Enhanced Gut Microbiome Capacity for Amino Acid Metabolism is associated with Peanut Oral Immunotherapy Failure.

Peanut Oral Immunotherapy (POIT) holds promise for remission of peanut allergy, though treatment is protracted and successful in only a subset of patients. Because the gut microbiome is linked to food allergy, we sought to identify fecal microbial predictors of POIT efficacy and to develop mechanistic insights into treatment response. Longitudinal functional analysis of the fecal microbiome of children (n=79) undergoing POIT in a first double-blind, placebo-controlled clinical trial, identified five microbial-derived bile acids enriched in fecal samples prior to POIT initiation that predicted treatment efficacy (AUC 0.71). Failure to induce disease remission was associated with a distinct fecal microbiome with enhanced capacity for bile acid deconjugation, amino acid metabolism, and increased peanut peptide degradation in vitro . Thus, microbiome mechanisms of POIT failure appear to include depletion of immunomodulatory secondary bile and amino acids and the antigenic peanut peptides necessary to promote peanut allergy desensitization and remission.

12,13-diHOME Promotes Inflammatory Macrophages and Epigenetically Modifies Their Capacity to Respond to Microbes and Allergens.

Elevated infant fecal concentrations of the bacterial-derived lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) increase the risk for childhood atopy and asthma. However, the mechanisms by which this lipid contributes to disease development are largely unknown. We hypothesized that macrophages, which are key to both antimicrobial and antigen responses, are functionally and epigenetically modified by 12,13-diHOME leading to short- and long-term dysfunction with consequences for both antimicrobial and antigenic responses. Macrophages exposed to 12,13-diHOME are skewed toward inflammatory IL-1ß highCD206low cells, a phenomenon that is further amplified in the presence of common microbial-, aero-, and food-allergens. These IL-1ß highCD206low macrophages also exhibit reduced bacterial phagocytic capacity. In primary immune cell coculture assays involving peanut allergen stimulation, 12,13-diHOME promotes both IL-1ß and IL-6 production, memory B cell expansion, and increased IgE production. Exposure to 12,13-diHOME also induces macrophage chromatin remodeling, specifically diminishing access to interferon-stimulated response elements resulting in reduced interferon-regulated gene expression upon bacterial lipopolysaccharide stimulation. Thus 12,13-diHOME reprograms macrophage effector function, B-cell interactions and promotes epigenetic modifications that exacerbate inflammatory response to allergens and mutes antimicrobial response along the interferon axis. These observations offer plausible mechanisms by which this lipid promotes early-life pathogenic microbiome development and innate immune dysfunction associated with childhood allergic sensitization.

The gut-airway microbiome axis in health and respiratory diseases.

Communication between the gut and remote organs, such as the brain or the cardiovascular system, has been well established and recent studies provide evidence for a potential bidirectional gut-airway axis. Observations from animal and human studies indicate that respiratory insults influence the activity of the gut microbiome and that microbial ligands and metabolic products generated by the gut microbiome shape respiratory immunity. Information exchange between these two large mucosal surface areas regulates microorganism-immune interactions, with significant implications for the clinical and treatment outcomes of a range of respiratory conditions, including asthma, chronic obstructive pulmonary disease and lung cancer. In this Review, we summarize the most recent data in this field, offering insights into mechanisms of gut-airway crosstalk across spatial and temporal gradients and their relevance for respiratory health.

Fecal microbiota is associated with extraintestinal manifestations in inflammatory bowel disease.

INTRODUCTION: A large proportion of patients with inflammatory bowel disease (IBD) experience IBD-related inflammatory conditions outside of the gastrointestinal tract, termed extraintestinal manifestations (EIMs) which further decreases quality of life and, in extreme cases, can be life threatening. The pathogenesis of EIMs remains unknown, and although gut microbiota alterations are a well-known characteristic of patients with IBD, its relationship with EIMs remains sparsely investigated. This study aimed to compare the gut microbiota of patients with IBD with and without EIMs. METHODS: A total of 131 Danish patients with IBD were included in the study, of whom 86 had a history of EIMs (IBD-EIM) and 45 did not (IBD-C). Stool samples underwent 16S rRNA sequencing. Amplicon sequence variants (ASVs) were mapped to the Silva database. Diversity indices and distance matrices were compared between IBD-EIM and IBD-C. Differentially abundant ASVs were identified using a custom multiple model statistical analysis approach, and modules of co-associated bacteria were identified using sparse correlations for compositional data (SparCC) and related to patient EIM status. RESULTS: Patients with IBD and EIMs exhibited increased disease activity, body mass index, increased fecal calprotectin levels and circulating monocytes and neutrophils. Microbiologically, IBD-EIM exhibited lower fecal microbial diversity than IBD-C (Mann-Whitney's test, p = .01) and distinct fecal microbiota composition (permutational multivariate analysis of variance; weighted UniFrac, R2 = 0.018, p = .01). A total of 26 ASVs exhibited differential relative abundances between IBD-EIM and IBD-C, including decreased Agathobacter and Blautia and increased Eggerthella lenta in the IBD-EIM group. SparCC analysis identified 27 bacterial co-association modules, three of which were negatively related to EIM (logistic regression, p < .05) and included important health-associated bacteria, such as Agathobacter and Faecalibacterium. CONCLUSIONS: The fecal microbiota in IBD patients with EIMs is distinct from that in IBD patients without EIM and could be important for EIM pathogenesis.

Early-life nasal microbiota dynamics relate to longitudinal respiratory phenotypes in urban children.

BACKGROUND: Five distinct respiratory phenotypes based on latent classes of longitudinal patterns of wheezing, allergic sensitization. and pulmonary function measured in urban children from ages from 0 to 7 years have previously been described. OBJECTIVE: Our aim was to determine whether distinct respiratory phenotypes are associated with early-life upper respiratory microbiota development and environmental microbial exposures. METHODS: Microbiota profiling was performed using 16S ribosomal RNA-based sequencing of nasal samples collected at age 12 months (n = 120) or age 36 months (n = 142) and paired house dust samples collected at 3 months (12-month, n = 73; 36-month, n = 90) from all 4 centers in the Urban Environment and Childhood Asthma (URECA) cohort. RESULTS: In these high-risk urban children, nasal microbiota increased in diversity between ages 12 and 36 months (ß = 2.04; P = .006). Age-related changes in microbiota evenness differed significantly by respiratory phenotypes (interaction P = .0007), increasing most in the transient wheeze group. At age 12 months, respiratory illness (R2 = 0.055; P = .0001) and dominant bacterial genus (R2 = 0.59; P = .0001) explained variance in nasal microbiota composition, and enrichment of Moraxella and Haemophilus members was associated with both transient and high-wheeze respiratory phenotypes. By age 36 months, nasal microbiota was significantly associated with respiratory phenotypes (R2 = 0.019; P = .0376), and Moraxella-dominated microbiota was associated specifically with atopy-associated phenotypes. Analysis of paired house dust and nasal samples indicated that 12 month olds with low wheeze and atopy incidence exhibited the largest number of shared bacterial taxa with their environment. CONCLUSION: Nasal microbiota development over the course of early childhood and composition at age 3 years are associated with longitudinal respiratory phenotypes. These data provide evidence supporting an early-life window of airway microbiota development that is influenced by environmental microbial exposures in infancy and associates with wheeze- and atopy-associated respiratory phenotypes through age 7 years.

3'UTR-Seq analysis of chicken abdominal adipose tissue reveals widespread intron retention in 3'UTR and provides insight into molecular basis of feed efficiency.

Feed efficiency (FE) is an important trait in the broiler industry due to its direct correlation to efficient muscle growth instead of fat deposition. The present study characterized and compared gene expression profiles in abdominal fat from broiler chickens of different FE levels to enhance the understanding of FE biology. Specifically, traditional whole-transcript RNA-sequencing (RNA-seq) and 3' UTR-sequencing (3' UTR-seq) were applied to 22 and 61 samples, respectively. Overall, these two sequencing techniques shared a high correlation (0.76) between normalized counts, although 3' UTR-seq showed a higher variance in sequencing and mapping performance statistics across samples and a lower rate of uniquely mapped reads. A higher percentage of 3' UTR-seq reads mapped to introns suggested the frequent presence of cleavage sites in introns, thus warranting future research to study its regulatory function. Differential expression analysis identified 1198 differentially expressed genes (DEGs) between high FE (HFE) and intermediate FE (IFE) chickens with False Discovery Rate < 0.05 and fold change > 1.2. The processes that were significantly enriched by the DEGs included extracellular matrix remodeling and mechanisms impacting gene expression at the transcriptional and translational levels. Gene ontology enrichment analysis suggested that the divergence in fat deposition and FE in broiler chickens could be associated with peroxisome and lipid metabolism possibly regulated by G0/G1 switch gene 2 (G0S2).

A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by cell-wall acetylation.

The mammalian microbiome encodes numerous secondary metabolite biosynthetic gene clusters; yet, their role in microbe-microbe interactions is unclear. Here, we characterized two polyketide synthase gene clusters (fun and pks) in the gut symbiont Limosilactobacillus reuteri. The pks, but not the fun, cluster encodes antimicrobial activity. Forty-one of 51 L. reuteri strains tested are sensitive to Pks products; this finding was independent of strains' host origin. Sensitivity to Pks was also established in intraspecies competition experiments in gnotobiotic mice. Comparative genome analyses between Pks-resistant and -sensitive strains identified an acyltransferase gene (act) unique to Pks-resistant strains. Subsequent cell-wall analysis of wild-type and act mutant strains showed that Act acetylates cell-wall components, providing resistance to Pks-mediated killing. Additionally, pks mutants lost their competitive advantage, while act mutants lost their Pks resistance in in vivo competition assays. These findings provide insight into how closely related gut symbionts can compete and co-exist in the gastrointestinal tract.

Prebiotic to Improve Calcium Absorption in Postmenopausal Women After Gastric Bypass: A Randomized Controlled Trial.

CONTEXT: The adverse skeletal effects of Roux-en-Y gastric bypass (RYGB) are partly caused by intestinal calcium absorption decline. Prebiotics, such as soluble corn fiber (SCF), augment colonic calcium absorption in healthy individuals. OBJECTIVE: We tested the effects of SCF on fractional calcium absorption (FCA), biochemical parameters, and the fecal microbiome in a post-RYGB population. METHODS: Randomized, double-blind, placebo-controlled trial of 20 postmenopausal women with history of RYGB a mean 5 years prior; a 2-month course of 20 g/day SCF or maltodextrin placebo was taken orally. The main outcome measure was between-group difference in absolute change in FCA (primary outcome) and was measured with a gold standard dual stable isotope method. Other measures included tolerability, adherence, serum calciotropic hormones and bone turnover markers, and fecal microbial composition via 16S rRNA gene sequencing. RESULTS: Mean FCA ± SD at baseline was low at 5.5 ± 5.1%. Comparing SCF to placebo, there was no between-group difference in mean (95% CI) change in FCA (+3.4 [-6.7, +13.6]%), nor in calciotropic hormones or bone turnover markers. The SCF group had a wider variation in FCA change than placebo (SD 13.4% vs 7.0%). Those with greater change in microbial composition following SCF treatment had greater increase in FCA (r2 = 0.72, P = 0.05). SCF adherence was high, and gastrointestinal symptoms were similar between groups. CONCLUSION: No between-group differences were observed in changes in FCA or calciotropic hormones, but wide CIs suggest a variable impact of SCF that may be due to the degree of gut microbiome alteration. Daily SCF consumption was well tolerated. Larger and longer-term studies are warranted.

Draft Genome Sequences of 12 Lactobacillus reuteri Strains of Rodent Origin.

Lactobacillus reuteri is a bacterial gut symbiont found in many vertebrate animals. The genetic heterogeneity of L. reuteri is likely to contribute to differences in ecological performance within a host. Here, we report the draft genome sequences of 12 L. reuteri strains of rodent origin.

Prophages in Lactobacillus reuteri Are Associated with Fitness Trade-Offs but Can Increase Competitiveness in the Gut Ecosystem.

The gut microbiota harbors a diverse phage population that is largely derived from lysogens, which are bacteria that contain dormant phages in their genome. While the diversity of phages in gut ecosystems is getting increasingly well characterized, knowledge is limited on how phages contribute to the evolution and ecology of their host bacteria. Here, we show that biologically active prophages are widely distributed in phylogenetically diverse strains of the gut symbiont Lactobacillus reuteri Nearly all human- and rodent-derived strains, but less than half of the tested strains of porcine origin, contain active prophages, suggesting different roles of phages in the evolution of host-specific lineages. To gain insight into the ecological role of L. reuteri phages, we developed L. reuteri strain 6475 as a model to study its phages. After administration to mice, L. reuteri 6475 produces active phages throughout the intestinal tract, with the highest number detected in the distal colon. Inactivation of recA abolished in vivo phage production, which suggests that activation of the SOS response drives phage production in the gut. In conventional mice, phage production reduces bacterial fitness as fewer wild-type bacteria survive gut transit compared to the mutant lacking prophages. However, in gnotobiotic mice, phage production provides L. reuteri with a competitive advantage over a sensitive host. Collectively, we uncovered that the presence of prophages, although associated with a fitness trade-off, can be advantageous for a gut symbiont by killing a competitor strain in its intestinal niche.IMPORTANCE Bacteriophages derived from lysogens are abundant in gut microbiomes. Currently, mechanistic knowledge is lacking on the ecological ramifications of prophage carriage yet is essential to explain the abundance of lysogens in the gut. An extensive screen of the bacterial gut symbiont Lactobacillus reuteri revealed that biologically active prophages are widely distributed in this species. L. reuteri 6475 produces phages throughout the mouse intestinal tract, but phage production is associated with reduced fitness of the lysogen. However, phage production provides a competitive advantage in direct competition with a nonlysogenic strain of L. reuteri that is sensitive to these phages. This combination of increased competition with a fitness trade-off provides a potential explanation for the domination of lysogens in gut ecosystem and how lysogens can coexist with sensitive hosts.

Draft Genome Sequence of Aryl Hydrocarbon Receptor Activator Strains Lactobacillus reuteri R2lc and 2010.

Lactobacillus reuteri R2lc and 2010 are pigmented rat intestinal isolates. L. reuteri R2lc has been studied in different animal disease models, including colitis and acute liver injury. We sequenced both isolates and identified novel secondary metabolite gene clusters, which may be linked to the probiotic potential of these strains.

Gut Symbionts Lactobacillus reuteri R2lc and 2010 Encode a Polyketide Synthase Cluster That Activates the Mammalian Aryl Hydrocarbon Receptor.

A mechanistic understanding of microbe-host interactions is critical to developing therapeutic strategies for targeted modulation of the host immune system. Different members of the gut symbiont species Lactobacillus reuteri modulate host health by, for example, reduction of intestinal inflammation. Previously, it was shown that L. reuteri activates the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that plays an important role in the mucosal immune system, by the production of tryptophan catabolites. Here, we identified a novel pathway by which L. reuteri activates AhR, which is independent of tryptophan metabolism. We screened a library of 36 L. reuteri strains and determined that R2lc and 2010, strains with a pigmented phenotype, are potent AhR activators. By whole-genome sequencing and comparative genomics, we identified genes unique to R2lc and 2010. Our analyses demonstrated that R2lc harbors two genetically distinct polyketide synthase (PKS) clusters, functionally unknown (fun) and pks, each carried by a multicopy plasmid. Inactivation of pks, but not fun, abolished the ability of R2lc to activate AhR. L. reuteri 2010 has a gene cluster homologous to the pks cluster in R2lc with an identical gene organization, which is also responsible for AhR activation. In conclusion, we identified a novel PKS pathway in L. reuteri R2lc and 2010 that is responsible for AhR activation.IMPORTANCE Temporary changes in the composition of the microbiota, for example, by oral administration of probiotics, can modulate the host immune system. However, the underlying mechanisms by which probiotics interact with the host are often unknown. Here, we show that Lactobacillus reuteri R2lc and 2010 harbor an orthologous PKS gene cluster that activates the aryl hydrocarbon receptor (AhR). AhR is a ligand-activated transcription factor that plays a key role in a variety of diseases, including amelioration of intestinal inflammation. Understanding the mechanism by which a bacterium modulates the immune system is critical for applying rational selection strategies for probiotic supplementation. Finally, heterologous and/or optimized expression of PKS is a logical next step toward the development of next-generation probiotics to prevent and treat disease.

d-Alanyl-d-Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria.

The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus Finally, our system represents the first "plug and play" counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)-an essential enzyme involved in peptidoglycan synthesis-increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

Gut Microbiota in Children Hospitalized with Oedematous and Non-Oedematous Severe Acute Malnutrition in Uganda.

BACKGROUND: Severe acute malnutrition (SAM) among children remains a major health problem in many developing countries. SAM manifests in both an oedematous and a non-oedematous form, with oedematous malnutrition in its most severe form also known as kwashiorkor. The pathogenesis of both types of malnutrition in children remains largely unknown, but gut microbiota (GM) dysbiosis has recently been linked to oedematous malnutrition. In the present study we aimed to assess whether GM composition differed between Ugandan children suffering from either oedematous or non-oedematous malnutrition. METHODOLOGY/PRINCIPAL FINDINGS: As part of an observational study among children hospitalized with SAM aged 6-24 months in Uganda, fecal samples were collected at admission. Total genomic DNA was extracted from fecal samples, and PCR amplification was performed followed by Denaturing Gradient Gel Electrophoresis (DGGE) and tag-encoded 16S rRNA gene-targeted high throughput amplicon sequencing. Alpha and beta diversity measures were determined along with ANOVA mean relative abundance and G-test of independence followed by comparisons between groups. Of the 87 SAM children included, 62% suffered from oedematous malnutrition, 66% were boys and the mean age was 16.1 months. GM composition was found to differ between the two groups of children as determined by DGGE (p = 0.0317) and by high-throughput sequencing, with non-oedematous children having lower GM alpha diversity (p = 0.036). However, beta diversity analysis did not reveal larger differences between the GM of children with oedematous and non-oedematous SAM (ANOSIM analysis, weighted UniFrac, R = -0.0085, p = 0.584; unweighted UniFrac, R = 0.0719, p = 0.011). CONCLUSIONS/SIGNIFICANCE: Our results indicate that non-oedematous SAM children have lower GM diversity compared to oedematous SAM children, however no clear compositional differences were identified.

Aflatoxin M1 in Tarhana chips.

Tarhana chips are a popular traditional fermented food consumed widely in the Kahramanmaras region of Turkey. Tarhana chips are different from many other types of fermented food in that they are produced in the form of tortilla chips. Cereal and yoghurt are the main ingredients in Tarhana chips. Aflatoxin M1 (AFM1) levels in dairy and dairy-based products are of concern for human health. To investigate AFM1 contamination, a total of 40 samples were collected from Kahramanmaras region and AFM1 levels were determined by competitive enzyme-linked immunosorbent assay (ELISA). Furthermore, physicochemical characteristics of Tarhana chips were investigated and compared with classic fried chips in terms of nutritional value. Based on data obtained from enzyme-linked immunosorbent assay, 21 (52.5%) out of 40 samples contained AFM1 in the range 0.5-36.6 ng/kg, so AFM1 levels of all samples were below the legal limit.