Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact ...mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.
The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4(+)FoxP3(+) regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4(+)FoxP3(+) regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer's patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.
Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.
A 1000-cow study across four European countries was undertaken to understand to what extent ruminant microbiomes can be controlled by the host animal and to identify characteristics of the host rumen ...microbiome axis that determine productivity and methane emissions. A core rumen microbiome, phylogenetically linked and with a preserved hierarchical structure, was identified. A 39-member subset of the core formed hubs in co-occurrence networks linking microbiome structure to host genetics and phenotype (methane emissions, rumen and blood metabolites, and milk production efficiency). These phenotypes can be predicted from the core microbiome using machine learning algorithms. The heritable core microbes, therefore, present primary targets for rumen manipulation toward sustainable and environmentally friendly agriculture.
The diversity of the methanogenic archaea associated with the six segments of the horse and donkey hindgut (caecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon, and ...rectum) was analyzed using 16S rDNA gene clone library. A total of 641 positive clones, 321 from the horse and 320 from the donkey hindgut, were examined by the RFLP, revealing 9 different ribotypes, 8 in the horse and 5 in the donkey hindgut. In both the animals Methanobacteriales (Methanobrevibacter-like sequences) and Methanomicrobiales (Methanocorpusculum-like sequences) were detected as the dominant orders followed by the uncultured Methanomassiliicoccales. The composition of the equine archaeal community was found to be dependent on the gut region. In both the two animals no Methanobrevibacter-like clones were detected in the caeca, which were instead inhabited by the Methanocorpusculum-like archeons. The Methanosarcinales were found only in distal regions of the horse hindgut.
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•The Methanobacterium-like sequences are the dominant archeons of the horse hindgut.•The Methanocorpusculum-like sequences are the dominant archeons of the donkey hindgut.•The Methanomassiliicoccales form an important part of the horse hindgut methanogens.•The methanogens diversity of the equine hindgut is both host animal and gut region dependent.•The equine caecum is inhabited mainly by the Methanocorpusculum-like methanogens.
Freezing is considered to be the best method for long-term storage of bacterial DNA from feces; however this method cannot be usually applied for samples of wild primates collected in the challenging ...conditions of the tropical forest. In order to find an alternative conservation method of fecal samples from wild great apes, we compared freezing with other fixation methods. Fecal samples from 11 captive gorillas (Gorilla gorilla gorilla) from three Czech Zoos were stored using freezing, RNA Stabilization Reagent (RNAlater), and 96% ethanol. Subsequently, the samples were examined using culture-independent methods (PCR-DGGE, and Real-time PCR) to qualitatively and quantitatively assess fecal microbiota composition and to compare differences among the storage methods. Noticeably, freezing samples resulted in the highest recoveries of DNA. No significant differences in DNA recovery were found between freezing and using RNAlater; however, significantly lower DNA concentrations were recovered from samples stored in 96% ethanol. Using PCR-DGGE we found that either 96% ethanol, RNAlater or freezing were suitable for preserving bacterial DNA; however fingerprints obtained from RNAlater storage were more similar to those obtained from the frozen method; in comparison to the patterns resulting from storing samples in ethanol. Using qPCR, frozen samples yielded the highest values of bacterial counts, with the exception of Enterobacteriaceae, which showed the highest numbers using samples stored in ethanol. Sequences of amplicons obtained from PCR-DGGE belonged to the families Clostridiaceae, Lactobacillaceae, Staphylococcaceae, and Lachnospiraceae, phylum Firmicutes; however most amplicons showed sequence similarity to previously uncultured microorganisms. Bacteria belonging to the phylum Firmicutes were the most frequently identified species in the fecal bacterial communities of captive western gorillas. The study showed that RNAlater is an optimal storage method when freezing is not possible.
► We compared freezing, RNAlater and ethanol for storage of fecal bacterial DNA. ► By PCR-DGGE and qPCR we assessed and compared fecal microbiota of captive gorillas. ► We recommend using RNAlater to preserve fecal samples, if freezing is not possible. ► Firmicutes were the most frequently identified species in gorilla fecal community.
The aim of this study was the comparison of label-free method with 2DE to other analytical method of bacterium Butyrivibrio fibrisolvens extracellular protein samples. Label-free quantification (LF) ...method performed in this work was compared with previously obtained results of proteomic analysis using two-dimensional electrophoresis (2DE) and nano-liquid chromatography coupled with mass spectroscopy (nLC/MS). B. fibrisolvens as an important plant fibre degrader was cultivated on four different carbon sources (including xylan, xylose, glucose, and a mixture of xylan with glucose). The impact of growth substrate on protein profile was assessed by six pair-wise comparisons evaluating the significantly differently abundant extracellular proteins. Gel-free and gel-based methods resulted in substantially dissimilar results. The LC-MS/MS approach detected substrate-dependent differences in transport and binding membrane proteins (TBP) and nucleotidase, while the 2DE approach detected substrate effect on proteins included in protein synthesis and butyrate synthesis. On the other hand, both methods observed differentially regulated proteins involved in the glycolytic pathway, however, the only shared enzyme (protein detected both by 2DE and LC-MS/MS approach) was fructose-bisphosphate aldolase. The LC-MS/MS approach detected a high abundance of separated peptides. However, it cannot be easily considered as supreme to 2DE analysis. Both methods differ in sample preparation, their advantages and limitations. The study findings indicate these methods can complement each other and together they elucidate better the metabolic functions of B. fibrisolvens.
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Comparison of LF and 2DE analysis showed limitations connected with a narrow optimised pH interval and number of affected proteins in 2DE in contrary to LF results.
Butyrivibrio fibrisolvens culture showed the same proteomic changes in the presence of different saccharides as the whole rumen fluid in vivo.
Selected proteins could be used for the monitoring of polysaccharides fermentation in the rumen and be used for optimisation hemicellulose degradation and overall feed utilisation.
The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen ...fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, −80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at −80 °C was found as the optimal method to study ruminal bacterial profile.
•Intra- and extra-cellular DNA bacterial profile of rumen liquor differed considerably.•Treatment of rumen fluid and DNA isolation method influenced bacterial community.•Storage temperature and cryoprotective additives had minor influence.•Intracellular DNA extraction is recommended.•Rumen content mixed with PBS-glycerol, stored at –80 °C is recommended.
The rumen microbiota is one of the most complex consortia of anaerobes, involving archaea, bacteria, protozoa, fungi and phages. They are very effective at utilizing plant polysaccharides, especially ...cellulose and hemicelluloses. The most important hemicellulose decomposers are clustered with the genus
. As the related species differ in their range of hydrolytic activities and substrate preferences,
was selected as one of the most effective isolates and thus suitable for proteomic studies on substrate comparisons in the extracellular fraction. The
genome is the biggest in the butyrivibria cluster and is focused on "environmental information processing" and "carbohydrate metabolism".
The study of the effect of carbon source on
3071 was based on cultures grown on four substrates: xylose, glucose, xylan, xylan with 25% glucose. The enzymatic activities were studied by spectrophotometric and zymogram methods. Proteomic study was based on genomics, 2D electrophoresis and nLC/MS (Bruker Daltonics) analysis.
Extracellular β-endoxylanase as well as xylan β-xylosidase activities were induced with xylan. The presence of the xylan polymer induced hemicellulolytic enzymes and increased the protein fraction in the interval from 40 to 80 kDa. 2D electrophoresis with nLC/MS analysis of extracellular
3071 proteins found 14 diverse proteins with significantly different expression on the tested substrates.
The comparison of four carbon sources resulted in the main significant changes in
proteome occurring outside the fibrolytic cluster of proteins. The affected proteins mainly belonged to the glycolysis and protein synthesis cluster.
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