Combined dietary supplementation of long chain inulin and Lactobacillus acidophilus W37 supports oral vaccination efficacy against Salmonella Typhimurium in piglets

Routine use of antibiotics in livestock animals strongly contributed to the creation of multidrug-resistant Salmonella Typhimurium strains (STM). Vaccination is an alternative to the use of antibiotics but often suffers from low efficacy. The present study investigated whether long-chain inulin (lcITF) and Lactobacillus acidophilus W37 (LaW37) can support vaccination efficacy against STM and if the interventions influence possible gut microbiota changes. Piglets received daily supplementation until sacrifice. Animals were vaccinated on day 25 after birth, one day after weaning, and were challenged with STM on days 52-54. Dietary intervention with lcITF/LaW37 enhanced vaccination efficacy by 2-fold during challenge and resulted in higher relative abundance of Prevotellaceae and lower relative abundance of Lactobacillaceae in faeces. Although strongest microbial effects were observed post STM challenge on day 55, transient effects of the lcITF/LaW37 intervention were also detected on day 10 after birth, and post-weaning on day 30 where increased relative abundance of faecal lactobacilli was correlated with higher faecal consistency. LcITF treatment increased post-weaning feed efficiency and faecal consistency but did not support vaccination efficacy. Vaccination in immune-immature young animals can be enhanced with functional additives which can simultaneously promote health in an ingredient-dependent fashion

Donor Fecal Microbiota Transplantation Alters Gut Microbiota and Metabolites in Obese Individuals With Steatohepatitis

The intestinal microbiota has been linked to the development and prevalence of steatohepatitis in humans. Interestingly, steatohepatitis is significantly lower in individuals taking a plant-based, low-animal-protein diet, which is thought to be mediated by gut microbiota. However, data on causality between these observations in humans is scarce. In this regard, fecal microbiota transplantation (FMT) using healthy donors is safe and is capable of changing microbial composition in human disease. We therefore performed a double-blind randomized controlled proof-of-principle study in which individuals with hepatic steatosis on ultrasound were randomized to two study arms: lean vegan donor (allogenic n = 10) or own (autologous n = 11) FMT. Both were performed three times at 8-week intervals. A liver biopsy was performed at baseline and after 24 weeks in every subject to determine histopathology (Nonalcoholic Steatohepatitis Clinical Research Network) classification and changes in hepatic gene expression based on RNA sequencing. Secondary outcome parameters were changes in intestinal microbiota composition and fasting plasma metabolomics. We observed a trend toward improved necro-inflammatory histology, and found significant changes in expression of hepatic genes involved in inflammation and lipid metabolism following allogenic FMT. Intestinal microbial community structure changed following allogenic FMT, which was associated with changes in plasma metabolites as well as markers of .Conclusion:Allogenic FMT using lean vegan donors in individuals with hepatic steatosis shows an effect on intestinal microbiota composition, which is associated with beneficial changes in plasma metabolites and markers of steatohepatitis.Peer reviewe

Formalin-fixed paraffin embedded colorectal cancer and healthy colonic tissue sample collection 2

Formalin-fixed paraffin embedded (FFPE) tissues from colorectal cancer patients (n=14) and healthy controls (n=13) were obtained from pathology archives for 16S rRNA amplicon sequencing. The FFPE tissue collection includes n=11 colorectal cancer (CRC), n=14 normal adjacent (ADJ) and n=13 healthy control tissues. Controls included mock controls (n=2), PCR negatives (n=2) and paired empty paraffin samples (n=21) which were concurrently processed with the tissues. Additional samples consisted of DNA extraction negatives including blank (n=3) and water (n=3) controls

Physiology of γ-aminobutyric acid production by <i>Akkermansia muciniphila</i>

Gut bacteria hold the potential to produce a broad range of metabolites that can modulate human functions, including molecules with neuroactive potential. One such molecule is gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system in animals. Metagenomic analyses suggest that the genomes of many gut bacteria encode glutamate decarboxylase (GAD), the enzyme that catalyzes GABA production. The genome of Akkermansia muciniphila, a mucin specialist and potential next-generation probiotic from the human gut, is predicted to encode GAD, suggesting a contributing role in GABA production in the human gut. In this study, A. muciniphila was grown in batch cultures with and without pH control. In both experiments, A. muciniphila was found to produce GABA as a response to acid (pH <5.5), although only when GABA precursors, either glutamate or glutamine, were present in the medium. Proteomic analysis comparing A. muciniphila grown with and without precursors at pH 4 did not show a difference in GAD expression, suggesting that it is expressed regardless of the presence of GABA precursors. To further investigate the function of A. muciniphila GAD, we heterologously expressed the gad gene (encoded by locus tag Amuc_0372) with a His tag in Escherichia coli and purified the GAD protein. Enzyme assays showed GAD activity in a pH range between 4 and 6, with the highest specific activity at pH 5 of 144 +/- 16 mu M GABA/min/mg. Overall, our results demonstrate the ability of A. muciniphila to produce GABA as an acid response and unravel the conditions under which GABA production in A. muciniphila occurs.Peer reviewe

Eight-week exercise training in humans with obesity : Marked improvements in insulin sensitivity and modest changes in gut microbiome

Objective: Obesity is associated with impaired gut microbiota diversity, which has been linked to the development of type 2 diabetes. This study aims to examine the effects of an 8-week aerobic exercise intervention on insulin sensitivity, visceral adiposity, and gut microbiota diversity and composition in participants with obesity. Methods: Fourteen participants (mean [SD], age 51 [11] years; BMI 34.9 [4.9] kg/m2) performed an 8-week exercise intervention (2 to 4 times/week on 65% to 85% of heart rate reserve). Insulin sensitivity (hyperinsulemic euglycemic clamp), cardiorespiratory fitness (maximal oxygen uptake), visceral adiposity (dual-energy X-ray absorptiometry scan) and gut microbiota composition (16S rRNA gene sequencing) were measured before and after the intervention. Results: Insulin sensitivity showed a significant increase (pre: 3.8 [1.9] mg/min/kg; post: 4.5 [1.7] mg/min/kg; p = 0.007) after training, whereas visceral adiposity decreased (pre: 959 [361] cm3; post: 897 [364] cm3; p = 0.02). No change in gut microbiota α- or β-diversity was found. At the genus level, the abundance of Ruminococcus gauvreauii (p = 0.02); Lachnospiraceae FCS020 group (p = 0.04), and Anaerostipes (p = 0.04) significantly increased after exercise training. Significant positive correlations were present for M-value (R. gauvreauii) and VO2 max (R. gauvreauii and Anaerostipes). Conclusions: Eight-week exercise training in humans with obesity leads to marked improvements in insulin sensitivity and body composition and is accompanied by modest changes in 3 gut microbiome genera, all belonging to the Firmicutes phylum

The Effect of Nutritional Intervention with Lactoferrin, Galactooligosacharides and Vitamin D on the Gut Microbiota Composition of Healthy Elderly Women

Background: Nutritional supplements, such as bovine lactoferrin (bLF), have been studied for their immunomodulatory properties, but little is known of their effect on the gut microbiota composition of the elderly when supplemented alone or combined with other nutritional supplements such as prebiotics and micronutrients. In the present study, fecal samples from a doubleblind, placebo‐controlled nutritional intervention study were analysed. At baseline (T1), 25 elderly women were distributed into two groups receiving dietary intervention (n = 12) or placebo treatment (n = 13) for 9 weeks. During the first 3 weeks of the study (T2), the intervention group consumed 1 g/day bLF, followed by 3 weeks (T3) of 1 g/day bLF and 2.64 g/day active galactooligosaccharides (GOS), and 3 weeks (T4) of 1 g/day bLF, 2.64 g/day GOS and 20 μg/day of vitamin D. The placebo group received maltodextrin, in dosages matching those of the intervention group. Fecal bacterial composition was profiled using partial 16S rRNA gene amplicon sequencing. Short‐chain fatty acids (SCFA) were determined in fecal water as were levels of calprotectin, zonulin, and alpha‐ 1‐antitrypsin, as markers of gastrointestinal barrier and inflammation. Results: A significant increase was observed in the relative abundance of the genus Holdemanella (p < 0.01) in the intervention group compared to the placebo at T1. During T2, Bifidobacterium relative abundance increased significantly (p < 0.01) in the intervention group compared to the placebo, and remained significantly higher until the end of the study. No other effect was reported during T3. Furthermore, concentrations of SCFAs and calprotectin, zonulin and alpha‐1‐antitrypsin did not change during the intervention, although zonulin levels increased significantly within the placebo group by the end of the intervention. Conclusions: We conclude that supplementation of bLF enhanced the relative abundance of Holdemanella in the fecal microbiota of healthy elderly women, and further addition of GOS enhanced the relative abundance of Bifidobacterium

Formalin-fixed paraffin embedded colorectal cancer and healthy colonic tissue sample collection 1

Formalin-fixed paraffin embedded (FFPE) tissues from colorectal cancer patients (n=14) and healthy controls (n=13) were obtained from pathology archives for 16S rRNA amplicon sequencing. The FFPE tissue collection includes n=14 colorectal cancer (CRC), n=14 normal adjacent (ADJ) and n=13 healthy control tissues. DNA extraction negatives consisted of blank (n=3) and water (n=3) controls that were concurrently processed with the tissues. Additional controls consisted of empty paraffin samples (n=6)

Combined dietary supplementation of long chain inulin and Lactobacillus acidophilus W37 supports oral vaccination efficacy against Salmonella Typhimurium in piglets

(1) Background: Routine use of antibiotics in livestock animals strongly contributed to the creation of multidrug-resistant Salmonella Typhimurium strains (STM). Vaccination is an alternative to the use of antibiotics but often suffers from low efficacy. (2) Methods: Here we investigated whether long-chain inulin (lcITF) and Lactobacillus acidophilus W37 (LaW37) can support vaccination efficacy against STM and if changes can be seen at the level of gut microbiota. Piglets received daily supplementation until sacrifice. Animals were vaccinated on day 25 after birth one day after weaning, and animals were challenged with STM on days 52-54. (3) Results: LcITF/LaW37 enhanced vaccination efficacy by 2-fold. Diarrhea was significantly lower in lcITF/LaW37 and lcITF groups. Feed efficiency post-weaning was higher for piglets that received lcITF. The LcITF/LaW37 group was characterized by higher relative abundance of members of the family Prevotellaceae and lower relative abundance of Lactobacillaceae in feces. Although strongest microbial effects were observed post STM challenge on day 55, the transient effects of the dietary interventions were also detected in early life, on day 10 after birth, and post-weaning on day 30. (4) Conclusions: These results show that vaccination in immune-immature animals can be enhanced with functional foods and can simultaneously promote piglet health in an ingredient-dependent fashion. Effects on microbiota of the interventions were observed during stress periods only and were associated mainly with diarrhea

Technical challenges regarding the use of formalin-fixed paraffin embedded (FFPE) tissue specimens for the detection of bacterial alterations in colorectal cancer

Background: Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Results: Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Conclusions: Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants

No interplay between gut microbiota composition and the lipopolysaccharide-induced innate immune response in humans in vivo

Objective: Animal studies have demonstrated the extensive interplay between the gut microbiota and immunity. Moreover, in critically ill patients, who almost invariably suffer from a pronounced immune response, a shift in gut microbiota composition is associated with infectious complications and mortality. We examined the relationship between interindividual differences in gut microbiota composition and variation in the in vivo cytokine response induced by bacterial lipopolysaccharide (LPS). Furthermore, we evaluated whether an LPS challenge alters the composition of the gut microbiota. Methods: Healthy male volunteers received an intravenous bolus of 2 ng kg−1 LPS (n = 70) or placebo (n = 8). Serial plasma concentrations of tumor necrosis factor-α, interleukin (IL)-6, IL-8 and IL-10 were measured, and subjects were divided into high and low cytokine responders. Gut microbiota composition was determined using 16s RNA gene sequencing of faecal samples obtained 1 day before (baseline) and 1 day and 7 days following the LPS challenge. Results: Baseline microbiota composition, analysed by principal coordinate analysis and random forest analysis, did not differ between high and low responders for any of the four measured cytokines. Furthermore, baseline microbiota diversity (Shannon and Chao indices) was similar in high and low responders. No changes in microbiota composition or diversity were observed at 1 and 7 days following the LPS challenge. Conclusion: Our results indicate that existing variation in gut microbiota composition does not explain the observed variability in the LPS-induced innate immune response. These findings strongly argue against the interplay between the gut microbiota composition and the innate immune response in humans.</p