PCR to profile Ace2 expression in germ-free mice, conventional raised specific pathogen-free mice, and gnotobiotic mice colonized with different microbiota. Intestinal Ace2 expression levels were significantly higher in germ-free mice compared to conventional mice. A similar trend was observed in the respiratory tract. Intriguingly, microbiota depletion via antibiotics partially recapitulated the germ-free phenotype, suggesting potential for microbiome-mediated regulation of Ace2 expression. Variability in intestinal Ace2 expression was observed in gnotobiotic mice colonized with different microbiota, partially attributable to differences in microbiome-encoded proteases and peptidases. Together, these data suggest that the microbiome may be one modifiable factor determining COVID-19 infection risk and disease severity.
Ret
is implicated in colorectal cancer (CRC) as both a proto-oncogene and a tumor suppressor. We asked whether RET signaling regulates tumorigenesis in an
Apc
-deficient preclinical model of CRC. We ...observed a sex-biased phenotype:
Apc
Min
/+
Ret
+/- females had significantly greater tumor burden than
Apc
Min
/+
Ret
+/- males, a phenomenon not seen in
Apc
Min
/+
mice, which had equal distributions by sex. Dysfunctional RET signaling was associated with gene expression changes in diverse tumor signaling pathways in tumors and normal-appearing colon. Sex-biased gene expression differences mirroring tumor phenotypes were seen in 26 genes, including the
Apc
tumor suppressor gene.
Ret
and
Tlr4
expression were significantly correlated in tumor samples from female but not male
Apc
Min
/+
Ret
+/- mice. Antibiotics resulted in reduction of tumor burden, inverting the sex-biased phenotype such that microbiota-depleted
Apc
Min
/+
Ret
+/- males had significantly more tumors than female littermates. Reconstitution of the microbiome rescued the sex-biased phenotype. Our findings suggest that RET represents a sexually dimorphic microbiome-mediated “switch” for regulation of tumorigenesis.
The oral and colonic microbiota are distinct in healthy individuals. However, this distinction is diminished in common diseases such as colon cancer and inflammatory bowel disease, suggesting a ...potential pathogenic role for oral bacteria when ectopically colonized in the gut. A key mechanism for the segregation of oral and colonic microbiota niches is thought to be microbiota-mediated colonization resistance whereby the commensal gut microbiota outcompete and eliminate the ingested oral bacteria.
We tested this theory by analyzing exact amplicon sequence variants generated from concurrent fecal and oral samples from healthy volunteers exposed to a brief course of a single antibiotic (cohort 1), acute leukemia patients (cohort 2), and stem cell transplant recipients (cohort 3). Cohorts 2 and 3 represent extreme clinical scenarios with respect to antibiotic pressure and severity of gut microbiota injury.
While mild antibiotic exposure in cohort 1 was not sufficient for colonization of any oral bacteria in the gut, even with extreme antibiotic pressure and severe gut microbiota disruptions in cohorts 2 and 3, only one oral species in each cohort colonized the gut.
Colonization resistance is dispensable for segregation of oral and colonic microbiota in humans. This finding implies that the presence of oral bacteria in the distal gut in diseases such as colon cancer and inflammatory bowel disease is not driven by impaired colonization resistance.
The multitude of barriers between the mouth and colon may eliminate swallowed oral bacteria. Ascertaining the presence of the same bacteria in the mouth and colon is methodologically challenging ...partly because 16S rRNA gene sequencing - the most commonly used method to characterize the human microbiota - has low confidence in taxonomic assignments deeper than genus for most bacteria. As different species of the same genus can have low-level variation across the same 16S rRNA gene region, shotgun sequencing is needed to identify a true overlap. We analyzed a curated, multi-cohort, shotgun metagenomic database with species-level taxonomy and clade-specific marker genes to fill this knowledge gap. Using 500 paired fecal/oral (4 oral sites) samples from 4 healthy adult cohorts, we found a minute overlap between the two niches. Comparing marker genes between paired oral and fecal samples with species-level overlap, the pattern of overlap in only 7 individuals was consistent with same-strain colonization. These findings argue against ectopic colonization of oral bacteria in the distal gut in healthy adults.
Gut motility is regulated by the microbiome via mechanisms that include bile acid metabolism. To localize the effects of microbiome-generated bile acids, we colonized gnotobiotic mice with different ...synthetic gut bacterial communities that were metabolically phenotyped using a functional in vitro screen. Using two different marker-based assays of gut transit, we inferred that bile acids exert effects on colonic transit. We validated this using an intra-colonic bile acid infusion assay and determined that these effects were dependent upon signaling via the bile acid receptor, TGR5. The intra-colonic bile acid infusion experiments further revealed sex-biased bile acid-specific effects on colonic transit, with lithocholic acid having the largest pro-motility effect. Transcriptional responses of the enteric nervous system (ENS) were stereotypic, regional, and observed in response to different microbiota, their associated bile acid profiles, and even to a single diet ingredient, evidencing exquisite sensitivity of the ENS to environmental perturbations.
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•Gut microbiome-generated bile acids regulate colonic transit via TGR5.•Lithocholic acid had the largest colonic pro-motility effect.•Bile acids exert sex-biased effects on gut transit times.•Enteric nervous system transcriptional responses are regional and microbiome-specific.
Microbiology; Microbiome; Gastroenterology
Gastrointestinal motility is regulated by a variety of environmental factors including gut microbes and metabolites. The ability to interrogate mouse models of gut motility has enabled elucidation of ...these relationships. Here we describe integration of the red carmine dye and fluorescence isothiocyanate-dextran marker-based assays for characterizing gut transit with spatial resolution, along with an optional intracolonic infusion protocol for studying the effects of metabolites on colonic transit. These protocols can be adapted for use in gnotobiotic and conventional mouse models.
For complete details on the use and execution of this protocol, please refer to Li et al. (2021).
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•Visual and fluorescent markers can characterize gut transit with spatial resolution•Identify metabolites’ effects on colonic transit via intra-colonic metabolite infusion•Transit assays can interfere with each other, so an interval between each use is required
Gastrointestinal motility is regulated by a variety of environmental factors including gut microbes and metabolites. The ability to interrogate mouse models of gut motility has enabled elucidation of these relationships. Here we describe integration of the red carmine dye and fluorescence isothiocyanate-dextran marker-based assays for characterizing gut transit with spatial resolution, along with an optional intracolonic infusion protocol for studying the effects of metabolites on colonic transit. These protocols can be adapted for use in gnotobiotic and conventional mouse models.
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