From Alpha Diversity to Zzz: Interactions among sleep, the brain, and gut microbiota in the first year of life.

Sleep disorders have been linked to alterations of gut microbiota composition in adult humans and animal models, but it is unclear how this link develops. With longitudinal assessments in 162 healthy infants, we present a so far unrecognized sleep-brain-gut interrelationship. First, we report a link between sleep habits and gut microbiota: daytime sleep is associated with bacterial diversity, and nighttime sleep fragmentation and variability are linked with bacterial maturity and enterotype. Second, we demonstrate a sleep-brain-gut link: bacterial diversity and enterotype are associated with sleep neurophysiology. Third, we show that the sleep-brain-gut link is relevant in development: sleep habits and bacterial markers predict behavioral-developmental outcomes. Our results demonstrate the dynamic interplay between sleep, gut microbiota, and the maturation of brain and behavior during infancy, which aligns with the newly emerging concept of a sleep-brain-gut axis. Importantly, sleep and gut microbiota represent promising health targets since both can be modified non-invasively. As many adult diseases root in early childhood, leveraging protective factors of adequate sleep and age-appropriate gut microbiota in infancy could constitute a health promoting factor across the entire human lifespan.

International clinical placement - Experiences of nursing students' cultural, personal and professional development; a qualitative study.

The purpose was to capture the experiences of cultural, personal and professional development during International Clinical Placement (ICP) among nursing students from three European countries. The paper presents findings based on the analysis of 23 reflections written by students immediately after returning from their ICP. The design builds on a qualitative study using a phenomenological approach and meaning condensation inspired by Kirsti Malterud. The analysis revealed four themes: Communication and barriers to be overcome, Culture as a serious business, Personal and professional achievements and Challenges and the importance of preceptorship. The ICP impacted on the participants' personal as well as professional way of understanding themselves as students and future nurses. A profound difference was seen between the achieved learning outcomes of participants completing an ICP in a high- or low-income country, respectively. Language barriers, the local culture and different nursing cultures were often challenging and pushed participants out of their comfort zone. All participants developed their cultural understanding in accordance with the Papadopoulos, Tilki and Taylor Model for Developing Cultural Competence. Findings indicate that educational institutions should establish well-planned exchange opportunities that adopt a two-way reciprocal (Erasmus) exchange programmes and be aware of the value of an appointed preceptor in the host country.

Pre-clinical evaluation of the effect of co-medication with antibiotics and oral steroids in Göttingen Minipigs on the biological activity of the probiotic medicinal product TSO (Trichuris suis ova).

The probiotic medicinal product TSO (Trichuris suis ova) is administered to patients with active ulcerative colitis in an ongoing clinical phase IIb trial where the typical co-medications are steroids (prednisolone or budesonide) and antibiotics (e.g., phenoxymethylpenicillin). The present pre-clinical study evaluates the effects of these co-medications on the biological activity of TSO in Göttingen Minipigs. This translationally relevant pre-clinical model allows administration of TSO with and without oral steroids or antibiotics in a manner similar to the administration to patients, followed by quantification of the biological activity of TSO. The biological activity of TSO was not affected by oral steroids but was reduced by oral antibiotics. Fecal calprotectin, the common marker of intestinal inflammation in patients with UC, did not differ between groups.

Development of the equine gut microbiota.

Shortly after birth the mammalian gut is colonized, by a transient microbiota, highly susceptible to environment and diet, that eventually stabilizes and becomes the resident gut microbiota. In a window of opportunity during the colonization, oral tolerance is established towards resident bacteria. In this study, the development of the equine gut microbiota was investigated in ten foals from parturition until post weaning. We found great differences in the core species of the gut microbiota composition between time-matched samples on Day 7 and 20 post-partum. Between day 20 and Day 50 post-partum, we saw the gut microbiota became increasingly dominated by fiber fermenting species. After Day 50, no significant changes in species abundance were observed. Gene expression analysis of pro- and anti-inflammatory cytokines in the blood revealed no significant changes before and after weaning. In summary, relative stability of the gut microbiota was reached within 50 days post-partum and, weaning did not have a major impact on the microbial composition.

Expression of immune regulatory genes correlate with the abundance of specific Clostridiales and Verrucomicrobia species in the equine ileum and cecum.

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFß, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

The Smallest Intestine (TSI)-a low volume in vitro model of the small intestine with increased throughput.

There is a growing interest in understanding the fate and behaviour of probiotic microorganisms and bioactive compounds during passage of the human gastrointestinal tract (GIT). Here, we report the development of a small volume in vitro model called The smallest Intestine (TSI) with increased throughput focusing on simulating passage through the stomach and small intestine (SI). The basic TSI module consists of five reactors, with a working volume of 12 ml each. During the simulated passage through the SI, bile is absorbed and pH is adjusted to physiologically relevant values for duodenum, jejunum and ileum. A consortium of seven representative bacterial members of the ileum microbiota is included in the ileal stage of the model. The behaviour of three putative probiotic Lactobacillus strains during in vitro simulated upper GIT passage was tested in the model and results were compared to previous studies describing probiotic survival. It was found, that probiotic persistence is strongly related to whether food was ingested, but also to presence of the ileal microbiota, which significantly impacted probiotic survival. In conclusion, TSI allows testing a substantial number of samples, at low cost and short time, and is thus suitable as an in vitro screening platform.

The half-life and exposure of cefuroxime varied in newborn infants after a Caesarean section.

AIM: No information was available on how fast intravenous cefuroxime administered to pregnant women before a Caesarean section was cleared in newborn infants. This study investigated the drug's half-life and the exposure of healthy newborn infants after their mothers received the drug. METHODS: Healthy mothers received a single dose of cefuroxime 15-60 minutes before skin incision. One blood sample was drawn from the umbilical cord, and two blood samples were drawn from the infant after delivery. Total plasma cefuroxime (µg/mL) was measured using high-pressure liquid chromatography. RESULTS: Cefuroxime was given to 22 mothers, including two who had twins. The concentration of cefuroxime varied significantly among infants (p < 0.001), while the rate of decline did not (p = 0.24). The median cefuroxime half-life was 3.5 hours (range 2.9-5.5), which was approximately three times longer than in normal adults and seemed to clear within 24 hours. The median area under the concentration-time curve was 65.0 hour µg/mL (range 31.7-162.4). CONCLUSION: We found that the cefuroxime half-life after a Caesarean section varied among infants and was longer than in normal adults but cleared within 24 hours. Exposure to cefuroxime in newborn infants may influence the gut microbiota and should be investigated further.

Gut microbial markers are associated with diabetes onset, regulatory imbalance, and IFN-γ level in NOD mice.

Gut microbiota regulated imbalances in the host's immune profile seem to be an important factor in the etiology of type 1 diabetes (T1D), and identifying bacterial markers for T1D may therefore be useful in diagnosis and prevention of T1D. The aim of the present study was to investigate the link between the early gut microbiota and immune parameters of non-obese diabetic (NOD) mice in order to select alleged bacterial markers of T1D. Gut microbial composition in feces was analyzed with 454/FLX Titanium (Roche) pyro-sequencing and correlated with diabetes onset age and immune cell populations measured in diabetic and non-diabetic mice at 30 weeks of age. The early gut microbiota composition was found to be different between NOD mice that later in life were classified as diabetic or non-diabetic. Those differences were further associated with changes in FoxP3(+) regulatory T cells, CD11b(+) dendritic cells, and IFN-γ production. The model proposed in this work suggests that operational taxonomic units classified to S24-7, Prevotella, and an unknown Bacteriodales (all Bacteroidetes) act in favor of diabetes protection whereas members of Lachnospiraceae, Ruminococcus, and Oscillospira (all Firmicutes) promote pathogenesis.

The effect of Lactobacillus paracasei subsp. paracasei L. casei W8® on blood levels of triacylglycerol is independent of colonisation.

Gut microbiota (GM) dysbiosis has been linked to obesity and its metabolic complications such as cardiovascular disease (CVD). The risk of developing CVD increases with elevated concentration of serum triacylglycerol (TAG). In a blinded, randomised two-arm parallel human intervention study we have previously found that four weeks of supplementation with Lactobacillus paracasei subsp. paracasei L. casei W8® (L. casei W8) compared to placebo reduced the concentration of TAG in 64 young healthy adults, an effect, likely mediated by a decreased stearoyl- CoA desaturase-1 (SCD1) activity. In the present study we analysed faecal samples obtained during the intervention study to investigate whether this effect was related to the ability of L. casei W8 to colonise the human gut after supplementation of L. casei W8 (1010 cfu daily) as determined by qPCR specific for L. paracasei and L. casei (L. casei group); whether L. casei W8 consumption affected GM composition as determined by 16S rRNA gene targeted 454/FLX amplicon sequencing; and whether these changes were associated with changes in TAG concentration and SCD1 activity. Faecal samples were collected at baseline, after four weeks supplementation and two weeks after the supplementation was ended, and fasting blood samples were collected at baseline and after 4 weeks. Four weeks supplementation with L. casei W8 did not affect the overall composition of the GM; however, an increase in the relative abundance of the L. casei group from 8.48×10-6% of the total GM compared to 2.83×10-3% at baseline (P<0.001) was observed. Two weeks after supplementation ended, the relative abundance of the L. casei group was still increased 14 times compared to before the intervention (P<0.01). However, neither the increase in the abundance of the L. casei group nor overall GM composition correlated with changes in blood lipids or SCD1 activity.

Growth/no growth models for Zygosaccharomyces rouxii associated with acidic, sweet intermediate moisture food products.

The most notorious spoilage organism of sweet intermediate moisture foods (IMFs) is Zygosaccharomyces rouxii, which can grow at low water activity, low pH and in the presence of organic acids. Together with an increased consumer demand for preservative free and healthier food products with less sugar and fat and a traditionally long self-life of sweet IMFs, the presence of Z. rouxii in the raw materials for IMFs has made assessment of the microbiological stability a significant hurdle in product development. Therefore, knowledge on growth/no growth boundaries of Z. rouxii in sweet IMFs is important to ensure microbiological stability and aid product development. Several models have been developed for fat based, sweet IMFs. However, fruit/sugar based IMFs, such as fruit based chocolate fillings and jams, have lower pH and aw than what is accounted for in previously developed models. In the present study growth/no growth models for acidified sweet IMFs were developed with the variables aw (0.65-0.80), pH (2.5-4.0), ethanol (0-14.5% (w/w) in water phase) and time (0-90 days). Two different strains of Z. rouxii previously found to show pronounced resistance to the investigated variables were included in model development, to account for strain differences. For both strains data sets with and without the presence of sorbic acid (250 ppm on product basis) were built. Incorporation of time as an exploratory variable in the models gave the possibility to predict the growth/no growth boundaries at each time between 0 and 90 days without decreasing the predictive power of the models. The influence of ethanol and aw on the growth/no growth boundary of Z. rouxii was most pronounced in the first 30 days and 60 days of incubation, respectively. The effect of pH was almost negligible in the range of 2.5-4.0. The presence of low levels of sorbic acid (250 ppm) eliminated growth of both strains at all conditions tested. The two strains tested have previously been shown to have similar tolerance towards the single stress factors included in the study, but when the stress factors were combined the two strains showed difference in their ability to grow illustrating the importance of including more strains when developing growth/no growth models. The developed models can be useful tools for development of new acidic sweet IMFs.

TL1A regulates TCRγδ+ intraepithelial lymphocytes and gut microbial composition.

TL1A is a proinflammatory cytokine, which is prevalent in the gut. High TL1A concentrations are present in patients with inflammatory bowel disease (IBD) and in IBD mouse models. However, the role of TL1A during steady-state conditions is relatively unknown. Here, we used TL1A knockout (KO) mice to analyse the impact of TL1A on the intestinal immune system and gut microbiota. The TL1A KO mice showed reduced amounts of small intestinal intraepithelial TCRγδ(+) and CD8(+) T cells, and reduced expression of the activating receptor NKG2D. Moreover, the TL1A KO mice had significantly reduced body weight and visceral adipose tissue deposits, as well as lower levels of leptin and CXCL1, compared with wild-type mice. Analysis of the gut microbial composition of TL1A KO mice revealed a reduction of caecal Clostridial cluster IV, a change in the Firmicutes/Bacteroidetes ratio in caecum and less Lactobacillus spp. in the mucosal ileum. Our results show that TL1A deficiency impacts on the gut microbial composition and the mucosal immune system, especially the intraepithelial TCRγδ(+) T-cell subset, and that TL1A is involved in the establishment of adipose tissue. This research contributes to a broader understanding of TL1A inhibition, which is increasingly considered for treatment of IBD.

Investigating the long-term effect of subchronic phencyclidine-treatment on novel object recognition and the association between the gut microbiota and behavior in the animal model of schizophrenia.

Subchronic phencyclidine (subPCP) treatment induces schizophrenic-like behavior in rodents, including cognitive deficits and increased locomotor sensitivity towards acute administration of PCP. Evidence is accumulating that the gut microbiota (GM) influences behavior through modulation of the microbiota-gut-brain axis, and hence, part of the variation within this animal model may derive from variation in the GM. The aims of this study was to investigate first, the duration of subPCP-induced cognitive impairment in the novel object recognition test, and second, the possible effect of subchronic PCP-treatment on the GM, and the association between the GM and the behavioral parameters. The association was further investigated by antibiotic reduction of the GM. Male Lister Hooded rats were dosed twice daily i.p. with either 5mg/kg PCP or sterile isotonic saline for seven days followed by a seven-day washout period. Rats were tested in the novel object recognition and the locomotor activity assays immediately after, three weeks after, or six weeks after washout, and the fecal GM was analyzed by high throughput sequencing. Antibiotic- and control-treated rats were tested in the same manner following washout. In conclusion, subPCP-treatment impaired novel object recognition up to three weeks after washout, whereas locomotor sensitivity was increased for at least six weeks after washout. Differences in the core gut microbiome immediately after washout suggested subPCP treatment to alter the GM. GM profiles correlated to memory performance. Administration of ampicillin abolished the subPCP-induced memory deficit. It thus seems reasonable to speculate that the GM influences memory performance, contributing to variation within the model.

Strategies to increase the stability of intermediate moisture foods towards Zygosaccharomyces rouxii: the effect of temperature, ethanol, pH and water activity, with or without the influence of organic acids.

Intermediate moisture foods (IMF) are in general microbiologically stable products. However, due to health concerns consumer demands are increasingly forcing producers to lower the fat, sugar and preservatives content, which impede the stability of the IMF products. One of the strategies to counteract these problems is the storage of IMF products at lower temperatures. Thorough knowledge on growth/no growth boundaries of Zygosaccharomyces rouxii in IMF products, also at different storage temperatures is an important tool for ensuring microbiologically stability. In this study, growth/no growth models for Z. rouxii, developed by Vermeulen et al. (2012) were further extended by incorporating the factor temperature. Three different data sets were build: (i) without organic acids, (ii) with acetic acid (10,000 ppm on product basis) and (iii) with sorbic acid (1500 ppm on product basis). For each of these data sets three different growth/no growth models were developed after 30, 60 and 90 days. The results show that the influence of temperature is only significant in the lower temperature range (8-15 °C). Also, the effect of pH is negligible (pH 5.0-6.2) unless organic acids are present. More specific, acetic acid had only an additive effect to ethanol and aw at low pH, whereas sorbic acid had also an additive effect at the higher pH values. For incubation periods longer than 30 days the growth/no growth boundary remained stable but enlarged gradually between day 60 and 90, except for the lower temperature range (<12 °C) where the boundary shifts to more stringent environmental conditions.

Characterization of the gut microbiota in leptin deficient obese mice - Correlation to inflammatory and diabetic parameters.

Gut microbiota have been implicated as a relevant factor in the development of type 2 diabetes mellitus (T2DM), and its diversity might be a cause of variation in animal models of T2DM. In this study, we aimed to characterise the gut microbiota of a T2DM mouse model with a long term vision of being able to target the gut microbiota to reduce the number of animals used in experiments. Male B6.V-Lep(ob)/J mice were characterized according to a number of characteristics related to T2DM, inflammation and gut microbiota. All findings were thereafter correlated to one another in a linear regression model. The total gut microbiota profile correlated to glycated haemoglobin, and high proportions of Prevotellaceae and Lachnospiraceae correlated to impaired or improved glucose intolerance, respectively. In addition, Akkermansia muciniphila disappeared with age as glucose intolerance worsened. A high proportion of regulatory T cells correlated to the gut microbiota and improved glucose tolerance. Furthermore, high levels of IL-10, IL-12 and TNF-α correlated to impaired glucose tolerance, blood glucose or glycated haemoglobin. The findings indicate that gut microbiota may contribute to variation in various disease read-outs in the B6.V-Lep(ob)/J model and considering them in both quality assurance and data evaluation for the B6.V-Lep(ob)/J model may have a reducing impact on the inter-individual variation.

Ampicillin-improved glucose tolerance in diet-induced obese C57BL/6NTac mice is age dependent.

Ampicillin has been shown to improve glucose tolerance in mice. We hypothesized that this effect is present only if treatment is initiated prior to weaning and that it disappears when treatment is terminated. High-fat fed C57BL/6NTac mice were divided into groups that received Ampicillin at different ages or not at all. We found that both diet and Ampicillin significantly changed the gut microbiota composition in the animals. Furthermore, there was a significant improvement in glucose tolerance in Ampicillin-treated, five-week-old mice compared to nontreated mice in the control group. At study termination, expressions of mRNA coding for tumor necrosis factor, serum amyloid A, and lactase were upregulated, while the expression of tumor necrosis factor (ligand) superfamily member 15 was downregulated in the ileum of Ampicillin-treated mice. Higher dendritic cell percentages were found systemically in high-fat diet mice, and a lower tolerogenic dendritic cell percentage was found both in relation to high-fat diet and late Ampicillin treatment. The results support our hypothesis that a "window" exists early in life in which an alteration of the gut microbiota affects glucose tolerance as well as development of gut immunity and that this window may disappear after weaning.

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga, an alkaline fermented food.

AIMS: To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed-based medium. Further, to characterize the antimicrobial substances produced. METHODS AND RESULTS: The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram-positive and Gram-negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed-based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra-high-performance liquid chromatography-time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin. CONCLUSIONS: The Bacillus amyloliquefaciens ssp. plantarum exhibited broad-spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.

Pinned, optically aligned diagnostic dock for use on the Z facility.

The pinned optically aligned diagnostic dock (PODD) is a multi-configuration diagnostic platform designed to measure x-ray emission on the Z facility. The PODD houses two plasma emission acquisition (PEA) systems, which are aligned with a set of precision machined pins. The PEA systems are modular, allowing a single diagnostic housing to support several different diagnostics. The PEA configurations fielded to date include both time-resolved and time-integrated, 1D spatially resolving, elliptical crystal spectrometers, and time-integrated, 1D spatially resolving, convex crystal spectrometers. Additional proposed configurations include time-resolved, monochromatic mirrored pinhole imagers and arrays of filtered x-ray diodes, diamond photo-conducting diode detectors, and bolometers. The versatility of the PODD system will allow the diagnostic configuration of the Z facility to be changed without significantly adding to the turn-around time of the machine. Additionally, the PODD has been designed to allow instrument setup to be completed entirely off-line, leaving only a refined alignment process to be performed just prior to a shot, which is a significant improvement over the instrument the PODD replaces. Example data collected with the PODD are presented.

Early life treatment with vancomycin propagates Akkermansia muciniphila and reduces diabetes incidence in the NOD mouse.

AIMS/HYPOTHESIS: Increasing evidence suggests that environmental factors changing the normal colonisation pattern in the gut strongly influence the risk of developing autoimmune diabetes. The aim of this study was to investigate, both during infancy and adulthood, whether treatment with vancomycin, a glycopeptide antibiotic specifically directed against Gram-positive bacteria, could influence immune homeostasis and the development of diabetic symptoms in the NOD mouse model for diabetes. METHODS: Accordingly, one group of mice received vancomycin from birth until weaning (day 28), while another group received vancomycin from 8 weeks of age until onset of diabetes. Pyrosequencing of the gut microbiota and flow cytometry of intestinal immune cells was used to investigate the effect of vancomycin treatment. RESULTS: At the end of the study, the cumulative diabetes incidence was found to be significantly lower for the neonatally treated group compared with the untreated group, whereas the insulitis score and blood glucose levels were significantly lower for the mice treated as adults compared with the other groups. Mucosal inflammation was investigated by intracellular cytokine staining of the small intestinal lymphocytes, which displayed an increase in cluster of differentiation (CD)4(+) T cells producing pro-inflammatory cytokines in the neonatally treated mice. Furthermore, bacteriological examination of the gut microbiota composition by pyrosequencing revealed that vancomycin depleted many major genera of Gram-positive and Gram-negative microbes while, interestingly, one single species, Akkermansia muciniphila, became dominant. CONCLUSIONS/INTERPRETATION: The early postnatal period is a critical time for microbial protection from type 1 diabetes and it is suggested that the mucolytic bacterium A. muciniphila plays a protective role in autoimmune diabetes development, particularly during infancy.