Dietary Isomalto/Malto-Polysaccharides Increase Fecal Bulk and Microbial Fermentation in Mice

Scope: The prevalence of metabolic-syndrome-related disease has strongly increased. Nutritional intervention strategies appear attractive, particularly with novel prebiotics. Isomalto/malto-polysaccharides (IMMPs) represent promising novel prebiotics that promote proliferation of beneficial bacteria in vitro. The present study investigates for the first time the in vivo effects of IMMP in mice. Methods and results: C57BL/6 wild-type mice received control or IMMP-containing (10%, w/w) diets for 3 weeks. IMMP leads to significantly more fecal bulk (+26%, p < 0.05), higher plasma non-esterified fatty acids (colorimetric assay, +10%, p < 0.05), and lower fecal dihydrocholesterol excretion (mass spectrometry, −50%, p < 0.05). Plasma and hepatic lipid levels (colorimetric assays following lipid extraction) are not influenced by dietary IMMP, as are other parameters of sterol metabolism, including bile acids (gas chromatography/mass spectrometry). IMMP is mainly fermented in the cecum and large intestine (high-performance anion exchange chromatography). Next-generation sequencing demonstrates higher relative abundance of Bacteroides and butyrate producers (Lachnospiraceae, Roseburia Odoribacter) in the IMMP group. Conclusion: The combined results demonstrate that IMMP administration to mice increases fecal bulk and induces potentially beneficial changes in the intestinal microbiota. Further studies are required in disease models to substantiate potential health benefits.</p

Long-Term beta-galacto-oligosaccharides Supplementation Decreases the Development of Obesity and Insulin Resistance in Mice Fed a Western-Type Diet

Scope: The gut microbiota might critically modify metabolic disease development. Dietary fibers such as galacto-oligosaccharides (GOS) presumably stimulate bacteria beneficial for metabolic health. This study assesses the impact of GOS on obesity, glucose, and lipid metabolism. Methods and results: Following Western-type diet feeding (C57BL/6 mice) with or without β-GOS (7% w/w, 15 weeks), body composition, glucose and insulin tolerance, lipid profiles, fat kinetics and microbiota composition are analyzed. GOS reduces body weight gain (p < 0.01), accumulation of epididymal (p < 0.05), perirenal (p < 0.01) fat, and insulin resistance (p < 0.01). GOS-fed mice have lower plasma cholesterol (p < 0.05), mainly within low-density lipoproteins, lower intestinal fat absorption (p < 0.01), more fecal neutral sterol excretion (p < 0.05) and higher intestinal GLP-1 expression (p < 0.01). Fecal bile acid excretion is lower (p < 0.01) in GOS-fed mice with significant compositional differences, namely decreased cholic, α-muricholic, and deoxycholic acid excretion, whereas hyodeoxycholic acid increased. Substantial changes in microbiota composition, conceivably beneficial for metabolic health, occurred upon GOS feeding. Conclusion: GOS supplementation to a Western-type diet improves body weight gain, dyslipidemia, and insulin sensitivity, supporting a therapeutic potential of GOS for individuals at risk of developing metabolic syndrome

The association between breastmilk oligosaccharides and faecal microbiota in healthy breastfed infants at two, six, and twelve weeks of age

Several factors affect gut microbiota development in early life, among which breastfeeding plays a key role. We followed 24 mother-infant pairs to investigate the associations between concentrations of selected human milk oligosaccharides (HMOs) in breastmilk, infant faeces, and the faecal microbiota composition in healthy, breastfed infants at two, six and 12 weeks of age. Lactation duration had a significant effect on breastmilk HMO content, which decreased with time, except for 3-fucosyllactose (3FL) and Lacto-N-fucopentaose III (LNFP III). We confirmed that microbiota composition was strongly influenced by infant age and was associated with mode of delivery and breastmilk LNFP III concentration at two weeks, with infant sex, delivery mode, and concentrations of 3′sialyllactose (3′SL) in milk at six weeks, and infant sex and Lacto-N-hexaose (LNH) in milk at 12 weeks of age. Correlations between levels of individual breastmilk HMOs and relative abundance of OTUs found in infant faeces, including the most predominant Bifidobacterium OTUs, were weak and varied with age. The faecal concentration of HMOs decreased with age and were strongly and negatively correlated with relative abundance of OTUs within genera Bifidobacterium, Parabacteroides, Escherichia-Shigella, Bacteroides, Actinomyces, Veillonella, Lachnospiraceae Incertae Sedis, and Erysipelotrichaceae Incertae Sedis, indicating the likely importance of these taxa for HMO metabolism in vivo.</p

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

Effects of pectin on fermentation characteristics, carbohydrate utilization, and microbial community composition in the gastrointestinal tract of weaning pigs

Scope: We aimed to investigate the effects of three different soluble pectins on the digestion of other consumed carbohydrates, and the consequent alterations of microbiota composition and SCFA levels in the intestine of pigs. Methods and results: Piglets were fed a low-methyl esterified pectin enriched diet (LMP), a high-methyl esterified pectin enriched diet (HMP), a hydrothermal treated soybean meal enriched diet (aSBM) or a control diet (CONT). LMP significantly decreased the ileal digestibility of starch resulting in more starch fermentation in the proximal colon. In the ileum, low-methyl esterified pectin present was more efficiently fermented by the microbiota than high-methyl esterified pectin present which was mainly fermented by the microbiota in the proximal colon. Treated soybean meal was mainly fermented in the proximal colon and shifted the fermentation of cereal dietary fiber to more distal parts, resulting in high SCFA levels in the mid colon. LMP, HMP, and aSBM decreased the relative abundance of the genus Lactobacillus and increased that of Prevotella in the colon. Conclusion: The LMP, HMP, and aSBM, differently affected the digestion processes compared to the control diet and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominating community, with potential health-promoting effects.</p

Human gastrointestinal microbiota modulation with dietary prebiotics : an intimate interaction throughout life

The colon is the most densely colonized area within the human gastrointestinal (GI) tract, and diet is one of the most fundamental modulators of the GI microbiota in both infants and adults. The colonic microbiota can be considered an “invisible organ” influencing metabolism, normal immune and nervous system functions, and the overall health and well-being of the host. During infancy the microbial colonization progresses through a sequence of well-orchestrated events leading to the establishment of a stable microbial ecosystem adapted for milk digestion. In breastfed infants, breastmilk is the sole source of nourishment and the source of microbes and bioactive components, such as free human milk oligosaccharides (HMOs). HMOs are believed to have evolved specifically to facilitate microbial colonization of the GI tract and to act as prebiotics by promoting growth and activity of bacterial species that are beneficial to a developing infant. We investigated faecal microbiota composition in 2-12 week old infants and detected high variability in their microbial profiles. We stratified the observed microbial patterns into three distinct microbial cluster types. As infants developed, their microbiota had a tendency to progress from a mixed community type towards faecal microbial clusters enriched in either Bifidobacterium and Bacteroides or only Bifidobacterium, and the ability of these infants to utilize HMOs also increased. GI microbiota composition differs between breastfed and formula fed infants and today’s infant formulas are often fortified with prebiotics (mainly galactooligosaccharides (scGOS) and/or fructooligosaccharides (lcFOS)) to better mimic the functional properties of human milk with respect to its effect on GI microbiota composition and function. We investigated the composition of faecal microbiota in infants receiving either breastmilk, infant formulas fortified with prebiotics, or mixed feeding. We compared these results with results obtained from infants fed traditional formulas, which did not contain prebiotics. Infants who received formulas fortified with prebiotics showed faecal profiles that closely resembled those of healthy breastfed infants, in particular with regard to the levels of Bifidobacterium and Lactobacillus, whereas this was not observed in infants fed traditional formula. Infants fed prebiotic fortified formulas also showed an accelerated transition towards the Bifidobacterium rich faecal microbial cluster type, as compared to their breastfed counterparts. This effect was not noted in infants which received mixed feeding, for whom the transition was delayed. Thus, the type and extent of prebiotic supplementation (breastmilk HMOs vs. prebiotics in infant formula) had an impact on both speed and direction of microbial colonization. Knowing that the HMO composition in breastmilk varies between mothers and across lactation stages, we investigated the link between maternal breastmilk HMO profiles and the microbiota composition in the faeces of corresponding infants. We did not detect strong and consistent correlations between specific breastmilk HMOs and microbial species that colonized the infant GI tract. It is likely that the microbial community in early life is shaped through a combined effect of many different milk HMOs, as well as other modulatory factors in breastmilk, including breastmilk’s own microbiota. Even though we could not predict infant microbiota profiles based on the HMO composition in maternal milk, we detected strong correlations between infant faecal microbial composition and an infant’s ability to utilize specific HMOs. There was a significant association between the cluster assignment and an infant’s ability to degrade breastmilk HMOs, with complete degradation of HMOs associated with Bifidobacterium dominated clusters and non-specific degradation associated with the mixed cluster. Constrained multivariate redundancy analysis indicated a significant association between faecal microbiota composition and gastrointestinal degradation of 2′-Fucosyllactose, Lacto-N-tetraose and Lacto-N-neotetraose, difucosyllactose, 6'Sialyllactose, Lacto-N-hexaose, Lacto-N-fucopentaose II and Lacto-N-fucopentaose III (FDRBifidobacterium, Parabacteroides, Escherichia-Shigella, Bacteroides, Actinomyces, Veillonella, Lachnospiraceae Incertae Sedis, and Erysipelotrichaceae Incertae Sedis, and to lesser extent lactobacilli. Thus, members of these taxa might play important roles in the intestinal microbial communities of infants as they were also identified as key groups in the analysis of microbial co-occurrence networks. The exact mode of action and effect of most prebiotic supplementations on GI microbiota community structure and function is still largely unknown. There is accumulating evidence suggesting that microbial species and strains show a high degree of specificity in their preference to utilize different prebiotic compounds. This specificity, together with the advances in glycosciences, offer leads for developing prebiotic supplementations for targeted approaches in modulating gut microbiota function for a particular health, preventative, or therapeutic purpose. Isomalto/malto-polysaccharides (IMMPs) are a novel group of starch-derived, slowly-fermentable fibres with a prebiotic potential. We investigated the fermentation behavior and modulatory effect of IMMPs on adult faecal microbiota using an in vitro batch fermentation model. The IMMPs tested included IMMP-94 (94% α-(1→6) glycosidic linkages), IMMP-96, IMMP-27 and IMMP-dig27 (after an enzymatic removal of digestible starch segments from IMMP-27) and varied in the percentage of α-(1→6) glycosidic linkages and chain length distribution. We showed that these differences had an effect on the speed of degradation and the dynamics within the microbial community. The fermentation of α-(1→6) glycosidic linkages in IMMP-94, IMMP-96 and IMMP-dig27 started after 12 h and finished within 48 h. IMMP-27 fermentation started directly after inoculation utilizing α-(1→4) glycosidic linkages, however, the utilization of α-(1→6) linked glucoses was delayed and started only after depletion of α-(1→4) linked glucose moieties. The IMMP fermentation was accompanied by a significant increase in overall microbial diversity and the relative abundance of Bifidobacterium and Lactobacillus, as well as increased production of short chain fatty acids (SCFAs) with acetic acid and succinic acids being the major products next to propionic acid and butyric acid. During the fermentation, polysaccharide fraction was degraded into isomalto-oligosaccharides (IMOs) mainly by extracellular enzymes. The smaller IMOs were further degraded by cell-associated enzymes. Using metatranscriptome data we showed that microbial community dynamics during fermentation also varied depending on the type of IMMP used and that the observed changes were reflected in the community gene expression profiles. Members of Bacteroides, Lactobacillus and Bifidobacterium were the predominant degraders of IMMPs, and the increased activity of these bacteria correlated with the presence of high amounts of α-(1→6) glycosidic linkages in the IMMPs tested. Furthermore, the metabolic changes as shown by the accumulation of SCFAs in the fermentation media, and the corresponding decrease in pH correlated with the microbiota activity, as measured at the metatranscriptome level. The research presented here shows how natural prebiotics (HMOs) and prebiotic supplementations (IMMPs, scGOS/lcFOS) can influence human GI microbiota structure and function during infancy and adulthood. Developments in the field of glycosciences together with a better understanding of the mode of action of prebiotics with regard to microbial community structure and function could eventually lead to development of substrates offering attractive and safe ways to modulate microbiota to achieve specific health outcomes. Our research provided insights into both, the infant and adult large intestinal ecosystems, but additional studies are needed and should also address the long-term effects of the prebiotic supplementation on human health.</p

Dietary Isomalto/Malto-Polysaccharides Increase Fecal Bulk and Microbial Fermentation in Mice

Scope: The prevalence of metabolic-syndrome-related disease has strongly increased. Nutritional intervention strategies appear attractive, particularly with novel prebiotics. Isomalto/malto-polysaccharides (IMMPs) represent promising novel prebiotics that promote proliferation of beneficial bacteria in vitro. The present study investigates for the first time the in vivo effects of IMMP in mice. Methods and results: C57BL/6 wild-type mice received control or IMMP-containing (10%, w/w) diets for 3 weeks. IMMP leads to significantly more fecal bulk (+26%, p &lt; 0.05), higher plasma non-esterified fatty acids (colorimetric assay, +10%, p &lt; 0.05), and lower fecal dihydrocholesterol excretion (mass spectrometry, −50%, p &lt; 0.05). Plasma and hepatic lipid levels (colorimetric assays following lipid extraction) are not influenced by dietary IMMP, as are other parameters of sterol metabolism, including bile acids (gas chromatography/mass spectrometry). IMMP is mainly fermented in the cecum and large intestine (high-performance anion exchange chromatography). Next-generation sequencing demonstrates higher relative abundance of Bacteroides and butyrate producers (Lachnospiraceae, Roseburia Odoribacter) in the IMMP group. Conclusion: The combined results demonstrate that IMMP administration to mice increases fecal bulk and induces potentially beneficial changes in the intestinal microbiota. Further studies are required in disease models to substantiate potential health benefits.</p

A combined enrichment and aptamer pulldown assay for Francisella tularensis detection in food and environmental matrices.

Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1-106 CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology

Characterization and Differential Gene Expression between Two Phenotypic Phase Variants in <em>Salmonella enterica</em> Serovar Typhimurium

<div><p><em>Salmonella enterica</em> serovar Typhimurium strain 798 has previously been shown to undergo phenotypic phase variation. One of the phenotypes expresses virulence traits such as adhesion, while the other phenotype does not. Phenotypic phase variation appears to correlate with the ability of this strain to cause persistent, asymptomatic infections of swine. A new method to detect cells in either phenotypic phase was developed using Evans Blue-Uranine agar plates. Using this new assay, rates of phenotypic phase variation were obtained. The rate of phase variation from non-adhesive to adhesive phenotype was approximately 10⁻⁴ per cell per generation while phase variation from the adhesive to the non-adhesive phenotype was approximately 10⁻⁶ per cell per generation. Two highly virulent <em>S.</em> Typhimurium strains, SL1344 and ATCC 14028, were also shown to undergo phase variation. However, while the rate from adhesive to non-adhesive phenotype was approximately the same as for strain 798, the non-adhesive to adhesive phenotype shift was 37-fold higher. Differential gene expression was measured using RNA-Seq. Eighty-three genes were more highly expressed by 798 cells in the adhesive phenotype compared to the non-adhesive cells. Most of the up-regulated genes were in virulence genes and in particular all genes in the <em>Salmonella</em> pathogenicity island 1 were up-regulated. When compared to the virulent strain SL1344, expression of the virulence genes was approximately equal to those up-regulated in the adhesive phenotype of strain 798. A comparison of invasive ability demonstrated that strain SL1344 was the most invasive followed by the adhesive phenotype of strain 798, then the non-adhesive phenotype of strain 798. The least invasive strain was ATCC 14028. The genome of strain 798 was sequenced and compared to SL1344. Both strains had very similar genome sequences and gene deletions could not readily explain differences in the rates of phase variation from non-adhesive to the adhesive phenotype.</p> </div

Growth of i518 and i519 in LB With 1% Salt and LB Without Added Salt.

<p>Growth curves of i518 and i519 were repeated on three separate occasions. Spectrophotometric readings were taken every thirty minutes. i518 grown in LB-0: ⧫ i518 grown in LB-H: ▴, i519 grown in LB-0: ▪, i519 grown in LB-H: • Shown is a single representative graph.</p