Genotypic and Phenotypic comparison of Bacteroides ovatus, B. thetaiotaomicron, and B. xylanisolvens isolates obtained from cow, goat, human, and pig feces

ABSTRACT Gastrointestinal Bacteroides play an important role in the health of their hosts. Do these Bacteroides perform the same functions in hosts that have different diets or gastrointestinal physiology? Do they show any co-evolution with the host environment? Within this study103 isolates of B. ovatus, B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence based-PCR (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high percent sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species, but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine and xanthine, purine derivatives, were utilized by most isolates, followed by a few amino sugars. Only rep-PCR analysis demonstrated host specific patterns, indicating that genomic changes due to co-evolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. In the second part of the study 24 isolates, from the original 103, of B. ovatus, B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using whole genome sequencing to determine possible genotypic differences among Bacteroides species and among same species from different host origins. The GC% content and gene numbers of the isolates compared to their type strain were similar but variation in overall genome size was seen in isolates of the same species. A broad analysis of the Clusters of Orthologous groups (COGs) did not show host specificity. A closer look at 4 COG categories (amino acid, carbohydrate, cell wall, and signal transduction) was done. At this level of analysis some differences between species and isolates from differing hosts was evident. Polysaccharide utilization loci (PULs) were compared for homology against the type strains of the isolates. There was variation in the number of homologous and non-homologous PULs and total number of PULs in the isolates. Differences among glycoside hydrolase (GH) families of the isolates of different Bacteroides species were found; comparison of the Bacteroides xylanisolvens isolates demonstrated host related differences for pig isolates compared to other host origins. Differences among homologous capsular polysaccharide (CPS) loci were seen in the downstream gene content of some CPS loci in isolates from different hosts. The results suggest that many of the genetic functions of isolates in this Bacteroides clade are conserved but that there are some differences in genomes by Bacteroides species and mammalian host origin

Interleukin-13 and interleukin-33 mRNA are underexpressed in the duodenal mucosa of German Shepherd dogs with chronic enteropathy

Background A recent genome‐wide association study in German Shepherd dogs (GSDs) with chronic enteropathy (CE) has identified polymorphisms in the Th2 cytokine genes. Hypothesis/objective To determine if the expression of the Th2 cytokines, interleukin‐13 (IL‐13) and interleukin‐33 (IL‐33), is altered in the duodenal mucosa of GSDs with CE compared to non‐GSDs with CE and healthy dogs. Animals Twenty client‐owned dogs diagnosed with CE (10 GSDs and 10 non‐GSDs) at the Bristol Veterinary School and 8 healthy Beagle dogs from the Iowa State University Service Colony. Methods Retrospective study using archived paraffin‐embedded duodenal biopsy samples. A novel RNA in situ hybridization technology (RNAscope) was used to hybridize IL‐13 and IL‐33 mRNA probes onto at least 10 sections from duodenal biopsy samples for each dog. RNAscope signals were visualized using a microscope and semi‐quantitative assessment was performed by a single operator. Results Based on duodenal villus, subvillus, epithelial, and lamina propria average expression scores, GSDs with CE had significantly lower IL‐13 and IL‐33 mRNA expression compared to non‐GSDs with CE (IL‐13, P \u3c .04; IL‐33, P \u3c .02) and healthy Beagle dogs (IL‐13, P \u3c .02; IL‐33, P \u3c .004). Conclusions and Clinical Importance Similar to human patients with ulcerative colitis, a subtype of human inflammatory bowel disease, these data indicate that Th2 cytokines may be involved in the pathogenesis of CE in GSDs

Indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-1) mRNA is over-expressed in the duodenal mucosa and is negatively correlated with serum tryptophan concentrations in dogs with protein-losing enteropathy

Introduction Dogs with protein-losing enteropathy (PLE) have decreased serum tryptophan concentrations, which may contribute to disease pathogenesis. Indoleamine-pyrrole 2,3-dioxygenase-1 (IDO-1) expression is associated with low serum tryptophan concentrations and is increased in the gastrointestinal tract of humans with inflammatory bowel disease (IBD). Therefore, the objective of our study was to determine if the mRNA expression of IDO-1 is increased in the duodenal mucosa of dogs with PLE as compared to dogs with chronic enteropathy (CE) and healthy dogs, and whether this expression is correlated with changes in serum tryptophan concentration. Methods Our study was a retrospective study using archived paraffin-embedded duodenal biopsy specimens from 8 healthy Beagle dogs from the Iowa State University Canine Service Colony and 18 and 6 client-owned dogs diagnosed with CE and PLE, respectively at the Bristol Veterinary School. A novel RNA in situ hybridization (ISH) technology, RNAscope, was used to identify IDO-1 mRNA mucosal expression in duodenal tissues. An IDO-1 specific probe was hybridized onto 10 duodenal biopsy sections from each dog whereby RNAscope signal (mRNA expression) was quantified by a single operator using light microscopy. Results Dogs with PLE had significantly higher mRNA expression of IDO-1 in the duodenal mucosa compared to healthy dogs (mucosal percentage IDO-1 positive: P = 0.0093, (mean ± S.D) control: 19.36 ± 7.08, PLE: 34.12 ± 5.98, average fold difference: 1.76 and mucosal IDO-1 H-score: P = 0.0356, (mean ± S.D) control: 45.26 ± 19.33, PLE: 84.37 ± 19.86, average fold difference: 1.86). The duodenal mucosal mRNA expression of IDO-1 was negatively correlated with serum tryptophan concentrations in dogs with PLE (mucosal IDO-1 H-score: Spearman’s rank correlation coefficient = -0.94, P = 0.0048). Conclusions In conclusion, our study suggests that decreased serum tryptophan concentrations in dogs with PLE is associated with increased intestinal IDO-1 expression. Further studies are needed to determine potential inflammatory pathways responsible for increased expression of IDO-1 in the intestinal tract of dogs with PLE

Helicobacter bilis Infection Alters Mucosal Bacteria and Modulates Colitis Development in Defined Microbiota Mice

Background: Helicobacter bilis infection of C3H/HeN mice harboring the altered Schaedler flora (ASF) triggers progressive immune responsiveness and the development of colitis. We sought to investigate temporal alterations in community structure of a defined (ASF-colonized) microbiota in normal and inflamed murine intestines and to correlate microbiota changes to histopathologic lesions. Methods: The colonic mucosal microbiota of healthy mice and ASF mice colonized with H. bilis for 3, 6, or 12 weeks were investigated by fluorescence in situ hybridization targeting the 16S ribosomal RNA genes of total bacteria, group-specific organisms, and individual ASF bacterial species. Microbial profiling of ASF and H. bilis abundance was performed on cecal contents. Results: Helicobacter bilis–colonized mice developed colitis associated with temporal changes in composition and spatial distribution of the mucosal microbiota. The number of total bacteria, ASF519, and helicobacter-positive bacteria were increased (P , 0.05), whereas ASF360/361-positive bacteria were decreased (P , 0.05) versus controls. Adherent biofilms in colitic mice were most often (P , 0.05) composed of total bacteria, ASF457, and H. bilis. Total numbers of ASF519 and H. bilis bacteria were positively correlated (P ¼ 0.03, r ¼ 0.39 and P , 0.0001, r ¼ 0.73), and total numbers of ASF360/361 bacteria were negatively correlated (P ¼ 0.003, r ¼ 20.53) to histopathologic score. Differences in cecal abundance of ASF members were not observed. Conclusions: Altered community structure with murine colitis is characterized by distinct ASF bacteria that interact with the colonic mucosa, by formation of an isolating interlaced layer, by attachment, or by invasion, and this interaction is differentially expressed over time

Phenotypic and Genomic Diversification in Complex Carbohydrate-Degrading Human Gut Bacteria

Symbiotic bacteria are responsible for the majority of complex carbohydrate digestion in the human colon. Since the identities and amounts of dietary polysaccharides directly impact the gut microbiota, determining which microorganisms consume specific nutrients is central for defining the relationship between diet and gut microbial ecology. Using a custom phenotyping array, we determined carbohydrate utilization profiles for 354 members of the Bacteroidetes, a dominant saccharolytic phylum. There was wide variation in the numbers and types of substrates degraded by individual bacteria, but phenotype-based clustering grouped members of the same species indicating that each species performs characteristic roles. The ability to utilize dietary polysaccharides and endogenous mucin glycans was negatively correlated, suggesting exclusion between these niches. By analyzing related Bacteroides ovatus/Bacteroides xylanisolvens strains that vary in their ability to utilize mucin glycans, we addressed whether gene clusters that confer this complex, multilocus trait are being gained or lost in individual strains. Pangenome reconstruction of these strains revealed a remarkably mosaic architecture in which genes involved in polysaccharide metabolism are highly variable and bioinformatics data provide evidence of interspecies gene transfer that might explain this genomic heterogeneity. Global transcriptomic analyses suggest that the ability to utilize mucin has been lost in some lineages of B. ovatus and B. xylanisolvens, which harbor residual gene clusters that are involved in mucin utilization by strains that still actively express this phenotype. Our data provide insight into the breadth and complexity of carbohydrate metabolism in the microbiome and the underlying genomic events that shape these behaviors

Glucocorticoid and dietary effects on mucosal microbiota in canine inflammatory bowel disease

The pathogenesis of canine inflammatory bowel disease (IBD) involves complex interactions between mucosal immunity and the intestinal microbiota. Glucocorticoids are commonly administered to reduce mucosal inflammation and gastrointestinal signs. The study objective was to evaluate the effects of diet and oral prednisone on the spatial distribution of mucosal bacteria in IBD dogs. Eight dogs diagnosed with IBD were treated with immunosuppressive doses of prednisone. The mucosal microbiota from endoscopic biopsies of IBD dogs and healthy controls (HC; n = 15 dogs) was evaluated by fluorescence in situ hybridization (FISH) targeting the 16S rRNA genes of total bacteria and bacterial species relevant in canine/human IBD. Apicaljunction protein (AJP) expression using immunohistochemistry investigated the effect of medical therapy on intestinal barrier integrity. All IBD dogs had a reduction in GI signs following diet and prednisone therapy compared with baseline CIBDAI scores (P \u3c 0.05). The mucosal microbiota of HC and diseased dogs was most abundant in free and adherent mucus. Only Lactobacilli were increased (P \u3c 0.05) in the adherent mucus of IBD dogs compared to HC. The spatial distribution of mucosal bacteria was significantly different (P \u3c 0.05) in IBD dogs following prednisone therapy, with higher numbers of Bifidobacteria and Streptococci detected across all mucosal compartments and increased numbers of Bifidobacterium spp., Faecalibacterium spp., and Streptococcus spp. present within adherent mucus. Differences in intestinal AJPs were detected with expression of occludin increased (P \u3c 0.05) in IBD dogs versus HC. The expressions of occludin and E-cadherin were increased but zonulin decreased (P \u3c 0.05 for each) in IBD dogs following prednisone therapy. In conclusion, the spatial distribution of mucosal bacteria differs between IBD and HC dogs, and in response to diet and glucocorticoid administration. Medical therapy was associated with beneficial changes in microbial community structure and enhanced mucosal epithelial AJP expression

Randomized, controlled trial evaluating the effect of multi-strain probiotic on the mucosal microbiota in canine idiopathic inflammatory bowel disease

The intestinal microbiota is increasingly linked to the pathogenesis of idiopathic inflammatory bowel disease (IBD) in dogs. While studies have reported alterations in fecal (luminal) microbial populations, only limited information is available about the mucosal microbiota of IBD dogs at diagnosis and following medical therapy. Our aim was to characterize the mucosal microbiota and determine the clinical, microbiological, and mucosal homeostatic effects of probiotic treatment in dogs with IBD. Thirty four IBD dogs were randomized to receive standard therapy (ST = diet + prednisone) with or without probiotic. Tissue sections from endoscopic biopsies were evaluated by fluorescence in situ hybridization (FISH) on a quantifiable basis. Disease activity and changes in mucosal microbiota and tight junction protein (TJP) expression were assessed before and after 8 weeks of IBD therapy. ST and ST/probiotic therapy modulated the number of mucosal bacteria of IBD dogs in a similar fashion. Both treatments increased the numbers of total bacteria and individual species residing within adherent mucus, with ST therapy increasing Bifidobacterium spp. and ST/probiotic therapy increasing Lactobacillus spp (P \u3c 0.05 for both), respectively. Both treatments were associated with rapid clinical remission but not improvement in histopathologic inflammation. Probiotic therapy was associated with upregulated (P \u3c 0.05) expression of TJPs E-cadherin, occludin, and zonulin versus ST. The probiotic effect on mucosal bacteria is similar to that of IBD dogs receiving ST. IBD dogs fed probiotic had increased TJP expression suggesting that probiotic may have beneficial effects on mucosal homeostasis

Cultivation and sequencing of rumen microbiome members from the Hungate1000 Collection

Productivity of ruminant livestock depends on the rumen microbiota, which ferment indigestible plant polysaccharides into nutrients used for growth. Understanding the functions carried out by the rumen microbiota is important for reducing greenhouse gas production by ruminants and for developing biofuels from lignocellulose. We present 410 cultured bacteria and archaea, together with their reference genomes, representing every cultivated rumen-associated archaeal and bacterial family. We evaluate polysaccharide degradation, short-chain fatty acid production and methanogenesis pathways, and assign specific taxa to functions. A total of 336 organisms were present in available rumen metagenomic data sets, and 134 were present in human gut microbiome data sets. Comparison with the human microbiome revealed rumen-specific enrichment for genes encoding de novo synthesis of vitamin B12, ongoing evolution by gene loss and potential vertical inheritance of the rumen microbiome based on underrepresentation of markers of environmental stress. We estimate that our Hungate genome resource represents ?75% of the genus-level bacterial and archaeal taxa present in the rumen.publishersversionPeer reviewe

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a ‘selfish’ model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet