Indigenous Populations of a Biological Control Agent in Agricultural Field Soils Predicted Suppression of a Plant Pathogen.

The nematophagous fungus Hyalorbilia oviparasitica and relatives (Hyalorbilia spp.) are known to parasitize several endoparasitic nematodes. In this project, we hypothesized that indigenous populations of this fungus could be used to predict nematode suppression in agricultural field soils. We quantified Hyalorbilia spp. in soil samples from 44 different sugar beet fields in the Imperial Valley of California. Seven soils harboring Hyalorbilia spp. and two that tested negative for the fungi were examined for nematode suppressive activity. Untreated and autoclaved portions of each soil were planted with cabbage and infested with sugar beet cyst nematode (Heterodera schachtii) juveniles. Females and cysts of H. schachtii were enumerated after 12 weeks. In the seven soils harboring Hyalorbilia spp., females and cysts in the untreated soils were reduced by 61 to 82% compared with the autoclaved controls. Soils with no detectable Hyalorbilia spp. exhibited no nematode suppression. Two novel Hyalorbilia strains, HsImV25 and HsImV27, were isolated from H. schachtii females reared in field soil using an enrichment and double-baiting cultivation technique. Both strains suppressed H. schachtii populations by more than 80% in soil-based assays, confirming that Hyalorbilia spp. are the likely causal agents of the nematode suppression in these soils. This study demonstrated that indigenous populations of a hyperparasite (Hyalorbilia spp.) in agricultural field soils predicted suppressive activity against a soilborne plant pathogen (H. schachtii). To our knowledge, this is the first report to demonstrate this capability. We anticipate that this research will provide a blueprint for other similar studies, thereby advancing the field of soilborne biological control.

Impact of various high fat diets on gene expression and the microbiome across the mouse intestines.

High fat diets (HFDs) have been linked to several diseases including obesity, diabetes, fatty liver, inflammatory bowel disease (IBD) and colon cancer. In this study, we examined the impact on intestinal gene expression of three isocaloric HFDs that differed only in their fatty acid composition-coconut oil (saturated fats), conventional soybean oil (polyunsaturated fats) and a genetically modified soybean oil (monounsaturated fats). Four functionally distinct segments of the mouse intestinal tract were analyzed using RNA-seq-duodenum, jejunum, terminal ileum and proximal colon. We found considerable dysregulation of genes in multiple tissues with the different diets, including those encoding nuclear receptors and genes involved in xenobiotic and drug metabolism, epithelial barrier function, IBD and colon cancer as well as genes associated with the microbiome and COVID-19. Network analysis shows that genes involved in metabolism tend to be upregulated by the HFDs while genes related to the immune system are downregulated; neurotransmitter signaling was also dysregulated by the HFDs. Genomic sequencing also revealed a microbiome altered by the HFDs. This study highlights the potential impact of different HFDs on gut health with implications for the organism as a whole and will serve as a reference for gene expression along the length of the intestines.

Impact of Various High Fat Diets on Gene Expression and the Microbiome Across the Mouse Intestines.

High fat diets (HFDs) have been linked to several diseases including obesity, diabetes, fatty liver, inflammatory bowel disease (IBD) and colon cancer. In this study, we examined the impact on intestinal gene expression of three isocaloric HFDs that differed only in their fatty acid composition - coconut oil (saturated fats), conventional soybean oil (polyunsaturated fats) and a genetically modified soybean oil (monounsaturated fats). Four functionally distinct segments of the mouse intestinal tract were analyzed using RNA-seq - duodenum, jejunum, terminal ileum and proximal colon. We found considerable dysregulation of genes in multiple tissues with the different diets, including those encoding nuclear receptors and genes involved in xenobiotic and drug metabolism, epithelial barrier function, IBD and colon cancer as well as genes associated with the microbiome and COVID-19. Network analysis shows that genes involved in metabolism tend to be upregulated by the HFDs while genes related to the immune system are downregulated; neurotransmitter signaling was also dysregulated by the HFDs. Genomic sequencing also revealed a microbiome altered by the HFDs. This study highlights the potential impact of different HFDs on gut health with implications for the organism as a whole and will serve as a reference for gene expression along the length of the intestines.

Diet high in linoleic acid dysregulates the intestinal endocannabinoid system and increases susceptibility to colitis in Mice.

Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro. These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

Crohn's disease in endoscopic remission, obesity, and cases of high genetic risk demonstrates overlapping shifts in the colonic mucosal-luminal interface microbiome.

BACKGROUND: Crohn's disease (CD) patients demonstrate distinct intestinal microbial compositions and metabolic characteristics compared to unaffected controls. However, the impact of inflammation and underlying genetic risk on these microbial profiles and their relationship to disease phenotype are unclear. We used lavage sampling to characterize the colonic mucosal-luminal interface (MLI) microbiome of CD patients in endoscopic remission and unaffected controls relative to obesity, disease genetics, and phenotype. METHODS: Cecum and sigmoid colon were sampled from 110 non-CD controls undergoing screening colonoscopy who were stratified by body mass index and 88 CD patients in endoscopic remission (396 total samples). CD polygenic risk score (GRS) was calculated using 186 known CD variants. MLI pellets were analyzed by 16S ribosomal RNA gene sequencing, and supernatants by untargeted liquid chromatography-mass spectrometry. RESULTS: CD and obesity were each associated with decreased cecal and sigmoid MLI bacterial diversity and distinct bacterial composition compared to controls, including expansion of Escherichia/Shigella. Cecal and sigmoid dysbiosis indices for CD were significantly greater in obese controls than non-overweight controls. CD, but not obesity, was characterized by altered biogeographic relationship between the sigmoid and cecum. GRS was associated with select taxonomic shifts that overlapped with changes seen in CD compared to controls including Fusobacterium enrichment. Stricturing or penetrating Crohn's disease behavior was characterized by lower MLI bacterial diversity and altered composition, including reduced Faecalibacterium, compared to uncomplicated CD. Taxonomic profiles including reduced Parasutterella were associated with clinical disease progression over a mean follow-up of 3.7 years. Random forest classifiers using MLI bacterial abundances could distinguish disease state (area under the curve (AUC) 0.93), stricturing or penetrating Crohn's disease behavior (AUC 0.82), and future clinical disease progression (AUC 0.74). CD patients showed alterations in the MLI metabolome including increased cholate:deoxycholate ratio compared to controls. CONCLUSIONS: Obesity, CD in endoscopic remission, and high CD genetic risk have overlapping colonic mucosal-luminal interface (MLI) microbiome features, suggesting a shared microbiome contribution to CD and obesity which may be influenced by genetic factors. Microbial profiling during endoscopic remission predicted Crohn's disease behavior and progression, supporting that MLI sampling could offer unique insight into CD pathogenesis and provide novel prognostic biomarkers.

Fungi associated with the potato taste defect in coffee beans from Rwanda.

BACKGROUND: Potato taste defect (PTD) of coffee is characterized by a raw potato like smell that leads to a lower quality taste in the brewed coffee, and harms the commercial value of some East African coffees. Although several causes for PTD have been proposed, none of them have been confirmed. Recently, high throughput sequencing techniques and bioinformatic analysis have shown great potential for identifying putative causal agents of plant diseases. Toward the goal of determining the cause of PTD, we examined raw coffee beans from Rwanda exhibiting varying PTD scores using an Illumina-based sequence analysis of the fungal rRNA ITS region. RESULTS: Six fungal amplicon sequence variants (ASVs) with high relative abundances correlated with coffee taste scores. Four of these ASVs exhibited negative correlations - Aspergillus versicolor, Penicillium cinnamopurpureum, Talaromyces radicus, and Thermomyces lanuginosus - indicating that they might be causing PTD. Two of these fungi exhibited positive correlations - Kazachstania humilis and Clavispora lusitaniae - indicating that they might be inhibiting organisms that cause PTD. CONCLUSIONS: This study addressed PTD causality from a new angle by examining fungi with high throughput sequencing. To our knowledge, this is the first study characterizing fungi associated with PTD, providing candidates for both causality and biocontrol.

Particle Radiation Side-Effects: Intestinal Microbiota Composition Shapes Interferon-γ-Induced Osteo-Immunogenicity.

Microbiota can both negatively and positively impact radiation-induced bone loss. Our prior research showed that compared to mice with conventional gut microbiota (CM), mice with restricted gut microbiota (RM) reduced inflammatory tumor necrosis factor (TNF) in bone marrow, interleukin (IL)-17 in blood, and chemokine (C-C motif) ligand 20 (CCL20) in bone marrow under anti-IL-17 treatment. We showed that Muribaculum intestinale was more abundant in intestinal epithelial cells (IECs) from the small intestine of female RM mice and positively associated with augmented skeletal bone structure. Female C57BL/6J pun RM mice, which were injected with anti-IL-17 antibody one day before exposure to 1.5 Gy 28Si ions of 850 MeV/u, showed high trabecular numbers in tibiae at 6 weeks postirradiation. Irradiated CM mice were investigated for lower interferon-γ and IL-17 levels in the small intestine than RM mice. IL-17 blockage resulted in bacterial indicator phylotypes being different between both microbiota groups before and after irradiation. Analysis of the fecal bacteria were performed in relation to bone quality and body weight, showing reduced tibia cortical thickness in irradiated CM mice (-15%) vs. irradiated RM mice (-9.2%). Correlation analyses identified relationships among trabecular bone parameters (TRI-BV/TV, Tb.N, Tb.Th, Tb.Sp) and Bacteroides massiliensis, Muribaculum sp. and Prevotella denticola. Turicibacter sp. was found directly correlated with trabecular separation in anti-IL-17 treated mice, whereas an unidentified Bacteroidetes correlated with trabecular thickness in anti-IL-17 neutralized and radiation-exposed mice. We demonstrated radiation-induced osteolytic damage to correlate with bacterial indicator phylotypes of the intestinal microbiota composition, and these relationships were determined from the previously discovered dose-dependent particle radiation effects on cell proliferation in bone tissue. New translational approaches were designed to investigate dynamic changes of gut microbiota in correlation with conditions of treatment and disease as well as mechanisms of systemic side-effects in radiotherapy.

Early-life effects of juvenile Western diet and exercise on adult gut microbiome composition in mice.

Alterations to the gut microbiome caused by changes in diet, consumption of antibiotics, etc., can affect host function. Moreover, perturbation of the microbiome during critical developmental periods potentially has long-lasting impacts on hosts. Using four selectively bred high runner and four non-selected control lines of mice, we examined the effects of early-life diet and exercise manipulations on the adult microbiome by sequencing the hypervariable internal transcribed spacer region of the bacterial gut community. Mice from high runner lines run ∼3-fold more on wheels than do controls, and have several other phenotypic differences (e.g. higher food consumption and body temperature) that could alter the microbiome, either acutely or in terms of coevolution. Males from generation 76 were given wheels and/or a Western diet from weaning until sexual maturity at 6 weeks of age, then housed individually without wheels on standard diet until 14 weeks of age, when fecal samples were taken. Juvenile Western diet reduced bacterial richness and diversity after the 8-week washout period (equivalent to ∼6 human years). We also found interactive effects of genetic line type, juvenile diet and/or juvenile exercise on microbiome composition and diversity. Microbial community structure clustered significantly in relation to both line type and diet. Western diet also reduced the relative abundance of Muribaculum intestinale These results constitute one of the first reports of juvenile diet having long-lasting effects on the adult microbiome after a substantial washout period. Moreover, we found interactive effects of diet with early-life exercise exposure, and a dependence of these effects on genetic background.

Herpes simplex virus infection, Acyclovir and IVIG treatment all independently cause gut dysbiosis.

Herpes simplex virus 1 (HSV) is a ubiquitous human virus resident in a majority of the global population as a latent infection. Acyclovir (ACV), is the standard of care drug used to treat primary and recurrent infections, supplemented in some patients with intravenous immunoglobulin (IVIG) treatment to suppress infection and deleterious inflammatory responses. As many diverse medications have recently been shown to change composition of the gut microbiome, we used Illumina 16S rRNA gene sequencing to determine the effects of ACV and IVIG on the gut bacterial community. We found that HSV, ACV and IVIG can all independently disrupt the gut bacterial community in a sex biased manner when given to uninfected C57BL/6 mice. Treatment of HSV infected mice with ACV or IVIG alone or together revealed complex interactions between these drugs and infection that caused pronounced sex biased dysbiosis. ACV reduced Bacteroidetes levels in male but not female mice, while levels of the Anti-inflammatory Clostridia (AIC) were reduced in female but not male mice, which is significant as these taxa are associated with protection against the development of graft versus host disease (GVHD) in hematopoietic stem cell transplant (HSCT) patients. Gut barrier dysfunction is associated with GVHD in HSCT patients and ACV also decreased Akkermansia muciniphila, which is important for maintaining gut barrier functionality. Cumulatively, our data suggest that long-term prophylactic ACV treatment of HSCT patients may contribute to GVHD and also potentially impact immune reconstitution. These data have important implications for other clinical settings, including HSV eye disease and genital infections, where ACV is given long-term.

High Levels of Oxidative Stress Create a Microenvironment That Significantly Decreases the Diversity of the Microbiota in Diabetic Chronic Wounds and Promotes Biofilm Formation.

Diabetics chronic wounds are characterized by high levels of oxidative stress (OS) and are often colonized by biofilm-forming bacteria that severely compromise healing and can result in amputation. However, little is known about the role of skin microbiota in wound healing and chronic wound development. We hypothesized that high OS levels lead to chronic wound development by promoting the colonization of biofilm-forming bacteria over commensal/beneficial bacteria. To test this hypothesis, we used our db/db-/- mouse model for chronic wounds where pathogenic biofilms develop naturally after induction of high OS immediately after wounding. We sequenced the bacterial rRNA internal transcribed spacer (ITS) gene of the wound microbiota from wound initiation to fully developed chronic wounds. Indicator species analysis, which considers a species' fidelity and specificity, was used to determine which bacterial species were strongly associated with healing wounds or chronic wounds. We found that healing wounds were colonized by a diverse and dynamic bacterial microbiome that never developed biofilms even though biofilm-forming bacteria were present. Several clinically relevant species that are present in human chronic wounds, such as Cutibacterium acnes, Achromobacter sp., Delftia sp., and Escherichia coli, were highly associated with healing wounds. These bacteria may serve as bioindicators of healing and may actively participate in the processes of wound healing and preventing pathogenic bacteria from colonizing the wound. In contrast, chronic wounds, which had high levels of OS, had low bacterial diversity and were colonized by several clinically relevant, biofilm-forming bacteria such as Pseudomonas aeruginosa, Enterobacter cloacae, Corynebacterium frankenforstense, and Acinetobacter sp. We observed unique population trends: for example, P. aeruginosa associated with aggressive biofilm development, whereas Staphylococcus xylosus was only present early after injury. These findings show that high levels of OS in the wound significantly altered the bacterial wound microbiome, decreasing diversity and promoting the colonization of bacteria from the skin microbiota to form biofilm. In conclusion, bacteria associated with non-chronic or chronic wounds could function as bioindicators of healing or non-healing (chronicity), respectively. Moreover, a better understanding of bacterial interactions between pathogenic and beneficial bacteria within an evolving chronic wound microbiota may lead to better solutions for chronic wound management.

The autoimmune susceptibility gene, PTPN2, restricts expansion of a novel mouse adherent-invasive E. coli.

Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation - suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if PTPN2 deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2, we identified increased abundance of a novel mouse AIEC (mAIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, mAIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, mAIEC infection increased the severity of, and prevented recovery from, induced colitis. Although mAIEC genome sequence analysis showed >90% similarity to LF82, mAIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of mAIEC. We show for the first time that an IBD susceptibility gene, PTPN2, modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.

Intestinal bacterial indicator phylotypes associate with impaired DNA double-stranded break sensors but augmented skeletal bone micro-structure.

Intestinal microbiota are considered a sensor for molecular pathways, which orchestrate energy balance, immune responses, and cell regeneration. We previously reported that microbiota restriction promoted higher levels of systemic radiation-induced genotoxicity, proliferative lymphocyte activation, and apoptotic polarization of metabolic pathways. Restricted intestinal microbiota (RM) that harbors increased abundance of Lactobacillus johnsonii (LBJ) has been investigated for bacterial communities that correlated radiation-induced genotoxicity. Indicator phylotypes were more abundant in RM mice and increased in prevalence after whole body irradiation in conventional microbiota (CM) mice, while none of the same ten most abundant phylotypes were different in abundance between CM mice before and after heavy ion irradiation. Muribaculum intestinale was detected highest in female small intestines in RM mice, which were lacking Ureaplasma felinum compared with males, and thus these bacteria could be contributing to the differential amounts of radiation-induced systemic genotoxicity between the CM and RM groups. Helicobacter rodentium and M.intestinale were found in colons in the radiation-resistant CM phenotype. While the expression of interferon-γ was elevated in the small intestine, and lower in blood in CM mice, high-linear energy transfer radiation reduced transforming growth factor-ß with peripheral interleukin (IL)-17 in RM mice, particularly in females. We found that female RM mice showed improved micro-architectural bone structure and anti-inflammatory radiation response compared with CM mice at a delayed phase 6 weeks postexposure to particle radiation. However, microbiota restriction reduced inflammatory markers of tumor necrosis factor in marrow, when IL-17 was reduced by intraperitoneal injection of IL-17 neutralizing antibody.

High Levels of Oxidative Stress and Skin Microbiome are Critical for Initiation and Development of Chronic Wounds in Diabetic Mice.

A balanced redox state is critical for proper healing. Although human chronic wounds are characterized by high levels of oxidative stress (OS), whether OS levels are critical for chronic wound development is not known. For these studies, we used our chronic wound model in diabetic mice that has similar characteristics as human chronic wounds, including naturally developed biofilm. We hypothesize that OS levels in wound tissues are critical for chronic wound initiation and development. We show that increased OS levels in the wound correlate with increased chronicity. Moreover, without increased OS levels, biofilm taken from chronic wounds and placed in new excision wounds do not create chronic wounds. Similarly, high OS levels in the wound tissue in the absence of the skin microbiome do not lead to chronic wounds. These findings show that both high OS levels and bacteria are needed for chronic wound initiation and development. In conclusion, OS levels in the wound at time of injury are critical for biofilm formation and chronic wound development and may be a good predictor of the degree of wound chronicity. Treating such wounds might be accomplished by managing OS levels with antioxidants combined with manipulation of the skin microbiome after debridement.

Dominant Role of the Gut Microbiota in Chemotherapy Induced Neuropathic Pain.

Chemotherapy induced peripheral neuropathy (CIPN), a toxic side effect of some cancer treatments, negatively impacts patient outcomes and drastically reduces survivor's quality of life (QOL). Uncovering the mechanisms driving chemotherapy-induced CIPN is urgently needed to facilitate the development of effective treatments, as currently there are none. Observing that C57BL/6 (B6) and 129SvEv (129) mice are respectively sensitive and resistant to Paclitaxel-induced pain, we investigated the involvement of the gut microbiota in this extreme phenotypic response. Reciprocal gut microbiota transfers between B6 and 129 mice as well as antibiotic depletion causally linked gut microbes to Paclitaxel-induced pain sensitivity and resistance. Microglia proliferated in the spinal cords of Paclitaxel treated mice harboring the pain-sensitive B6 microbiota but not the pain-resistant 129 microbiota, which exhibited a notable absence of infiltrating immune cells. Paclitaxel decreased the abundance of Akkermansia muciniphila, which could compromise barrier integrity resulting in systemic exposure to bacterial metabolites and products - that acting via the gut-immune-brain axis - could result in altered brain function. Other bacterial taxa that consistently associated with both bacteria and pain as well as microglia and pain were identified, lending support to our hypothesis that microglia are causally involved in CIPN, and that gut bacteria are drivers of this phenotype.

Bacterial Microbiome Dynamics in Post Pull-Through Hirschsprung-Associated Enterocolitis (HAEC): An Experimental Study Employing the Endothelin Receptor B-Null Mouse Model.

PURPOSE: Hirschsprung-associated enterocolitis (HAEC) is the most frequent potentially life-threatening complication in children with Hirschsprung disease (HSCR) even after definitive corrective surgery. Mounting evidence suggests that intestinal microbiota likely contribute to the etiology of enterocolitis, so the aim of this study was to use a mouse model of post pull-through HAEC to compare the fecal bacterial communities of animals which developed HAEC to those free of enterocolitis. METHODS: Ten Ednrb -/- and 8wild type mice underwent the microsurgical pull-through surgery, and stool was collected at the time of surgery, and then either at 2 and 4 weeks after the operation, or when the mice developed enterocolitis. The mid-colon of all animals was collected, prepared and histologically graded for enterocolitis. Fecal DNA was isolated and bacterial 16S rRNA genes analyzed using Illumina sequencing. RESULTS: Six Ednrb-/- mice developed HAEC with a mean enterocolitis score of 5.7, while the remaining 4 mutant and 8 WT mice remained free of enterocolitis by 4 weeks. The HAEC group had lower alpha diversity by Chao1 analysis compared with WT group, while the Ednrb-/- mice demonstrated distinct bacterial communities from WT mice on beta diversity analysis. The most striking finding was increased proportion of Akkermansia and reduced Bacteroidetes compared with the NO HAEC and WT groups, suggesting Akkermansia may contribute to development of enterocolitis while Bacteroidetes may be protective. Less abundant genera that were reduced in HAEC were Dysgonomas and Clostridium XIVa which may play a protective role. CONCLUSIONS: This is the first study to identify Akkermansia as potentially playing a role in HAEC, either as a pathobiont taxa contributing to pathogenesis of enterocolitis, or possibly a protective commensal taxa expanded in response to inflammation. These findings characterized the dynamic shifts in the gut microbial communities through the onset of post pull-through HAEC, and suggests that there may be identifiable bacterial community differences in HSCR patients that are high risk for developing HAEC.

Concurrent gut transcriptome and microbiota profiling following chronic ethanol consumption in nonhuman primates.

Alcohol use disorder (AUD) results in increased intestinal permeability, nutrient malabsorption, and increased risk of colorectal cancer (CRC). Our understanding of the mechanisms underlying these morbidities remains limited because studies to date have relied almost exclusively on short-term heavy/binge drinking rodent models and colonic biopsies/fecal samples collected from AUD subjects with alcoholic liver disease (ALD). Consequently, the dose- and site-dependent impact of chronic alcohol consumption in the absence of overt liver disease remains poorly understood. In this study, we addressed this knowledge gap using a nonhuman primate model of voluntary ethanol self-administration where rhesus macaques consume varying amounts of 4% ethanol in water for 12 months. Specifically, we performed RNA-Seq and 16S rRNA gene sequencing on duodenum, jejunum, ileum, and colon biopsies collected from 4 controls and 8 ethanol-consuming male macaques. Our analysis revealed that chronic ethanol consumption leads to changes in the expression of genes involved in protein trafficking, metabolism, inflammation, and CRC development. Additionally, we observed differences in the relative abundance of putatively beneficial bacteria as well as those associated with inflammation and CRC. Given that the animals studied in this manuscript did not exhibit signs of ALD or CRC, our data suggest that alterations in gene expression and bacterial communities precede clinical disease and could serve as biomarkers as well as facilitate future studies aimed at developing interventions to restore gut homeostasis.

Microbial, metabolomic, and immunologic dynamics in a relapsing genetic mouse model of colitis induced by T-synthase deficiency.

Intestinal dysbiosis is thought to confer susceptibility to inflammatory bowel disease (IBD), but it is unknown whether dynamic changes in the microbiome contribute to fluctuations in disease activity. We explored this question using mice with intestine-specific deletion of C1galt1 (also known as T-synthase) (Tsyn mice). These mice develop spontaneous microbiota-dependent colitis with a remitting/relapsing course due to loss of mucin core-1 derived O-glycans. 16S rRNA sequencing and untargeted metabolomics demonstrated age-specific perturbations in the intestinal microbiome and metabolome of Tsyn mice compare with littermate controls at weeks 3 (disease onset), 5 (during remission), and 9 (after relapse). Colitis remission corresponded to increased levels of FoxP3+RORγt+CD4+ T cells in the colonic lamina propria that were positively correlated with operational taxonomic units (OTUs) in the S24-7 family and negatively correlated with OTUs in the Clostridiales order. Relapse was characterized by marked expansion of FoxP3-RORγt+CD4+ T cells expressing IFNγ and IL17A, which were associated with Clostridiales OTUs distinct from those negatively correlated with FoxP3+RORγt+CD4+ T cells. Our findings suggest that colitis remission and relapse in the Tsyn model may reflect alterations in the microbiome due to reduced core-1 O-glycosylation that shift the balance of regulatory and pro-inflammatory T cell subsets. We investigated whether genetic variation in C1galt1 correlated with the microbiome in a cohort of 78 Crohn's disease patients and 101 healthy controls. Polymorphisms near C1galt1 (rs10486157) and its molecular chaperone, Cosmc (rs4825729), were associated with altered composition of the colonic mucosal microbiota, supporting the relevance of core-1 O-glycosylation to host regulation of the microbiome.

Chemopreventive Metabolites Are Correlated with a Change in Intestinal Microbiota Measured in A-T Mice and Decreased Carcinogenesis.

Intestinal microbiota play a significant role in nutrient metabolism, modulation of the immune system, obesity, and possibly in carcinogenesis, although the underlying mechanisms resulting in disease or impacts on longevity caused by different intestinal microbiota are mostly unknown. Herein we use isogenic Atm-deficient and wild type mice as models to interrogate changes in the metabolic profiles of urine and feces of these mice, which are differing in their intestinal microbiota. Using high resolution mass spectrometry approach we show that the composition of intestinal microbiota modulates specific metabolic perturbations resulting in a possible alleviation of a glycolytic phenotype. Metabolites including 3-methylbutyrolactone, kyneurenic acid and 3-methyladenine known to be onco-protective are elevated in Atm-deficient and wild type mice with restricted intestinal microbiota. Thus our approach has broad applicability to study the direct influence of gut microbiome on host metabolism and resultant phenotype. These results for the first time suggest a possible correlation of metabolic alterations and carcinogenesis, modulated by intestinal microbiota in A-T mice.

A Disease-Associated Microbial and Metabolomics State in Relatives of Pediatric Inflammatory Bowel Disease Patients.

BACKGROUND & AIMS: Microbes may increase susceptibility to inflammatory bowel disease (IBD) by producing bioactive metabolites that affect immune activity and epithelial function. We undertook a family based study to identify microbial and metabolic features of IBD that may represent a predisease risk state when found in healthy first-degree relatives. METHODS: Twenty-one families with pediatric IBD were recruited, comprising 26 Crohn's disease patients in clinical remission, 10 ulcerative colitis patients in clinical remission, and 54 healthy siblings/parents. Fecal samples were collected for 16S ribosomal RNA gene sequencing, untargeted liquid chromatography-mass spectrometry metabolomics, and calprotectin measurement. Individuals were grouped into microbial and metabolomics states using Dirichlet multinomial models. Multivariate models were used to identify microbes and metabolites associated with these states. RESULTS: Individuals were classified into 2 microbial community types. One was associated with IBD but irrespective of disease status, had lower microbial diversity, and characteristic shifts in microbial composition including increased Enterobacteriaceae, consistent with dysbiosis. This microbial community type was associated similarly with IBD and reduced microbial diversity in an independent pediatric cohort. Individuals also clustered bioinformatically into 2 subsets with shared fecal metabolomics signatures. One metabotype was associated with IBD and was characterized by increased bile acids, taurine, and tryptophan. The IBD-associated microbial and metabolomics states were highly correlated, suggesting that they represented an integrated ecosystem. Healthy relatives with the IBD-associated microbial community type had an increased incidence of elevated fecal calprotectin. CONCLUSIONS: Healthy first-degree relatives can have dysbiosis associated with an altered intestinal metabolome that may signify a predisease microbial susceptibility state or subclinical inflammation. Longitudinal prospective studies are required to determine whether these individuals have a clinically significant increased risk for developing IBD.