Ryan Legge

Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genome-wide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn’s disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1–V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

Host genetics and diet, but not immunoglobulin A expression, converge to shape compositional features of the gut microbiome in an advanced intercross population of mice

BackgroundIndividuality in the species composition of the vertebrate gut microbiota is driven by a combination of host and environmental factors that have largely been studied independently. We studied the convergence of these factors in a G10 mouse population generated from a cross between two strains to search for quantitative trait loci (QTLs) that affect gut microbiota composition or ileal Immunoglobulin A (IgA) expression in mice fed normal or high-fat diets.ResultsWe found 42 microbiota-specific QTLs in 27 different genomic regions that affect the relative abundances of 39 taxa, including four QTL that were shared between this G10 population and the population previously studied at G4. Several of the G10 QTLs show apparent pleiotropy. Eight of these QTLs, including four at the same site on chromosome 9, show significant interaction with diet, implying that diet can modify the effects of some host loci on gut microbiome composition. Utilization patterns of IghV variable regions among IgA-specific mRNAs from ileal tissue are affected by 54 significant QTLs, most of which map to a segment of chromosome 12 spanning the Igh locus. Despite the effect of genetic variation on IghV utilization, we are unable to detect overlapping microbiota and IgA QTLs and there is no significant correlation between IgA variable pattern utilization and the abundance of any of the taxa from the fecal microbiota.ConclusionsWe conclude that host genetics and diet can converge to shape the gut microbiota, but host genetic effects are not manifested through differences in IgA production.

Dietary selenium affects host selenoproteome expression by influencing the gut microbiota.

Colonization of the gastrointestinal tract and composition of the microbiota may be influenced by components of the diet, including trace elements. To understand how selenium regulates the intestinal microflora, we used high‐throughput sequencing to examine the composition of gut microbiota of mice maintained on selenium‐deficient, selenium‐sufficient, and selenium‐enriched diets. The microbiota diversity increased as a result of selenium in the diet. Specific phylotypes showed differential effects of selenium, even within a genus, implying that selenium had unique effects across microbial taxa. Conventionalized germ‐free mice subjected to selenium diets gave similar results and showed an increased diversity of the bacterial population in animals fed with higher levels of selenium. Germ‐free mice fed selenium diets modified their selenoproteome expression similar to control mice but showed higher levels and activity of glutathione peroxidase 1 and methionine‐R‐sulfoxide reductase 1 in the liver, suggesting partial sequestration of selenium by the gut microorganisms, limiting its availability for the host. These changes in the selenium status were independent of the levels of other trace elements. The data show that dietary selenium affects both composition of the intestinal microflora and colonization of the gastrointestinal tract, which, in turn, influence the host selenium status and selenoproteome expression.—Kasaikina, M. V., Kravtsova, M. A., Lee, B. C., Seravalli, J., Peterson, D. A., Walter, J., Legge, R., Benson, A. K., Hatfield, D. L., Gladyshev, V. N. Dietary selenium affects host selenoproteome expression by influencing the gut microbiota. FASEB J. 25, 2492–2499 (2011). www.fasebj.org

Microbial Successions Are Associated with Changes in Chemical Profiles of a Model Refrigerated Fresh Pork Sausage during an 80-Day Shelf Life Study

ABSTRACT Fresh pork sausage is produced without a microbial kill step and therefore chilled or frozen to control microbial growth. In this report, the microbiota in a chilled fresh pork sausage model produced with or without an antimicrobial combination of sodium lactate and sodium diacetate was studied using a combination of traditional microbiological methods and deep pyrosequencing of 16S rRNA gene amplicons. In the untreated system, microbial populations rose from 102 to 106 CFU/g within 15 days of storage at 4°C, peaking at nearly 108 CFU/g by day 30. Pyrosequencing revealed a complex community at day 0, with taxa belonging to the Bacilli, Gammaproteobacteria, Betaproteobacteria, Actinobacteria, Bacteroidetes, and Clostridia. During storage at 4°C, the untreated system displayed a complex succession, with species of Weissella and Leuconostoc that dominate the product at day 0 being displaced by species of Pseudomonas (P. lini and P. psychrophila) within 15 days. By day 30, a second wave of taxa (Lactobacillus graminis, Carnobacterium divergens, Buttiauxella brennerae, Yersinia mollaretti, and a taxon of Serratia) dominated the population, and this succession coincided with significant chemical changes in the matrix. Treatment with lactate-diacetate altered the dynamics dramatically, yielding a monophasic growth curve of a single species of Lactobacillus (L. graminis), followed by a uniform selective die-off of the majority of species in the population. Of the six species of Lactobacillus that were routinely detected, L. graminis became the dominant member in all samples, and its origins were traced to the spice blend used in the formulation.

Molecular characterization of the intestinal microbiota in patients with and without abdominal bloating

Recent studies have demonstrated differences in the intestinal microbiota between patients with irritable bowel syndrome (IBS) and healthy controls (HC), suggesting a role for the intestinal microbiota in the pathogenesis of IBS. Alterations in the microbiota have also been implicated in the pathogenesis of abdominal bloating, a commonly reported symptom in IBS. We investigated the relationship between the intestinal microbiota, abdominal bloating, and altered bowel patterns in a cohort of patients with IBS and HC. The 16S rRNA gene from fresh fecal samples was amplified and pyrosequenced by using Roche-454 Titanium chemistry. A Core Measurable Microbiome (CMM) was generated for Operational Taxonomic Unit (OTU) detected in >75% of all samples and compositional features of CMM were compared between groups by Linear Discriminant Analysis (LDA). IBS differentiated from HC by LDA using continuous variation in the species/OTUs or the CMM genera. When subcategorized based on bloating symptoms and bowel characteristics, the same subjects were also well differentiated from one another and from HC. ANOVA analysis showed quantitative species/OTU differences between the subgroups including IBS with and without bloating, and subtypes based on bowel characteristics. The clear LDA differentiation and the significant microbial taxa differences between the groups imply a significant association of the microbiota with bloating symptoms and bowel characteristics in IBS. These changes in the microbiota may serve as a biomarker for IBS and its clinical subtypes and suggest a role for the intestinal microbiota in the pathogenesis of the main symptoms of the disorder.

Tu2090 Molecular Characterization of the Intestinal Microbiota Focusing on Subgroups of Patients With Irritable Bowel Syndrome.

Alterations in the intestinal microbiota have been implicated in the pathogenesis of irritable bowel syndrome (IBS). Recent studies using advanced molecular biology techniques have demonstratedmicrobiota compositional differences between IBS patients and healthy controls (HC). Many of these studies, however, are confounded by the fact that the study populations comprise a mixed group of patients with IBS or a specific subtype of IBS. Thus, there is a need for systematic comparison of microbiota composition between subjects with clinicallyrelevant subtypes of IBS. Aim: To investigate the differences in composition of the intestinal microbiota between clinically-relevant, well-defined subgroups of patients with IBS and HC using deep pyrosequencing of the 16S rRNA gene. Methods: DNA was isolated from fecal samples from 60 IBS patients (Rome III: C-IBS, D-IBS, M-IBS; n=20 per group) and 20 HC. The V1-V2 variable regions of the 16S rRNA gene were amplified using bar-coded fusion primers and pyrosequenced using Roche-454 Titanium chemistry. Sequences were processed through the Multi-CLASSIFIER algorithm for taxonomic assignment. Classified reads were assigned to a species/OTU-level status using BLAST pipeline. Unclassified readswere clustered into OTUs at 97% using CD-Hit. A Core Measurable Microbiome (CMM) was generated for OTUs detected in ≥75% of all samples. Compositional features of CMM were compared using Linear Discriminant Analysis (LDA). Quantitative differences in species/OTUs and taxonomic groups were tested by ANOVA with the Tukey correction for multiple testing. Results: Subjects with IBS differentiated from HC by LDA using continuous variation in the species/OTUs or the CMM genera as variables. When further subcategorized based on bowel characteristics, the same subjects were also well-differentiated from one another and from HC. ANOVA analysis showed quantitative species/OTUs differences between the study subgroups: I. a significant increase in members of the Actinobacteria phyla (in particular the genus Collinsiella) in the C-IBS subgroup compared to all other groups, II. a significant decrease in members of Firmicutes phyla ( Oscillibacter, Anaerovorax, Incertae sedis XIII, Streptococcus and Eubacteriaceae) in D-IBS and M-IBS compared to HC and C-IBS, and III. a significant lower levels of Lactobacillus in HC compared to C-IBS and D-IBS. Conclusion: We demonstrated clear differences in the intestinal microbiota between patients with clinically-relevant subtypes of IBS. While differences in many individual taxa were identified, there was little difference in the relative abundances of major taxonomic groups (e.g., ratio of Bacteriodetes;Firmicutes:Proteobacteria). Our results provide a rationale for further investigation of the causality and the role of these microbiota compositional alterations in the pathogenesis of IBS.