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1.
Microbiology (Reading) ; 162(9): 1608-1619, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27418066

RESUMO

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.


Assuntos
Fenômenos Fisiológicos Bacterianos , Translocação Bacteriana , Doença de Crohn/microbiologia , Mucosa Intestinal/microbiologia , Adulto , Idoso , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Doença de Crohn/patologia , Microbioma Gastrointestinal , Humanos , Mucosa Intestinal/patologia , Pessoa de Meia-Idade , Adulto Jovem
2.
J Clin Gastroenterol ; 47(7): 612-20, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23426447

RESUMO

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.


Assuntos
Bactérias/patogenicidade , Doença de Crohn/microbiologia , DNA Bacteriano/análise , Mucosa Intestinal/microbiologia , Adesinas Bacterianas/genética , Adulto , Idoso , Bactérias/genética , Proteínas de Bactérias/genética , Sequência de Bases , Doença de Crohn/classificação , Escherichia coli/genética , Escherichia coli/patogenicidade , Proteínas de Escherichia coli/genética , Feminino , Genoma Bacteriano/genética , Humanos , Masculino , Metagenoma/genética , Pessoa de Meia-Idade , Mycobacterium/genética , Mycobacterium/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Salmonella/genética , Salmonella/patogenicidade , Virulência/genética , Yersinia/genética , Yersinia/patogenicidade , Adulto Jovem
3.
Crit Rev Microbiol ; 38(1): 52-93, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22242906

RESUMO

It has been more than 25 years since Mycobacterium paratuberculosis was first proposed as an etiologic agent in Crohn's disease based on the isolation of this organism from several patients. Since that time, a great deal of information has been accumulated that clearly establishes an association between M. paratuberculosis and Crohn's disease. However, data are conflicting and difficult to interpret and the field has become divided into committed advocates and confirmed skeptics. This review is an attempt to provide a thorough and objective summary of current knowledge from both basic and clinical research from the views and interpretations of both the antagonists and proponents. The reader is left to draw his or her own conclusions related to the validity of the issues and claims made by the opposing views and data interpretations. Whether M. paratuberculosis is a causative agent in some cases or simply represents an incidental association remains a controversial topic, but current evidence suggests that the notion should not be so readily dismissed. Remaining questions that need to be addressed in defining the role of M. paratuberculosis in Crohn's disease and future implications are discussed.


Assuntos
Doença de Crohn/microbiologia , Infecções por Mycobacterium/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Doença de Crohn/complicações , Humanos , Infecções por Mycobacterium/complicações , Mycobacterium avium subsp. paratuberculosis/fisiologia
4.
J Med Microbiol ; 67(4): 549-559, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29458679

RESUMO

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.


Assuntos
Bactérias/isolamento & purificação , Doença de Crohn/microbiologia , Microbioma Gastrointestinal , Íleo/microbiologia , Intestinos/microbiologia , Adulto , Idoso , Bactérias/classificação , Bactérias/genética , Translocação Bacteriana , Feminino , Humanos , Mucosa Intestinal/microbiologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
6.
Gut Pathog ; 8: 24, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27239228

RESUMO

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.

7.
J Microbiol Methods ; 119: 239-42, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26569458

RESUMO

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.


Assuntos
Bactérias/isolamento & purificação , Íleo/microbiologia , Microbiota , RNA Ribossômico 16S/genética , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/genética , Humanos , Metagenômica
8.
PLoS One ; 10(7): e0134382, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26222621

RESUMO

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.


Assuntos
Doença de Crohn/microbiologia , Íleo/microbiologia , Mucosa Intestinal/microbiologia , Adulto , Idoso , Bactérias/genética , DNA Bacteriano/genética , Fezes/microbiologia , Feminino , Humanos , Masculino , Microbiota/genética , Pessoa de Meia-Idade , Adulto Jovem
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