scholarly journals Assessment of the Microbiota in Microdissected Tissues of Crohn's Disease Patients

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Gert De Hertogh ◽  
Bart Lemmens ◽  
Peter Verhasselt ◽  
Ronald de Hoogt ◽  
Xavier Sagaert ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Bacterial Pathogen ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Mucosal Healing ◽  
Rrna Gene ◽  
Excellent Method ◽  
Rrna Gene Sequencing ◽  
Laser Capture

The microbiota of the gastrointestinal tract is frequently mentioned as one of the key players in the etiopathogenesis of Crohn's disease (CD). Four hypotheses have been suggested: the single, still unknown bacterial pathogen, an abnormal overall composition of the bowel microbiota (“dysbiosis”), an abnormal immunological reaction to an essentially normally composed microbiota, and increased bacterial translocation. We propose that laser capture microdissection of selected microscopic structures, followed by broad-range 16S rRNA gene sequencing, is an excellent method to assess spatiotemporal alterations in the composition of the bowel microbiota in CD. Using this approach, we demonstrated significant changes of the composition, abundance, and location of the gut microbiome in this disease. Some of these abnormal findings persisted even after macroscopic mucosal healing. Further investigations along these lines may lead to a better understanding of the possible involvement of the bowel bacteria in the development of clinical Crohn's disease.

scholarly journals Metabonomics and the Gut Microbiome Associated With Primary Response to Anti-TNF Therapy in Crohn’s Disease

2020 ◽  
Vol 14 (8) ◽  
pp. 1090-1102 ◽  
Author(s):  
N S Ding ◽  
J A K McDonald ◽  
A Perdones-Montero ◽  
Douglas N Rees ◽  
S O Adegbola ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Bile Acid ◽  
Crohn's Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Primary Response ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background and Aims Anti-tumour necrosis factor [anti-TNF] therapy is indicated for treatment of moderate to severe inflammatory bowel disease [IBD], but has a primary non-response rate of around 30%. We aim to use metabonomic and metataxonomic profiling to identify predictive biomarkers of anti-TNF response in Crohn’s disease. Methods Patients with luminal Crohn’s disease, commencing anti-TNF therapy, were recruited with urine, faeces, and serum samples being collected at baseline and 3-monthly. Primary response was defined according to a combination of clinical and objective markers of inflammation. Samples were measured using three UPLC-MS assays: lipid, bile acid, and Hydrophillic Interaction Liquid Chromatography [HILIC] profiling with 16S rRNA gene sequencing of faeces. Results Samples were collected from 76 Crohn’s disease patients who were anti-TNF naïve and from 13 healthy controls. There were 11 responders, 37 non-responders, and 28 partial responders in anti-TNF-treated Crohn’s patients. Histidine and cysteine were identified as biomarkers of response from polar metabolite profiling [HILIC] of serum and urine. Lipid profiling of serum and faeces found phosphocholines, ceramides, sphingomyelins, and triglycerides, and bile acid profiling identified primary bile acids to be associated with non-response to anti-TNF therapy, with higher levels of phase 2 conjugates in non-responders. Receiver operating curves for treatment response demonstrated 0.94 +/ -0.10 [faecal lipid], 0.81 +/- 0.17 [faecal bile acid], and 0.74 +/- 0.15 [serum bile acid] predictive ability for anti-TNF response in Crohn’s disease. Conclusions This prospective, longitudinal cohort study of metabonomic and 16S rRNA gene sequencing analysis demonstrates that a range of metabolic biomarkers involving lipid, bile acid, and amino acid pathways may contribute to prediction of response to anti-TNF therapy in Crohn’s disease. Podcast This article has an associated podcast which can be accessed at https://academic.oup.com/ecco-jcc/pages/podcast

scholarly journals Influence of Enteral Nutrition on Gut Microbiota Composition in Patients with Crohn’s Disease: A Systematic Review

Nutrients ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2551 ◽  
Author(s):  
Paulina Horwat ◽  
Stanisław Kopeć ◽  
Aleksandra Garczyk ◽  
Iwona Kaliciak ◽  
Zuzanna Staręga ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Enteral Nutrition ◽  
Gut Microbiota ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Exclusive Enteral Nutrition ◽  
Faecal Samples ◽  
Hs Crp ◽  
Rrna Gene Sequencing

The aim of the study was to systematically and comprehensively evaluate whether exclusive enteral nutrition (EEN) has impact on gut microbiota in patients with Crohn’s disease (CD). The databases PUBMED (MEDLINE), SCOPUS and WEB OF SCIENCE were searched. Out of 232 studies, 9 met inclusion criteria. The combined analyzed population consists of 118 patients with CD and treated with EEN with a time of intervention of 2–12 weeks. Studies were conducted in children, with the exception of one study. All applied feeding formulas had similar energy value and composition. The microbiome analysis was based on 16S rRNA gene sequencing of faecal samples. In all studies, EEN treatment decreases inflammatory markers (i.e., hs-CRP and FCP). A change in abundance of numerous bacterial families (Clostridiaceae, Eubacteriaceae, Bacteroidaceae) was noticed, especially in Bacteroidaceae. An increase in families connected to the more severe clinical course (Fusobacteria, Prevotella, Lachnospiraceae) was observed in only 2.5% of CD patients. Our analyses suggest EEN has a beneficial influence on gut microbiome in patients with CD, which is interrelated with clinical patient’s improvement and time of disease remission.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

scholarly journals Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Janis R. Bedarf ◽  
Naiara Beraza ◽  
Hassan Khazneh ◽  
Ezgi Özkurt ◽  
David Baker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
False Positive ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

scholarly journals The growth response of rice (Oryza sativa L. var. FARO 44) in vitro after inoculation with bacterial isolates from a typical ferruginous ultisol

2021 ◽  
Vol 45 (1) ◽  
Author(s):  
Musa Saheed Ibrahim ◽  
Beckley Ikhajiagbe
Keyword(s):  
16S Rrna ◽  
Bacillus Cereus ◽  
Proteus Mirabilis ◽  
Growth Response ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rice Seedlings ◽  
Rrna Gene Sequencing

Abstract Background Rice forms a significant portion of food consumed in most household worldwide. Rice production has been hampered by soil factors such as ferruginousity which has limited phosphorus availability; an important mineral component for the growth and yield of rice. The presence of phosphate-solubilizing bacteria (PSB) in soils has been reported to enhance phosphate availability. In view of this, the present study employed three bacteria species (BCAC2, EMBF2 and BCAF1) that were previously isolated and proved P solubilization capacities as inocula to investigate the growth response of rice germinants in an in vitro setup. The bacteria isolates were first identified using 16S rRNA gene sequencing and then applied as inoculum. The inolula were prepared in three concentrations (10, 7.5 and 5.0 ml) following McFarland standard. Viable rice (var. FARO 44) seeds were sown in petri dishes and then inoculated with the three inocula at the different concentrations. The setup was studied for 28 days. Results 16S rRNA gene sequencing identified the isolates as: isolate BCAC2= Bacillus cereus strain GGBSU-1, isolate BCAF1= Proteus mirabilis strain TL14-1 and isolate EMBF2= Klebsiella variicola strain AUH-KAM-9. Significant improvement in rice germination, morphology, physiology and biomass parameters in the bacteria-inoculated setups was observed compared to the control. Germination percentage after 4 days was 100 % in the inoculated rice germinants compared to 65% in the control (NiS). Similarly, inoculation with the test isolates enhanced water-use efficiency by over 40%. The rice seedlings inoculated with Bacillus cereus strain GGBSU-1 (BiS) showed no signs of chlorosis and necrosis throughout the study period as against those inoculated with Proteus mirabilis strain TL14-1 (PiS) and Klebsiella variicola strain AUH-KAM-9 (KiS). Significant increase in chlorophyll-a, chlorophyll-b and alpha amylase was observed in the rice seedlings inoculated with BiS as against the NiS. Conclusion Inoculating rice seeds with Bacillus cereus strain GGBSU-1, Proteus mirabilis strain TL14-1 and Klebsiella variicola strain AUH-KAM-9 in an in vitro media significantly improved growth parameters of the test plant. Bacillus cereus strain GGBSU-1 showed higher efficiency due to a more improved growth properties observed.

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