Shallowater, TX, United States
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Dowd S.E.,Molecular Research Mr Dna | Davis B.,Texas Tech University Health Sciences Center | Galandiuk S.,University of Louisville | Chamberlin W.M.,Sisters of Charity of Leavenworth Health System | Kuenstner J.T.,Charleston Laboratories
Journal of Clinical Gastroenterology | Year: 2013

OBJECTIVE:: To determine whether bacterial pathogens can be detected within the diseased submucosal tissues of patients with Crohn's disease by molecular techniques independent of cultural methods. DESIGN:: We designed a quantitative polymerase chain reaction to detect 32 virulence genes and transposons within submucosal tissues of patients with Crohn's disease and controls and compared the microbiome of the submucosa with mucosal bacterial populations. RESULTS:: Within submucosal tissues, the bacterial invasion/adherence genes eaeA and invA were detected in 43% of patients (P=0.01 and 0.008 vs. mucosa and controls, respectively) and the Mycobacterium-specific IS900 and 251F genes detected in 50% of patients (P=0.03 vs. mucosa and controls). These findings were mutually exclusive: invasion/adhesion genes and Mycobacterium-associated transposons were not detected in the same patient. Metagenomic sequencing and quantitative polymerase chain reaction results confirmed effective separation of the submucosal and mucosal microbiome and the existence of a submucosal bacterial population within diseased tissues. CONCLUSIONS:: This study is the first to examine the microbial populations of submucosal tissues during intestinal disease and provide evidence of a distinct submucosal microbiome and biotypes within Crohn's disease. These data suggests that Crohn's disease may not be a single disease, but a spectrum that can be divided into distinct biotypes based on the presence of invasion/adherence genes or Mycobacterium-associated transposons. If corroborated by larger population studies, these findings could revolutionize the diagnosis, management, and treatment of Crohn's disease by the identification of patient biotypes and the application of targeted chemotherapeutic treatments that go beyond supportive in nature. Copyright © 2013 by Lippincott Williams & Wilkins.


Rogers G.B.,University of Queensland | Rogers G.B.,King's College London | Rogers G.B.,Flinders University | Kozlowska J.,King's College London | And 7 more authors.
Scientific Reports | Year: 2014

Despite the fundamental contribution of the gut microbiota to host physiology, the extent of its variation in genetically-identical animals used in research is not known. We report significant divergence in both the composition and metabolism of gut microbiota in genetically-identical adult C57BL/6 mice housed in separate controlled units within a single commercial production facility. The reported divergence in gut microbiota has the potential to confound experimental studies using mammalian models.


PubMed | Children's Healthcare Of Atlanta, University of Chicago, Baylor College of Medicine, Boston University and 8 more.
Type: Journal Article | Journal: Cell reports | Year: 2016

The relationship between the host and its microbiota is challenging to understand because both microbial communities and their environments are highly variable. We have developed a set of techniques based on population dynamics and information theory to address this challenge. These methods identify additional bacterial taxa associated with pediatric Crohn disease and can detect significant changes in microbial communities withfewer samples than previous statistical approaches required. We have also substantially improved the accuracy of the diagnosis based on the microbiota from stool samples, and we found that the ecological niche of a microbe predicts its role in Crohn disease. Bacteria typically residing in the lumen of healthy individuals decrease in disease, whereas bacteria typically residing on the mucosa of healthy individuals increase in disease. Our results also show that the associations with Crohn disease are evolutionarily conserved and provide a mutual information-based method to depict dysbiosis.


PubMed | University of Queensland, Molecular Research MR DNA, Charles River UK and King's College London
Type: | Journal: Scientific reports | Year: 2014

Despite the fundamental contribution of the gut microbiota to host physiology, the extent of its variation in genetically-identical animals used in research is not known. We report significant divergence in both the composition and metabolism of gut microbiota in genetically-identical adult C57BL/6 mice housed in separate controlled units within a single commercial production facility. The reported divergence in gut microbiota has the potential to confound experimental studies using mammalian models.


Kellermayer R.,Baylor College of Medicine | Mir S.A.V.,Baylor College of Medicine | Nagy-Szakal D.,Baylor College of Medicine | Cox S.B.,Research and Testing Laboratory | And 6 more authors.
Journal of Pediatric Gastroenterology and Nutrition | Year: 2012

Objectives: In patients with inflammatory bowel diseases (IBDs), the presence of noncaseating mucosal granuloma is sufficient for diagnosing Crohn disease (CD) and may represent a specific immune response or microbial-host interaction. The cause of granulomas in CD is unknown and their association with the intestinal microbiota has not been addressed with high-throughput methodologies. Methods: The mucosal microbiota from 3 different pediatric centers was studied with 454 pyrosequencing of the bacterial 16S rRNA gene and the fungal small subunit (SSU) ribosomal region in transverse colonic biopsy specimens from 26 controls and 15 treatment-naïve pediatric CD cases. Mycobacterium avium subspecies paratuberculosis (MAP) was tested with real-time polymerase chain reaction. The correlation of granulomatous inflammation with C-reactive protein was expanded to 86 treatment-naïve CD cases. RESULTS:: The CD microbiota separated from controls by distance-based redundancy analysis (P=0.035). Mucosal granulomata found in any portion of the intestinal tract associated with an augmented colonic bacterial microbiota divergence (P=0.013). The granuloma-based microbiota separation persisted even when research center bias was eliminated (P=0.04). Decreased Roseburia and Ruminococcus in granulomatous CD were important in this separation; however, principal coordinates analysis did not reveal partitioning of the groups. CRP levels >1mg/dL predicted the presence of mucosal granulomata (odds ratio 28 [6-134.32]; 73% sensitivity, 91% specificity). Conclusions: Granulomatous CD associates with microbiota separation and C-reactive protein elevation in treatment-naïve children; however, overall dysbiosis in pediatric CD appears rather limited. Geographical/center bias should be accounted for in future multicenter microbiota studies. © 2012 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.


Rice W.C.,U.S. Department of Agriculture | Galyean M.L.,Texas Tech University | Cox S.B.,Research and Testing Laboratories | Dowd S.E.,Molecular Research MR DNA | Cole N.A.,U.S. Department of Agriculture
BMC Microbiology | Year: 2012

Background: The high demand for ethanol in the U.S. has generated large stocks of wet distillers grains (DG), a byproduct from the manufacture of ethanol from corn and sorghum grains. Little is known, however, about the potential influence of dietary DG on fecal microbial community structure. A better understanding of the microbial population in beef cattle feces could be an important monitoring tool to facilitate goals of improving nutrient management, increasing animal growth performance and decreasing odors and/or shedding of pathogens. Five diets consisting of a traditional diet fed to finishing beef cattle in the Southern High Plains of Texas-CON (steam-flaked corn control with 0% DG), and four concentrations of DG in the dietary dry matter; 10 C (10% corn-based DG), 5S (5% sorghum-based DG), 10S (10% sorghum DG), and 15S (15% sorghum DG) were fed to steers at the Texas Tech University Burnett Animal Center. Diets were essentially isonitrogenous with a formulated crude protein value of 13.5%. Results: Fecal grab samples were obtained from 20 steers (n = 4 per diet) and the barcoded DNA pyrosequencing method was used to generate 127,530 16S operational taxonomic units (OTUs). A total of 24 phyla were observed, distributed amongst all beef cattle on all diets, revealing considerable animal to animal variation, however only six phyla (core set) were observed in all animals regardless of dietary treatment. The average abundance and range of abundance, respectively of the core phyla were as follows: Firmicutes (61%, 19 to 83%), Bacteroidetes (28%, 11 to 63%), Proteobacteria (3%, 0.34 to 17.5%), Tenericutes (0.15%, 0.0 to 0.35%), Nitrospirae (0.11%, 0.03 to 0.22%), and Fusobacteria (0.086%, 0.017 to 0.38%). Feeding DG-based diets resulted in significant shifts in the fecal microbial community structure compared with the traditional CON. Four low abundance phyla significantly responded to dietary treatments: Synergistetes (p = 0.01), WS3 (p = 0.054), Actinobacteria (p = 0.06), and Spirochaetes (p = 0.06). Conclusions: This is, to our knowledge, the first study using this method to survey the fecal microbiome of beef cattle fed various concentrations of wet DG. Comparison of our results with other cattle DNA sequencing studies of beef and dairy cattle feces from a variety of geographical locations and different management practices identifies a core set of three phyla shared across all cattle. These three phyla, in order of relative abundance are; Firmicutes, Bacteroidetes, and Proteobacteria. The presence of large animal-to-animal variation in cattle microbiome was noted in our study as well as by others. © 2012 Rice et al; BioMed Central Ltd.


Davinic M.,Texas Tech University | Fultz L.M.,Texas Tech University | Acosta-Martinez V.,U.S. Department of Agriculture | Calderon F.J.,U.S. Department of Agriculture | And 5 more authors.
Soil Biology and Biochemistry | Year: 2012

This study integrated physical, chemical, and molecular techniques to assess relationships between soil bacterial community structures and the quantity and quality of soil organic carbon (SOC) at the soil microenvironment scale (e.g., within different aggregate size-fractions). To accomplish this goal, soil samples (0-5cm) were collected from the Texas High Plains region under a variety of dryland and irrigated cropping systems. The soil was separated into macroaggregates, microaggregates, and silt+clay fractions that were analyzed for (1) bacterial diversity via pyrosequencing of the 16s rRNA gene and (2) SOC quantity and quality using a combustion method and mid-infrared diffuse reflectance spectroscopy (mid-IR), respectively. Results from pyrosequencing showed that each soil microenvironment supported a distinct bacterial community. Similarly, mid-IR data revealed distinct spectral features indicating that these fractions were also distinguished by organic and mineral composition. Macroaggregates showed relatively high abundance of Actinobacteria (excluding order Rubrobacteriales) and α-Proteobacteria and contained the most SOC. Microaggregates showed high relative abundance of Rubrobacteriales and the least amount of SOC. Predominance within the soil microenvironment and correlations along the mid-IR spectra were different between members of the order Rubrobacteriales compared with all other members of the Actinobacteria phyla, suggesting they have different ecological niches. Mid-IR results revealed microaggregates had greater absorbance in the 1370-1450cm -1 region for phenolic and alkyl groups (possibly recalcitrant C). Silt+clay fractions were distinguished by Gemmatimonadetes and OP10 phyla, which positively correlated with spectral absorption in the1250-1150cm -1 range (indicating both degradable and recalcitrant C forms). In contrast to general diversity index measurements, distributions of the more rare bacterial phyla (phyla representing <6% of the identified population) were more important for differentiating between communities in soil microenvironments. To our knowledge, this is the first study to investigate soil bacterial communities among soil aggregates using pyrosequenging and to associate these communities to specific soil C chemistries as indicated by mid-IR absorbance. © 2011 Elsevier Ltd.


Shah R.,Baylor College of Medicine | Cope J.L.,Baylor College of Medicine | Nagy-Szakal D.,Baylor College of Medicine | Dowd S.,Molecular Research MR DNA | And 3 more authors.
Gut Microbes | Year: 2016

Inflammatory bowel diseases (IBD) are chronic intestinal inflammatory disorders characterized by a complex disruption of the physiologic interaction between the host immune system and intestinal microbes precipitated by environmental factors. Numerous observations indicate the altered composition and function of the intestinal microbiome of patients with ulcerative colitis (UC), a subtype of IBD. The accuracy of these results may be limited by confounding factors, such as concurrent medication use. To address these limitations, we examined the colonic mucosal microbiome of pediatric patients with UC prior to initiating treatment. Based on bacterial 16S rRNA gene sequencing, we identified a significant decrease in the phylum Verrucomicrobia in patients with UC. At the genus level, we observed a significant decrease in the short chain fatty acid producer Roseburia. Despite these compositional changes, we did not identify inferred gene content differences between the UC and control groups. To determine if microbial taxa may be associated with clinical outcomes, we retrospectively assessed the clinical course of the UC patients. Despite similar metrics of OTU richness and diversity, multiple OTU differences were observed between patients who responded to therapy and those who did not. Our observations regarding the mucosal microbiome and the associations with differential clinical outcomes support the contributions of gut microbes to disease onset and modulation. © 2016 Taylor & Francis


Glassing A.,Montana State University Billings | Dowd S.E.,Molecular Research Mr Dna | Galandiuk S.,University of Louisville | Davis B.,Texas Tech University Health Sciences Center | And 3 more authors.
Journal of Microbiological Methods | Year: 2015

Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass. © 2015 Elsevier B.V.


PubMed | University of Louisville, Texas Tech University Health Sciences Center, Molecular Research Mr Dna and Montana State University Billings
Type: | Journal: Journal of microbiological methods | Year: 2015

Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40-70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass.

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