Structure and Function of Bovine Whey Derived Oligosaccharides Showing Synbiotic Epithelial Barrier Protective Properties.

Commensal gut microbiota and probiotics have numerous effects on the host's metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure-function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS's and MOS's potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of ß1-6- and ß1-3-linked galactoses, and 3'- and 6'-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure-function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Linking Human Milk Oligosaccharides, Infant Fecal Community Types, and Later Risk To Require Antibiotics.

Human milk oligosaccharides (HMOs) may provide health benefits to infants partly by shaping the development of the early-life intestinal microbiota. In a randomized double-blinded controlled multicentric clinical trial, healthy term infants received either infant formula (control) or the same formula with two HMOs (2'-fucosyllactose and lacto-N-neotetraose; test) from enrollment (0 to 14 days) to 6 months. Then, all infants received the same follow-up formula without HMOs until 12 months of age. Breastfed infants (BF) served as a reference group. Stool microbiota at 3 and 12 months, analyzed by 16S rRNA gene sequencing, clustered into seven fecal community types (FCTs) with marked differences in total microbial abundances. Three of the four 12-month FCTs were likely precursors of the adult enterotypes. At 3 months, microbiota composition in the test group (n = 58) appeared closer to that of BF (n = 35) than control (n = 63) by microbiota alpha (within group) and beta (between groups) diversity analyses and distribution of FCTs. While bifidobacteriaceae dominated two FCTs, its abundance was significantly higher in one (FCT BiH for Bifidobacteriaceae at high abundance) than in the other (FCT Bi for Bifidobacteriaceae). HMO supplementation increased the number of infants with FCT BiH (predominant in BF) at the expense of FCT Bi (predominant in control). We explored the association of the FCTs with reported morbidities and medication use up to 12 months. Formula-fed infants with FCT BiH at 3 months were significantly less likely to require antibiotics during the first year than those with FCT Bi. Previously reported lower rates of infection-related medication use with HMOs may therefore be linked to gut microbiota community types. (This study has been registered at ClinicalTrials.gov under registration number NCT01715246.)IMPORTANCE Human milk is the sole and recommended nutrition for the newborn infant and contains one of the largest constituents of diverse oligosaccharides, dubbed human milk oligosaccharides (HMOs). Preclinical and clinical association studies indicate that HMOs have multiple physiological functions largely mediated through the establishment of the gut microbiome. Until recently, HMOs were not available to investigate their role in randomized controlled intervention trials. To our knowledge, this is the first report on the effects of 2 HMOs on establishing microbiota in newborn infants. We provide a detailed description of the microbiota changes observed upon feeding a formula with 2 HMOs in comparison to breastfed reference infants' microbiota. Then, we associate the microbiota to long-term health as assessed by prescribed antibiotic use.

Bangladeshi children with acute diarrhoea show faecal microbiomes with increased Streptococcus abundance, irrespective of diarrhoea aetiology.

We report streptococcal dysbiosis in acute diarrhoea irrespective of aetiology. Compared with 20 healthy local controls, 71 Bangladeshi children hospitalized with acute diarrhoea (AD) of viral, mixed viral/bacterial, bacterial and unknown aetiology showed a significantly decreased bacterial diversity with loss of pathways characteristic for the healthy distal colon microbiome (mannan degradation, methylerythritol phosphate and thiamin biosynthesis), an increased proportion of faecal streptococci belonging to the Streptococcus bovis and Streptococcus salivarius species complexes, and an increased level of E. coli-associated virulence genes. No enteropathogens could be attributed to a subgroup of patients. Elevated lytic coliphage DNA was detected in 2 out of 5 investigated enteroaggregative E. coli (EAEC)-infected patients. Streptococcal outgrowth in AD is discussed as a potential nutrient-driven consequence of glucose provided with oral rehydration solution.

Yeast, beef and pork extracts counteract Clostridium difficile toxin A enterotoxicity.

Clostridium difficile is responsible for a large proportion of nosocomial cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present study provides evidence that yeast, beef and pork extracts, ingredients commonly used to grow bacteria, can counteract C. difficile toxin A enterotoxicity in vitro and in vivo. In model intestinal epithelial cells the individual extracts could prevent the toxin A-induced decrease in epithelial barrier function and partially prevented actin disaggregation and cell rounding. Mice with ad libitum access to individual extracts for 1 week had almost complete reduction in toxin A-induced fluid secretion in intestinal loops. Concomitantly, the toxin A-induced expression of the essential proinflammatory mediator Cox-2 was normalized. Moreover this protective effect was also seen when mice received only two doses of extract by intragastric gavage within 1 week. These results show that yeast, beef and pork extracts have the potential to counteract the intestinal pathogenesis triggered by C. difficile toxin A.

Rapid identification of differentiation markers from whole epithelial cells by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and statistical analysis.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) was applied to identify markers for cellular differentiation. The differentiation of a human colon epithelial carcinoma T84 cell line was monitored over a period of 28 days by transepithelial electrical resistance (TER) measurements, alkaline phosphatase (AP) assay, and MALDI-TOF mass spectral fingerprints combined with statistical analysis. MALDI-MS generated specific mass spectral fingerprints characteristic of cell differentiation. Twenty-two ions were selected as diagnostic signals of fully differentiated T84 cells. Ten protein ion signals, detected by MALDI-MS and validated by statistical analysis, were proposed as T84 cell differentiation markers. Among these signals, ubiquitin was identified as a T84 cell differentiation marker by nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS). Moreover, depending on the concentration of the cells seeded on the growth support, it was possible to predict the timing of the exponential phase and of cellular differentiation by MALDI-MS-derived marker ions. MALDI-TOFMS was compared to other methods for the determination of cellular differentiation: TER measurements are rapid but yield limited information as to the cellular differentiation state. AP assays are more specific for the differentiation state but take more time. By contrast, MALDI-MS has been found to be a fast, sensitive and precise method for cell differentiation assessment and provides the opportunity for multiplexing and high throughput. Moreover, the consumable costs per assay are very low.