Dennis S. Nielsen

Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults

Background Recent evidence suggests that there is a link between metabolic diseases and bacterial populations in the gut. The aim of this study was to assess the differences between the composition of the intestinal microbiota in humans with type 2 diabetes and non-diabetic persons as control. Methods and Findings The study included 36 male adults with a broad range of age and body-mass indices (BMIs), among which 18 subjects were diagnosed with diabetes type 2. The fecal bacterial composition was investigated by real-time quantitative PCR (qPCR) and in a subgroup of subjects (N = 20) by tag-encoded amplicon pyrosequencing of the V4 region of the 16S rRNA gene. The proportions of phylum Firmicutes and class Clostridia were significantly reduced in the diabetic group compared to the control group (P = 0.03). Furthermore, the ratios of Bacteroidetes to Firmicutes as well as the ratios of Bacteroides-Prevotella group to C. coccoides-E. rectale group correlated positively and significantly with plasma glucose concentration (P = 0.04) but not with BMIs. Similarly, class Betaproteobacteria was highly enriched in diabetic compared to non-diabetic persons (P = 0.02) and positively correlated with plasma glucose (P = 0.04). Conclusions The results of this study indicate that type 2 diabetes in humans is associated with compositional changes in intestinal microbiota. The level of glucose tolerance should be considered when linking microbiota with metabolic diseases such as obesity and developing strategies to control metabolic diseases by modifying the gut microbiota.

Patterns of Early Gut Colonization Shape Future Immune Responses of the Host

The most important trigger for immune system development is the exposure to microbial components immediately after birth. Moreover, targeted manipulation of the microbiota can be used to change host susceptibility to immune-mediated diseases. Our aim was to analyze how differences in early gut colonization patterns change the composition of the resident microbiota and future immune system reactivity. Germ-free (GF) mice were either inoculated by single oral gavage of caecal content or let colonized by co-housing with specific pathogen-free (SPF) mice at different time points in the postnatal period. The microbiota composition was analyzed by denaturing gradient gel electrophoresis for 16S rRNA gene followed by principal component analysis. Furthermore, immune functions and cytokine concentrations were analyzed using flow cytometry, ELISA or multiplex bead assay. We found that a single oral inoculation of GF mice at three weeks of age permanently changed the gut microbiota composition, which was not possible to achieve at one week of age. Interestingly, the ex-GF mice inoculated at three weeks of age were also the only mice with an increased pro-inflammatory immune response. In contrast, the composition of the gut microbiota of ex-GF mice that were co-housed with SPF mice at different time points was similar to the gut microbiota in the barrier maintained SPF mice. The existence of a short GF postnatal period permanently changed levels of systemic regulatory T cells, NK and NKT cells, and cytokine production. In conclusion, a time window exists that enables the artificial colonization of GF mice by a single oral dose of caecal content, which may modify the future immune phenotype of the host. Moreover, delayed microbial colonization of the gut causes permanent changes in the immune system.

Viable Saccharomyces cerevisiae cells at high concentrations cause early growth arrest of non‐Saccharomyces yeasts in mixed cultures by a cell–cell contact‐mediated mechanism

The growth of Kluyveromyces thermotolerans and Torulaspora delbrueckii was examined in mixed cultures with Saccharomyces cerevisiae in YPD modified for wine fermentations. Although the three yeasts had similar maximum specific growth rates in these fermentations, K. thermotolerans and T. delbrueckii arrested growth earlier than S. cerevisiae, thereby obtaining lower stationary phase cell concentrations than S. cerevisiae. Various single and mixed culture fermentations with the three yeasts were carried out in order to find an explanation for this phenomenon. The early growth arrests of K. thermotolerans and T. delbrueckii were absent in single cultures of the two yeasts, and they seemed to be due neither to nutrient limitations nor to the presence of growth‐inhibitory compounds. Rather, they seemed to be due to a cell–cell contact mechanism dependent on the presence of viable S. cerevisiae cells at high concentrations. These results contribute to an increased understanding of why K. thermotolerans and T. delbrueckii arrest growth before S. cerevisiae during wine fermentations. Copyright

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE).

The yeast populations associated with the fermentation of Ghanaian cocoa were investigated using denaturing gradient gel electrophoresis (DGGE). Samples were collected at 12–24 h intervals from heap and tray fermentations, at three different fermentation sites and different periods during the season. Eukaryotic universal primers were used to amplify a fragment of the 26S rRNA gene. The DGGE profiles were relatively complex, underlining that the fermentation of cocoa is a complex microbial process. The identities of selected fragments in the denaturing gels were revealed by sequencing. Hanseniaspora guilliermondii, Candida krusei and Pichia membranifaciens were detected from most fermentations, indicating their possible important role in the fermentation of Ghanaian cocoa. Saccharomyces cerevisiae and Candida zemplinina were almost exclusively detected during tray fermentations. The developed DGGE protocol was compared with traditional culture‐based isolations. The results were comparable but slightly different, as one yeast species (C. zemplinina) was only detected using DGGE. On the other hand, Trichosporon asahii yielded only faint bands in the denaturing gels, despite the fact that it was detected using culture‐based methods. Analysis of pure cultures showed that the targeted region of the 26S rRNA gene was poorly amplified in T. asahii, whereas all other investigated isolates were amplified efficiently using the chosen PCR approach. Cluster analysis revealed that the DGGE profiles clustered according to fermentation method and fermentation site. Furthermore, clustering according to progress in the fermentation was observed. The DGGE technique therefore seems to offer a relatively fast and reliable method for studying yeast population dynamics during cocoa fermentations. The nucleotide sequences determined in this study have been assigned Genbank Accession Nos AY762330–AY762349. Copyright

Gut Microbiota Composition Is Correlated to Grid Floor Induced Stress and Behavior in the BALB/c Mouse

Stress has profound influence on the gastro-intestinal tract, the immune system and the behavior of the animal. In this study, the correlation between gut microbiota composition determined by Denaturing Grade Gel Electrophoresis (DGGE) and tag-encoded 16S rRNA gene amplicon pyrosequencing (454/FLX) and behavior in the Tripletest (Elevated Plus Maze, Light/Dark Box, and Open Field combined), the Tail Suspension Test, and Burrowing in 28 female BALB/c mice exposed to two weeks of grid floor induced stress was investigated. Cytokine and glucose levels were measured at baseline, during and after exposure to grid floor. Stressing the mice clearly changed the cecal microbiota as determined by both DGGE and pyrosequencing. Odoribacter, Alistipes and an unclassified genus from the Coriobacteriaceae family increased significantly in the grid floor housed mice. Compared to baseline, the mice exposed to grid floor housing changed the amount of time spent in the Elevated Plus Maze, in the Light/Dark Box, and burrowing behavior. The grid floor housed mice had significantly longer immobility duration in the Tail Suspension Test and increased their number of immobility episodes from baseline. Significant correlations were found between GM composition and IL-1α, IFN-γ, closed arm entries of Elevated Plus Maze, total time in Elevated Plus Maze, time spent in Light/Dark Box, and time spent in the inner zone of the Open Field as well as total time in the Open Field. Significant correlations were found to the levels of Firmicutes, e.g. various species of Ruminococccaceae and Lachnospiraceae. No significant difference was found for the evaluated cytokines, except an overall decrease in levels from baseline to end. A significant lower level of blood glucose was found in the grid floor housed mice, whereas the HbA1c level was significantly higher. It is concluded that grid floor housing changes the GM composition, which seems to influence certain anxiety-related parameters.

Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

ABSTRACT The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.

A Possible Link between Food and Mood: Dietary Impact on Gut Microbiota and Behavior in BALB/c Mice

Major depressive disorder is a debilitating disease in the Western World. A western diet high in saturated fat and refined sugar seems to play an important part in disease development. Therefore, this study is aimed at investigating whether saturated fat or sucrose predisposes mice to develop behavioral symptoms which can be interpreted as depression-like, and the possible influence of the gut microbiota (GM) in this. Fourty-two mice were randomly assigned to one of three experimental diets, a high-fat, a high-sucrose or a control diet for thirteen weeks. Mice on high-fat diet gained more weight (p = 0.00009), displayed significantly less burrowing behavior than the control mice (p = 0.034), and showed decreased memory in the Morris water maze test compared to mice on high-sucrose diet (p = 0.031). Mice on high-sucrose diet burrowed less goal-oriented, showed greater latency to first bout of immobility in the forced swim test when compared to control mice (p = 0.039) and high-fat fed mice (p = 0.013), and displayed less anxiety than mice on high-fat diet in the triple test (p = 0.009). Behavioral changes were accompanied by a significant change in GM composition of mice fed a high-fat diet, while no difference between diet groups was observed for sucrose preferences, LPS, cholesterol, HbA1c, BDNF and the cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12(p70), IL-17 and TNF-α. A series of correlations was found between GM, behavior, BDNF and inflammatory mediators. In conclusion, the study shows that dietary fat and sucrose affect behavior, sometimes in opposite directions, and suggests a possible association between GM and behavior.

Quantitatively different, yet qualitatively alike: a meta-analysis of the mouse core gut microbiome with a view towards the human gut microbiome.

Background A number of human diseases such as obesity and diabetes are associated with changes or imbalances in the gut microbiota (GM). Laboratory mice are commonly used as experimental models for such disorders. The introduction and dynamic development of next generation sequencing techniques have enabled detailed mapping of the GM of both humans and animal models. Nevertheless there is still a significant knowledge gap regarding the human and mouse common GM core and thus the applicability of the latter as an animal model. The aim of the present study was to identify inter- and intra-individual differences and similarities between the GM composition of particular mouse strains and humans. Methodology/Principal Findings A total of 1509428 high quality tag-encoded partial 16S rRNA gene sequences determined using 454/FLX Titanium (Roche) pyro-sequencing reflecting the GM composition of 32 human samples from 16 individuals and 88 mouse samples from three laboratory mouse strains commonly used in diabetes research were analyzed using Principal Coordinate Analysis (PCoA), nonparametric multivariate analysis of similarity (ANOSIM) and alpha diversity measures. A reliable cutoff threshold for low abundant taxa estimated on the basis of the present study is recommended for similar trials. Conclusions/Significance Distinctive quantitative differences in the relative abundance of most taxonomic groups between the examined categories were found. All investigated mouse strains clustered separately, but with a range of shared features when compared to the human GM. However, both mouse fecal, caecal and human fecal samples shared to a large extent not only representatives of the same phyla, but also a substantial fraction of common genera, where the number of shared genera increased with sequencing depth. In conclusion, the GM of mice and humans is quantitatively different (in terms of abundance of specific phyla and species) but share a large qualitatively similar core.

The biodiversity of predominant lactic acid bacteria in dolo and pito wort for the production of sorghum beer

Aim:  To quantify and identify the predominant lactic acid bacteria (LAB) in dolo and pito wort processing, and to examine their biodiversity at strain level.

The microbiology of alkaline-fermentation of indigenous seeds used as food condiments in Africa and Asia

Alkaline-fermented food condiments play an important role in the diets of many people in developing and a few developed countries. The rise in pH during production of these foods is due to the ability of the dominant microorganisms, Bacillus spp., to hydrolyze proteins into amino acids and ammonia. Studies have been undertaken which have investigated a number of these products like dawadawa, ugba, bikalga, kinema, natto, and thua-nao. In this review, current knowledge about the principal microbiological activities and biochemical modifications which occur during the processing of the alkaline condiments including nutritional, antimicrobial, and probiotic aspects are discussed. The current use of molecular biology methods in microbiological research has allowed unambiguous and more reliable identification of microorganisms involved in these fermentations generating sufficient knowledge for the selection of potential starter cultures for controlled and better production procedures for alkaline-fermented seeds condiments.