Williams Turpin

Association of host genome with intestinal microbial composition in a large healthy cohort

Intestinal microbiota is known to be important in health and disease. Its composition is influenced by both environmental and host factors. Few large-scale studies have evaluated the association between host genetic variation and the composition of microbiota. We recruited a cohort of 1,561 healthy individuals, of whom 270 belong in 123 families, and found that almost one-third of fecal bacterial taxa were heritable. In addition, we identified 58 SNPs associated with the relative abundance of 33 taxa in 1,098 discovery subjects. Among these, four loci were replicated in a second cohort of 463 subjects: rs62171178 (nearest gene UBR3) associated with Rikenellaceae, rs1394174 (CNTN6) associated with Faecalibacterium, rs59846192 (DMRTB1) associated with Lachnospira, and rs28473221 (SALL3) associated with Eubacterium. After correction for multiple testing, 6 of the 58 associations remained significant, one of which replicated. These results identify associations between specific genetic variants and the gut microbiome.

Lactobacillaceae and Cell Adhesion: Genomic and Functional Screening

The analysis of collections of lactic acid bacteria (LAB) from traditional fermented plant foods in tropical countries may enable the detection of LAB with interesting properties. Binding capacity is often the main criterion used to investigate the probiotic characteristics of bacteria. In this study, we focused on a collection of 163 Lactobacillaceace comprising 156 bacteria isolated from traditional amylaceous fermented foods and seven strains taken from a collection and used as controls. The collection had a series of analyses to assess binding potential for the selection of new probiotic candidates. The presence/absence of 14 genes involved in binding to the gastrointestinal tract was assessed. This enabled the detection of all the housekeeping genes (ef-Tu, eno, gap, groEl and srtA) in the entire collection, of some of the other genes (apf, cnb, fpbA, mapA, mub) in 86% to 100% of LAB, and of the other genes (cbsA, gtf, msa, slpA) in 0% to 8% of LAB. Most of the bacteria isolated from traditional fermented foods exhibited a genetic profile favorable for their binding to the gastrointestinal tract. We selected 30 strains with different genetic profiles to test their binding ability to non-mucus (HT29) and mucus secreting (HT29-MTX) cell lines as well as their ability to degrade mucus. Assays on both lines revealed high variability in binding properties among the LAB, depending on the cell model used. Finally, we investigated if their binding ability was linked to tighter cross-talk between bacteria and eukaryotic cells by measuring the expression of bacterial genes and of the eukaryotic MUC2 gene. Results showed that wild LAB from tropical amylaceous fermented food had a much higher binding capacity than the two LAB currently known to be probiotics. However their adhesion was not linked to any particular genetic equipment.

Assessment and Selection of Competing Models for Zero-Inflated Microbiome Data

Typical data in a microbiome study consist of the operational taxonomic unit (OTU) counts that have the characteristic of excess zeros, which are often ignored by investigators. In this paper, we compare the performance of different competing methods to model data with zero inflated features through extensive simulations and application to a microbiome study. These methods include standard parametric and non-parametric models, hurdle models, and zero inflated models. We examine varying degrees of zero inflation, with or without dispersion in the count component, as well as different magnitude and direction of the covariate effect on structural zeros and the count components. We focus on the assessment of type I error, power to detect the overall covariate effect, measures of model fit, and bias and effectiveness of parameter estimations. We also evaluate the abilities of model selection strategies using Akaike information criterion (AIC) or Vuong test to identify the correct model. The simulation studies show that hurdle and zero inflated models have well controlled type I errors, higher power, better goodness of fit measures, and are more accurate and efficient in the parameter estimation. Besides that, the hurdle models have similar goodness of fit and parameter estimation for the count component as their corresponding zero inflated models. However, the estimation and interpretation of the parameters for the zero components differs, and hurdle models are more stable when structural zeros are absent. We then discuss the model selection strategy for zero inflated data and implement it in a gut microbiome study of > 400 independent subjects.

Fecal microbiota manipulation prevents dysbiosis and alcohol-induced liver injury in mice

BACKGROUND & AIMS Alcoholic liver disease (ALD) is a leading cause of liver failure and mortality. In humans, severe alcoholic hepatitis is associated with key changes to intestinal microbiota (IM), which influences individual sensitivity to develop advanced ALD. We used the different susceptibility to ALD observed in two distinct animal facilities to test the efficiency of two complementary strategies (fecal microbiota transplantation and prebiotic treatment) to reverse dysbiosis and prevent ALD. METHODS Mice were fed alcohol in two distinct animal facilities with a Lieber DeCarli diet. Fecal microbiota transplantation was performed with fresh feces from alcohol-resistant donor mice to alcohol-sensitive receiver mice three times a week. Another group of mice received pectin during the entire alcohol consumption period. RESULTS Ethanol induced steatosis and liver inflammation, which were associated with disruption of gut homeostasis, in alcohol-sensitive, but not alcohol resistant mice. IM analysis showed that the proportion of Bacteroides was specifically lower in alcohol-sensitive mice (p<0.05). Principal coordinate analysis showed that the IM of sensitive and resistant mice clustered differently. We targeted IM using two different strategies to prevent alcohol-induced liver lesions: (1) pectin treatment which induced major modifications of the IM, (2) fecal microbiota transplantation which resulted in an IM very close to that of resistant donor mice in the sensitive recipient mice. Both methods prevented steatosis, liver inflammation, and restored gut homeostasis. CONCLUSIONS Manipulation of IM can prevent alcohol-induced liver injury. The IM should be considered as a new therapeutic target in ALD. LAY SUMMARY Sensitivity to alcoholic liver disease (ALD) is driven by intestinal microbiota in alcohol fed mice. Treatment of mice with alcohol-induced liver lesions by fecal transplant from alcohol fed mice resistant to ALD or with prebiotic (pectin) prevents ALD. These findings open new possibilities for treatment of human ALD through intestinal microbiota manipulation.

Determinants of Intestinal Permeability in Healthy First-Degree Relatives of Individuals with Crohnʼs Disease

Background:The Genetics, Environmental, Microbial Project is a multicenter study assessing etiological factors in Crohns disease by studying healthy first-degree relatives (FDRs) of individuals affected by Crohns disease. We aimed to evaluate the contribution of genetic, microbial, and environmental factors to the determination of intestinal permeability in healthy FDRs. Methods:IP was assessed using the lactulose-mannitol ratio (LacMan ratio). FDRs were genotyped for 167 inflammatory bowel disease-associated single nucleotide polymorphisms. Taxonomic profile of the fecal microbiota was determined by Illumina MiSeq pyrosequencing of 16S ribosomal RNA. The associations of LacMan ratio with demographic factors, inflammatory bowel disease-associated single nucleotide polymorphisms and the fecal microbiota were assessed. Results:One hundred ninety-six white FDRs were included. Eleven percent of FDRs had an elevated LacMan ratio (≥0.03). A multivariate analysis demonstrated that younger subjects and nonsmokers had higher LacMan ratios, P = 3.62 × 10−4 and P = 0.03, respectively. The LacMan ratio was not significantly heritable, H2r, 0.13, P = 0.13. There was no association between any of the 167 inflammatory bowel disease-associated risk variants and LacMan ratio nor was there a correlation between fecal microbial composition and the LacMan ratio. Conclusions:We did not find LacMan ratio to be significantly heritable suggesting that the contribution of genetic factors to the determination of intestinal permeability in healthy FDRs is modest. Environmental factors, such as smoking, are likely more important determinants. The effect of age on intestinal barrier function has been underappreciated.

Determination of expression and activity of genes involved in starch metabolism in Lactobacillus plantarum A6 during fermentation of a cereal-based gruel

Traditional fermented gruels prepared from cereals are widely used for complementary feeding of young children in Africa and usually have a low energy density. The amylase activity of some lactic acid bacteria (LAB) helps increase the energy content of gruels through partial hydrolysis of starch, thus enabling the incorporation of more starchy material while conserving the desired semi-liquid consistency for young children. Even if this ability is mainly related to the production of alpha-amylase (E.C. 3.2.1.1), in a recent molecular screening, genes coding for enzymes involved in starch metabolism were detected in the efficient amylolytic LAB Lactobacillus plantarum A6: alpha-glucosidase (E.C. 3.2.1.20), neopullulanase (E.C. 3.2.1.135), amylopectin phosphorylase (E.C. 2.4.1.1) and maltose phosphorylase (E.C. 2.4.1.8). The objective of this study was to investigate the expression of these genes in a model of starchy fermented food made from pearl millet (Pennisetum glaucum). Transcriptional and enzymatic analyses were performed during the 18-h fermentation period. Liquefaction was mainly caused by an extracellular alpha amylase encoded by the amyA gene specific to the A6 strain among L. plantarum species and found in both Lactobacillus amylovorus and Lactobacillus manihotivorans. The second most active enzyme was neopullulanase. Other starch metabolizing enzymes were less often detected. The dynamic detection of transcripts of gene during starch fermentation in pearl millet porridge suggests that the set of genes we investigated was not expressed continuously but transiently.

Behavior of lactobacilli isolated from fermented slurry (ben-saalga) in gnotobiotic rats.

Most bacterial strains, which have been studied so far for their probiotic functions, are extensively used by manufacturers in developed countries. In our work, we sought to study a mix (called BSL) comprising three strains belonging to Lactobacillus fermentum, L. paraplantarum and L. salivarius, that were isolated from a traditional African pearl millet based fermented slurry. Our objective was to study this BSL cocktail in gnotobiotic rats, to evaluate their survival and their behavior in the digestive tract conditions. After a single oral inoculation of germfree rats with BSL, the species established stably in the digestive tract with the following hierarchy of abundance: L. salivarius> L. plantarum> L. fermentum. BSL cocktail was metabolically active since it produced 50 mM lactate and it expressed genes involved in binding mechanism in the caecum. Furthermore, the global morphology of the colon epithelium was not disturbed by the BSL cocktail. BSL cocktail did not modify mucus content and host mucus-related genes (MUC1, MUC2, MUC3 or resistin-like molecule β). The cocktail of lactobacilli enhanced the proliferating cell nuclear antigen (PCNA) at a level comparable to what was observed in conventional rats. PCNA was involved in proliferation and DNA repair, but the presence of the cocktail did not provoke proliferative events (with Ki67 as indicator), so we suppose BSL may help gut preservation. This work is the first step towards the selection of strains that are derived from traditional fermented food to formulate new probiotic mixture.

Microbiome analysis - from technical advances to biological relevance.

The development of culture-independent techniques and next-generation sequencing has led to a staggering rise in the number of microbiome studies over the last decade. Although it remains important to identify the taxa of microbes present in a variety of environmental samples, including the gut microbiomes of healthy and diseased individuals, the next stage of microbiome research will need to focus on uncovering the role of the microbiome rather than its mere composition. Here, we introduce techniques that go beyond identifying the taxa present within a sample and examine the biological function of the microbiome or the host-microbiome interaction.

FUT2 genotype and secretory status are not associated with fecal microbial composition and inferred function in healthy subjects

ABSTRACT Heritability analysis of the microbiota has demonstrated the importance of host genotype in defining the human microbiota. The alpha (1,2)-fucosyltransferase 2 encoded by FUT2 is involved in the formation of the H antigen and the SNP, rs601338 is associated with ABO histo-blood group antigen secretion in the intestinal mucosa. Previous studies have provided non replicated results for the association of this polymorphism with the composition and inferred function of intestinal microbiota. We aimed to assess this relationship in a large cohort of 1,190 healthy individuals. Genotyping was performed using the HumanCoreEXOME chip, microbial composition was addressed by 16S rRNA gene sequencing. Firmicutes, Bacteroidetes, and Actinobacteria were the dominant phyla in this cohort. Although we have sufficient power to detect significant associations of FUT2 genotype/ inferred phenotype with the microbiota, our data demonstrate that FUT2 genotype and secretor status is not associated with microbial alpha diversity, microbial composition or inferred microbial function after correction for multiple testing. Thus, FUT2 genotype and inferred phenotype are not associated with human fecal microbial composition and imputed function.

Determinants of IBD Heritability: Genes, Bugs, and More

Defining the etiology of inflammatory bowel disease (IBD) continues to elude researchers, in part due to the possibility that there may be different triggers for a spectrum of disease phenotypes that are currently classified as either Crohns disease (CD) or ulcerative colitis (UC). What is clear is that genetic susceptibility plays an important role in the development of IBD, and large genome-wide association studies using case-control approaches have identified more than 230 risk alleles. Many of these identified risk alleles are located in a variety of genes important in host-microbiome interactions. In spite of these major advances, the mechanisms behind the genetic influence on disease development remain unknown. In addition, the identified genetic risks have thus far failed to fully define the hereditability of IBD. Host genetics influence host interactions with the gut microbiota in maintaining health through a balance of regulated immune responses and coordinated microbial composition and function. What remains to be defined is how alterations in these interactions can lead to disease. The nature and cause of changes in the microbiota in patients with IBD are poorly understood. In spite of the large catalog of alterations in the microbiota of IBD patients, inflammation itself can alter the microbiota, leaving open the question of which is cause or effect. The composition and function of the gut microbiota are influenced by many factors, including environmental factors, dietary factors, and, as recent studies have shown, host genetic makeup. More than 200 loci have shown potential to influence the microbiota, but replication and larger studies are still required to validate these findings. It would seem reasonable to consider the combination of both host genetic makeup and the inheritance of the microbiota as interdependent heritable forces that could explain the nature of an individuals susceptibility to IBD or indeed the actual cause of IBD. In this review, we will consider the contribution of the host genetics, the microbiome, and the influence of host genetics on the microbiota to the heritability of IBD.