scholarly journals Brussels sprouts, inulin and fermented milk alter the faecal microbiota of human microbiota-associated rats as shown by PCR-temporal temperature gradient gel electrophoresis using universal,Lactobacillus and Bifidobacterium16S rRNA gene primers

2005 ◽  
Vol 93 (5) ◽  
pp. 677-684 ◽  
Author(s):  
Christèle Humblot ◽  
Aurélia Bruneau ◽  
Malène Sutren ◽  
Evelyne F. Lhoste ◽  
Joël Doré ◽  
...  
Keyword(s):  
Control Diet ◽  
Fermented Milk ◽  
Initial Time ◽  
Rrna Gene ◽  
Final Time ◽  
Brussels Sprouts ◽  
Gradient Gel Electrophoresis ◽  
The Mean ◽  
Temperature Gradient Gel Electrophoresis ◽  
Degree Of Similarity

We investigated the effect of Brussels sprouts, inulin and a fermented milk on the faecal microbiota diversity of human microbiota-associated (HMA) rats by PCR-temporal temperature gradient gel electrophoresis (PCR-TTGE) using universal and group-specific 16S rRNA gene primers. The HMA rats were submitted to a control diet for 10 d (initial time), then switched to the experimental diets for 4 weeks (final time). Using universal primers, the mean degree of similarity between all faecal samples at initial time was 80·8 %. In the group consuming the control diet throughout the experiment, the mean degree of similarity between the PCR-TTGE profiles at initialv.final time was 76·8 %, reflecting a spontaneous temporal variation. The mean degree of similarity between control and experimental groups at final time was lower, 72·4 %, 74·4 % and 75·6 % for inulin, Brussels sprouts and fermented milk, respectively, indicating a dietary effect on the predominant populations. Using specific primers, bifidobacteria could be detected only in those rats that had consumed inulin, showing a specific increasing effect of this dietary compound. TheLactobacilluspopulation was very heterogeneous at initial time but tended to homogenize within each dietary group. At final time, caecal contents were collected for analysis of SCFA and β-glucuronidase activity. Inulin and Brussels sprouts increased the butyrate and acetate proportion, respectively, while the fermented milk did not modify the caecal biochemistry. This experiment shows for the first time that cruciferous vegetables are able to alter the diversity and the metabolic activities of the digestive microbiota in HMA rats.

scholarly journals Influence of Camembert consumption on the composition and metabolism of intestinal microbiota: a study in human microbiota-associated rats

2004 ◽  
Vol 92 (3) ◽  
pp. 429-438 ◽  
Author(s):  
Christophe Lay ◽  
Malène Sutren ◽  
Pascale Lepercq ◽  
Catherine Juste ◽  
Lionel Rigottier-Gois ◽  
...  
Keyword(s):  
Intestinal Microbiota ◽  
Dairy Products ◽  
Basal Diet ◽  
Fermented Milk ◽  
Metabolic Characteristics ◽  
Human Microbiota ◽  
Gradient Gel Electrophoresis ◽  
Metabolic Activities ◽  
Temperature Gradient Gel Electrophoresis ◽  
Micro Organisms

The objective of the present study was to evaluate the consequence of Camembert consumption on the composition and metabolism of human intestinal microbiota. Camembert cheese was compared with milk fermented by yoghurt starters andLactobacillus caseias a probiotic reference. The experimental model was the human microbiota-associated (HM) rat. HM rats were fed a basal diet (HMB group), a diet containing Camembert made from pasteurised milk (HMCp group) or a diet containing fermented milk (HMfm group). The level of micro-organisms from dairy products was measured in faeces using cultures on a specific medium and PCR–temporal temperature gradient gel electrophoresis. The metabolic characteristics of the caecal microbiota were also studied: SCFA, NH3, glycosidase and reductase activities, and bile acid degradations. The results showed that micro-organisms from cheese comprised 105–108bacteria/g faecal sample in the HMCp group.Lactobacillusspecies from fermented milk were detected in HMfm rats. Consumption of cheese and fermented milk led to similar changes in bacterial metabolism: a decrease in azoreductase activity and NH3concentration and an increase in mucolytic activities. However, specific changes were observed: in HMCp rats, the proportion of ursodeoxycholic resulting from chenodeoxycholic epimerisation was higher; in HMfm rats, α and β-galactosidases were higher than in other groups and both azoreductases and nitrate reductases were lower. The results show that, as for fermented milk, Camembert consumption did not greatly modify the microbiota profile or its major metabolic activities. Ingested micro-organisms were able to survive in part during intestinal transit. These dairy products exert a potentially beneficial influence on intestinal metabolism.

scholarly journals Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

2005 ◽  
Vol 71 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Anne-Ga�lle Le Bourhis ◽  
Katiana Saunier ◽  
Jo�l Dor� ◽  
Jean-Philippe Carlier ◽  
Jean-Fran�ois Chamba ◽  
...  
Keyword(s):  
Temperature Gradient ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Clostridial Species ◽  
The 16S Rrna Gene

ABSTRACT A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.

Temperature Gradient Gel Electrophoresis of the Amplified Product of a Small 16S rRNA Gene Fragment for the Identification of Listeria Species Isolated from Food

2000 ◽  
Vol 63 (5) ◽  
pp. 659-661 ◽  
Author(s):  
MARISA MANZANO ◽  
LUCA COCOLIN ◽  
CARLO CANTONI ◽  
GIUSEPPE COMI
Keyword(s):  
Polymerase Chain Reaction ◽  
Temperature Gradient ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gel Electrophoresis ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Gradient Gel Electrophoresis ◽  
Polymerase Chain ◽  
Temperature Gradient Gel Electrophoresis

The development of a rapid method for the identification of Listeria spp. is described. It is based on the polymerase chain reaction amplification of a small fragment from the 16S rRNA gene followed by temperature gradient gel electrophoresis. Forty-five strains of Listeria spp. (Listeria monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) were used for the optimization of the protocol. No differences were observed between the results of the identification of the strains tested using traditional methods and those obtained by polymerase chain reaction–temperature gradient gel electrophoresis analysis.

scholarly journals Assessing changes in composition of intestinal microbiota in neonatal BALB/c mice through cluster analysis of molecular markers

2008 ◽  
Vol 99 (6) ◽  
pp. 1174-1177 ◽  
Author(s):  
Reiko Fujiwara ◽  
Jun Watanabe ◽  
Kei Sonoyama
Keyword(s):  
Cluster Analysis ◽  
Intestinal Microbiota ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Control Diet ◽  
Group Method ◽  
Dice Similarity Coefficient ◽  
Rrna Gene ◽  
High Similarity ◽  
Pair Group ◽  
Gradient Gel Electrophoresis

The present study introduced a molecular biological approach to demonstrate changes in the composition of intestinal microbiota in neonatal mice. Female BALB/c mice were fed either a control diet or a diet supplemented with fructo-oligosaccharide (FOS) at 50 g/kg diet, and then mated to male mice. A cultivation-independent approach, denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene, was performed to characterise changes in intestinal microbial populations in pups at 0, 7, 14 and 21 d old and their dams. Comparisons of DGGE profiles were performed using the Dice similarity coefficient and the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis based on numbers, positions and intensities of bands. DGGE profiles differed between dams fed control and FOS-supplemented diets. Although profiles in pups on the day of birth showed a high similarity with dams, profiles in 7-d-old pups differed from dams and showed high similarity to littermates. In 14- and 21-d-old pups, profiles again showed high similarity with dams. DGGE profiles in pups were divided into two large clusters of control and FOS-supplemented diet groups in the range of 0- to 21-d-old, suggesting modulation of intestinal microbiota in infants by manipulation of microbiota in dams. The present study shows a useful technique for demonstrating changes in intestinal microbiota and provides a mouse model for modulation of intestinal microbiota in neonatal life.

scholarly journals Effects of orally administeredLactobacillus caseiDN-114 001 on the composition or activities of the dominant faecal microbiota in healthy humans

2006 ◽  
Vol 95 (2) ◽  
pp. 421-429 ◽  
Author(s):  
Violaine Rochet ◽  
Lionel Rigottier-Gois ◽  
Maléne Sutren ◽  
Marie-Noëlle Krementscki ◽  
Claude Andrieux ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fold Increase ◽  
Fermented Milk ◽  
Milk Product ◽  
Faecal Microbiota ◽  
Specific Primers ◽  
Healthy Humans ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

The composition and activities of the faecal microbiota in twelve healthy subjects analysed in a single open study were monitored before (1-week baseline step), during (10d supplementation step) and after (10d follow-up step) the ingestion of a fermented milk containingLactobacillus caseiDN-114001. Fluorescentin situhybridisation with group-specific DNA probes, real-time PCR usingL. paracaseigroup-specific primers and temporal temperature gradient gel electrophoresis (TTGE) using group-specific primers were carried out, together with bacterial enzyme activity and metabolite analyses to monitor the structure and activities of the faecal microbiota.L. caseiDNA was detected in the faeces of all of the subjects by TTGE after 10d supplementation. Its quantification by real-time PCR showed a 1000-fold increase during the test step compared with initial levels. No major modification in either the dominant members of the faecal microbiota or their activities was observed during the trial. In conclusion, the short-term consumption of a milk product containingL. caseiDN-114001 was accompanied by a high, transient increase in the quantity of this strain in the faeces of all of the subjects without markedly affecting biochemical or bacteriological factors.

scholarly journals Analysis of BIOLOG GN Substrate Utilization Patterns by Microbial Communities

1998 ◽  
Vol 64 (4) ◽  
pp. 1220-1225 ◽  
Author(s):  
Kornelia Smalla ◽  
Ute Wachtendorf ◽  
Holger Heuer ◽  
Wen-tso Liu ◽  
Larry Forney
Keyword(s):  
Activated Sludge ◽  
Microbial Communities ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Carbon Sources ◽  
Rrna Gene ◽  
Activated Sludge Reactor ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Biolog Gn

ABSTRACT BIOLOG GN plates are increasingly used to characterize microbial communities by determining the ability of the communities to oxidize various carbon sources. Studies were done to determine whether the BIOLOG GN plate assay accurately reflects the catabolic potential of the inoculum used. To gain insight into which populations of microbial communities contribute to the BIOLOG patterns, denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis (TGGE) were used to assess the diversity of ribotypes in the inocula and individual wells of BIOLOG plates following incubation. These studies were done with microbial communities from the rhizosphere of potatoes and an activated sludge reactor fed with glucose and peptone. TGGE analyses of BIOLOG wells inoculated with cell suspensions from the potato rhizosphere revealed that, compared with the inoculum, there was a decrease in the number of 16S rRNA gene fragments obtained from various wells, as well as a concomitant loss of populations that had been numerically dominant in the inoculum. The dominant fragments in TGGE gels could be assigned to the γ subclass of the classProteobacteria, suggesting that fast-growing bacteria adapted to high substrate concentrations were numerically dominant in the wells and may have been primarily responsible for the patterns of substrate use that were observed. Similarly, the community structure changed in wells inoculated with cells from activated sludge; one or more populations were enriched, but all dominant populations of the inoculum could be detected in at least one well. This study showed that carbon source utilization profiles obtained with BIOLOG GN plates do not necessarily reflect the functional potential of the numerically dominant members of the microbial community used as the inoculum.

scholarly journals Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis

2002 ◽  
Vol 68 (8) ◽  
pp. 3691-3701 ◽  
Author(s):  
Jean-Claude Ogier ◽  
Olivier Son ◽  
Alexandra Gruss ◽  
Patrick Tailliez ◽  
Agnes Delacroix-Buchet
Keyword(s):  
Temperature Gradient ◽  
Gel Electrophoresis ◽  
Dairy Products ◽  
Bacterial Species ◽  
Fermented Milk ◽  
Multiple Control ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Yeasts And Molds

ABSTRACT Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.

scholarly journals Application of temperature gradient gel electrophoresis to the characterization of a nitrifying bioaugmentation product

2003 ◽  
Vol 43 (2) ◽  
pp. 277-286 ◽  
Author(s):  
Melissa A. Fouratt ◽  
Jeremy S. Rhodes ◽  
Charles M. Smithers ◽  
Nancy G. Love ◽  
Ann M. Stevens
Keyword(s):  
Temperature Gradient ◽  
Gel Electrophoresis ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

scholarly journals A Grain-Based SARA Challenge Affects the Composition of Epimural and Mucosa-Associated Bacterial Communities throughout the Digestive Tract of Dairy Cows

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1658
Author(s):  
Jan C. Plaizier ◽  
Anne-Mette Danscher ◽  
Paula A. Azevedo ◽  
Hooman Derakhshani ◽  
Pia H. Andersen ◽  
...  
Keyword(s):  
Digestive Tract ◽  
Relative Abundance ◽  
Bacterial Communities ◽  
Dry Matter ◽  
Control Diet ◽  
Rrna Gene ◽  
Ruminal Acidosis ◽  
Clustering Patterns ◽  
The 16S Rrna Gene ◽  
Distal Jejunum

The effects of a subacute ruminal acidosis (SARA) challenge on the composition of epimural and mucosa-associated bacterial communities throughout the digestive tract were determined in eight non-lactating Holstein cows. Treatments included feeding a control diet containing 19.6% dry matter (DM) starch and a SARA-challenge diet containing 33.3% DM starch for two days after a 4-day grain step-up. Subsequently, epithelial samples from the rumen and mucosa samples from the duodenum, proximal, middle and distal jejunum, ileum, cecum and colon were collected. Extracted DNA from these samples were analyzed using MiSeq Illumina sequencing of the V4 region of the 16S rRNA gene. Distinct clustering patterns for each diet existed for all sites. The SARA challenge decreased microbial diversity at all sites, with the exception of the middle jejunum. The SARA challenge also affected the relative abundances of several major phyla and genera at all sites but the magnitude of these effects differed among sites. In the rumen and colon, the largest effects were an increase in the relative abundance of Firmicutes and a reduction of Bacteroidetes. In the small intestine, the largest effect was an increase in the relative abundance of Actinobacteria. The grain-based SARA challenge conducted in this study did not only affect the composition and cause dysbiosis of epimural microbiota in the rumen, it also affected the mucosa-associated microbiota in the intestines. To assess the extent of this dysbiosis, its effects on the functionality of these microbiota must be determined in future.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

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