Transitional and temporal changes in the mucosal and submucosal intestinal microbiota in advanced Crohn's disease of the terminal ileum.

PURPOSE: Crohn's disease is a chronic debilitating intestinal syndrome of unknown aetiology that is thought to result in part from an imbalance (dysbiosis) of the intestinal microbial populations, known as the microbiota. In this study we sought to compare the microbiota at the mucosal and submucosal levels at the resection margin in Crohn's disease to those in other intestinal dysbiotic disease controls to determine the level of bacterial translocation. METHODOLOGY: 16S microbiota sequencing was performed on DNA extracted from mucosal and submucosal samples from resected intestinal tissues from Crohn's disease and controls. RESULTS: Grossly normal appearing tissue at the resection margin showed early signs, suggesting bacterial translocation, with two bacterial families having penetrated the mucosal surfaces. In contrast, 4 and 13 bacterial families were present within submucosal tissues at the disease centre and disease margin, respectively. Although there was no significant difference in biodiversity, there was increased bacterial richness in the Crohn's disease group as compared to non-IBD controls. CONCLUSION: The presence and/or absence of certain bacteria suggested disease-specific ecological or micro-environmental pressures driving or excluding certain organisms in Crohn's disease. The data suggest that several of the dysbiotic conditions previously reported for Crohn's disease are not unique but common to general dysbiosis. The examination of multiple intestinal sites in advanced disease may provide a spectrum of disease from early onset at the resection margin to active disease at the disease margin and late-stage fibrostenotic disease at the centre of the lesion, and a unique etiopathogenic view of Crohn's disease.

The predominant site of bacterial translocation across the intestinal mucosal barrier occurs at the advancing disease margin in Crohn's disease.

Crohn's disease is characterized by increased permeability of the intestinal mucosal barriers and an abnormal or dysregulated immune response to specific and/or commensal bacteria arising from the intestinal lumen. To determine the types of bacteria that are transgressing the mucosal barrier and colonizing the intestinal submucosal tissues, we performed 16S rRNA gene microbiota sequencing of the submucosal and mucosal tissues at the advancing disease margin in ileal Crohn's disease. Microbial populations were compared between mucosa and submucosa and non-inflammatory bowel disease (non-IBD) controls, as well as to microbial populations previously found at the centre of the disease lesion. There was no significant increase in bacteria within the submucosa of non-IBD controls at any taxonomic level when compared to the corresponding superjacent mucosa, indicating an effective mucosal barrier within the non-IBD population. In contrast, there was a statistically significant increase in 13 bacterial families and 16 bacterial genera within the submucosa at the advancing disease margin in Crohn's disease when compared to the superjacent mucosa. Major increases within the submucosa included bacteria of the Families Sphingomonadaceae, Alicyclobacillaceae, Methylobacteriaceae, Pseudomonadaceae and Prevotellaceae. Data suggest that the primary site of bacterial translocation across the mucosal barrier occurs at the margin between diseased and normal tissue, the advancing disease margin. The heterogeneity of the bacterial populations penetrating the mucosal barrier and colonizing the submucosal intestinal tissues and, therefore, contributing to the inflammatory processes, suggests that bacterial translocation is secondary to a primary event leading to a breakdown of the mucosal barrier.

Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples.

BACKGROUND: The advent and use of highly sensitive molecular biology techniques to explore the microbiota and microbiome in environmental and tissue samples have detected the presence of contaminating microbial DNA within reagents. These microbial DNA contaminants may distort taxonomic distributions and relative frequencies in microbial datasets, as well as contribute to erroneous interpretations and identifications. RESULTS: We herein report on the occurrence of bacterial DNA contamination within commonly used DNA extraction kits and PCR reagents and the effect of these contaminates on data interpretation. When compared to previous reports, we identified an additional 88 bacterial genera as potential contaminants of molecular biology grade reagents, bringing the total number of known contaminating microbes to 181 genera. Many of the contaminants detected are considered normal inhabitants of the human gastrointestinal tract and the environment and are often indistinguishable from those genuinely present in the sample. CONCLUSIONS: Laboratories working on bacterial populations need to define contaminants present in all extraction kits and reagents used in the processing of DNA. Any unusual and/or unexpected findings need to be viewed as possible contamination as opposed to unique findings.

Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA.

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.

An improved Tn7-lux reporter for broad host range, chromosomally-integrated promoter fusions in Gram-negative bacteria.

An improved vector for chromosomally-integrated promoter-lux fusions is described. The modified vector was tested in parallel with the unmodified vector using the well-characterized Escherichia coli araBAD promoter in the Pseudomonas aeruginosa attTn7 site. The modified mini-Tn7 showed reduced background luminescence, and increased luminescence upon induction, giving >16-fold higher induction ratio.

Microbial Population Differentials between Mucosal and Submucosal Intestinal Tissues in Advanced Crohn's Disease of the Ileum.

Since Crohn's disease is a transmural disease, we hypothesized that examination of deep submucosal tissues directly involved in the inflammatory disease process may provide unique insights into bacterial populations transgressing intestinal barriers and bacterial populations more representative of the causes and agents of the disease. We performed deep 16s microbiota sequencing on isolated ilea mucosal and submucosal tissues on 20 patients with Crohn's disease and 15 non-inflammatory bowel disease controls with a depth of coverage averaging 81,500 sequences in each of the 70 DNA samples yielding an overall resolution down to 0.0001% of the bacterial population. Of the 4,802,328 total sequences generated, 98.9% or 4,749,183 sequences aligned with the Kingdom Bacteria that clustered into 8545 unique sequences with <3% divergence or operational taxonomic units enabling the identification of 401 genera and 698 tentative bacterial species. There were significant differences in all taxonomic levels between the submucosal microbiota in Crohn's disease compared to controls, including organisms of the Order Desulfovibrionales that were present within the submucosal tissues of most Crohn's disease patients but absent in the control group. A variety of organisms of the Phylum Firmicutes were increased in the subjacent submucosa as compared to the parallel mucosal tissue including Ruminococcus spp., Oscillospira spp., Pseudobutyrivibrio spp., and Tumebacillus spp. In addition, Propionibacterium spp. and Cloacibacterium spp. were increased as well as large increases in Proteobacteria including Parasutterella spp. and Methylobacterium spp. This is the first study to examine the microbial populations within submucosal tissues of patients with Crohn's disease and to compare microbial communities found deep within the submucosal tissues with those present on mucosal surfaces. Our data demonstrate the existence of a distinct submucosal microbiome and ecosystem that is not well reflected in the mucosa and/or downstream fecal material.

Role for ferredoxin:NAD(P)H oxidoreductase (FprA) in sulfate assimilation and siderophore biosynthesis in Pseudomonads.

Pyridine-2,6-bis(thiocarboxylate) (PDTC), produced by certain pseudomonads, is a sulfur-containing siderophore that binds iron, as well as a wide range of transition metals, and it affects the net hydrolysis of the environmental contaminant carbon tetrachloride. The pathway of PDTC biosynthesis has not been defined. Here, we performed a transposon screen of Pseudomonas putida DSM 3601 to identify genes necessary for PDTC production (Pdt phenotype). Transposon insertions within genes for sulfate assimilation (cysD, cysNC, and cysG [cobA2]) dominated the collection of Pdt mutations. In addition, two insertions were within the gene for the LysR-type transcriptional activator FinR (PP1637). Phenotypic characterization indicated that finR mutants were cysteine bradytrophs. The Pdt phenotype of finR mutants could be complemented by the known target of FinR regulation, fprA (encoding ferredoxin:NADP(+) oxidoreductase), or by Escherichia coli cysJI (encoding sulfite reductase). These data indicate that fprA is necessary for effective sulfate assimilation by P. putida and that the effect of finR mutation on PDTC production was due to deficient expression of fprA and sulfite reduction. fprA expression in both P. putida and P. aeruginosa was found to be regulated by FinR, but in a manner dependent upon reduced sulfur sources, implicating FinR in sulfur regulatory physiology. The genes and phenotypes identified in this study indicated a strong dependence upon intracellular reduced sulfur/cysteine for PDTC biosynthesis and that pseudomonads utilize sulfite reduction enzymology distinct from that of E. coli and possibly similar to that of chloroplasts and other proteobacteria.