Unveiling the impact of STEC infecting phages on the colon microbiota using an in vitro fermentation model

(Bacterio)phages are considered safe for humans consumption, being regard as excellent biocontrol tools to prevent foodborne pathogens spread. Phages major advantage is their inherent specificity towards a bacterial species, yet some reports have shown phages ability to evolve to infect different hosts when transiting the gastrointestinal tract (GIT). And so, it is of extreme importance to understand the safety outcome of using phages as biocontrol agents in food, with particular interest in the ones that target species from Enterobacteriaceae family, commonly found in the human GIT microbiota. In this study, the impact of a phage infecting Shiga toxin-producing Escherichia coli (STEC), named E. coli phage vB_EcoS_Ace (Ace), towards the colon microbiota was investigated. An in vitro batch fermentation model was used, and the inoculum was the fecal material of three healthy donors. Fermentations metabolome was analyzed through GC and HPLC, and the concentration of both phage Ace and STEC strain were monitored along time (up to 24h). The interference with the gut microbiota composition and functional potential was assessed by shot gun metagenomics. We observed an increase in phage titre only when the host was present, suggesting that there was no other suitable host within the different microbiotas used. Also, the microbiotas composition did not alter when phage Ace was added. Nevertheless, the attenuated version of STEC strain did indeed create some perturbation in the microbiota, which led to different functional potential. This was corroborated by the differences observed for both gas and short chain fatty acid acids dynamics. The microbiotas individuality was an important factor for the observed perturbations. Moreover, phage Ace revealed to be a safe phage when intended to be used as a biocontrol agent for food products. Also, we concluded that the in vitro fermentation model is a reliable, easy, and nonexpensive safety screening methodology for phages.info:eu-repo/semantics/publishedVersio

An in vitro fermentation model to study the impact of bacteriophages targeting shiga toxin-encoding escherichia coli on the colonic microbiota

Lytic bacteriophages are considered safe for human consumption as biocontrol agents against foodborne pathogens, in particular in ready-to-eat foodstuffs. Phages could, however, evolve to infect different hosts when passing through the gastrointestinal tract (GIT). This underlines the importance of understanding the impact of phages towards colonic microbiota, particularly towards bacterial families usually found in the colon such as the Enterobacteriaceae. Here we propose in vitro batch fermentation as model for initial safety screening of lytic phages targeting Shiga toxin-producing Escherichia coli (STEC). As inoculum we used faecal material of three healthy donors. To assess phage safety, we monitored fermentation parameters, including short chain fatty acid production and gas production/intake by colonic microbiota. We performed shotgun metagenomic analysis to evaluate the outcome of phage interference with colonic microbiota composition and functional potential. During the 24h incubation, concentrations of phage and its host were also evaluated. We found the phage used in this study, named E. coli phage vB_EcoS_Ace (Ace), to be safe towards human colonic microbiota, independently of the donors faecal content used. This suggests that individuality of donor faecal microbiota did not interfere with phage effect on the fermentations. However, the model revealed that the attenuated STEC strain used as phage host perturbed the faecal microbiota as based on metagenomic analysis, with potential differences in metabolic output. We conclude that the in vitro batch fermentation model used in this study is a reliable safety screening for lytic phages intended to be used as biocontrol agents.info:eu-repo/semantics/publishedVersio

Molecular ecology of the yet uncultured bacterial Ct85-cluster in the mammalian gut

In our previous studies on irritable bowel syndrome (IBS) –associated microbiota by molecular methods, we demonstrated that a particular 16S rRNA gene amplicon was more abundant in the feces of healthy subjects or mixed type IBS (IBS-M) –sufferers than in the feces of individuals with diarrhea-type IBS (IBS-D). In the current study, we demonstrated that this, so called Ct85-amplicon, consists of a cluster of very heterogeneous 16S rRNA gene sequences, and defined six 16S rRNA gene types, a to f, within this cluster, each representing a novel species-, genus- or family level taxon. We then designed specific PCR primers for these sequence types, mapped the distribution of the Ct85-cluster sequences and that of the newly defined sequence types in several animal species and compared the sequence types present in the feces of healthy individuals and IBS sufferers using two IBS study cohorts, Finnish and Dutch. Various Ct85-cluster sequence types were detected in the fecal samples of several companion and production animal species with remarkably differing prevalences and abundances. The Ct85 sequence type composition of swine closely resembled that of humans. One of the five types (d) shared between humans and swine was not present in any other animals tested, while one sequence type (b) was found only in human samples. In both IBS study cohorts, one type (e) was more prevalent in healthy individuals than in the IBS-M group. By revealing various sequence types in the widespread Ct85-cluster and their distribution, the results improve our understanding of these uncultured bacteria, which is essential for future efforts to cultivate representatives of the Ct85-cluster and reveal their roles in IBS.Peer reviewe

Chicory inulin enhances fermentation of 2'-fucosyllactose by infant fecal microbiota and differentially influences immature dendritic cell and T-cell cytokine responses under normal and Th2-polarizing conditions

Scope: Non-digestible carbohydrates (NDCs) such as native chicory inulin and 2′-fucosyllactose (2′-FL) are added to infant formula to mimic some of the human milk oligosaccharide (HMO) functions. It is unknown whether combining inulin and 2′-FL influences their fermentation kinetics and whether the immune-modulatory effects of these NDCs are different under normal and inflammatory-prone Th2-polarizing conditions. Methods and results: We investigated the in vitro fermentation of 2′-FL and native chicory inulin, fermented individually and combined, using fecal inocula of 8-week-old infants. Native inulin was fermented in a size-dependent fashion and expedited the fermentation of 2′-FL. Fermentation of both native inulin and 2′FL increased the relative abundance of Bifidobacterium, which coincided with the production of acetate and lactate. The fermentation digesta of all fermentations differentially influenced both dendritic cell and T-cell cytokine responses under normal culture conditions or in presence of the Th2-polarizing cytokines IL-33 and TSLP, with the most pronounced effect for IL-1β in the presence of TSLP. Conclusions: Our findings show that native inulin can expedite the fermentation of 2′-FL by infant fecal microbiota and that these NDC fermentation digesta have different effects under normal and Th2-polarizing conditions, indicating that infants with different immune backgrounds might benefit from tailored NDC formulations

A novel technique capable of taking 'protected' biopsies for reliable assessment of the distribution of microbiota along the colonic mucosa

We evaluated a novel 'protected' biopsy method to reliably ascertain the spatial distribution of the mucosa-adherent colonic microbiota. Apart from minor differences at genus level, overall similarities along the colon were high between the various areas, irrespective of protected or unprotected sampling.Peer reviewe

Endo-1,3(4)-β-Glucanase-Treatment of Oat β-Glucan Enhances Fermentability by Infant Fecal Microbiota, Stimulates Dectin-1 Activation and Attenuates Inflammatory Responses in Immature Dendritic Cells

Background: Non-digestible carbohydrates are added to infant formula to mimic the effects of human milk oligosaccharide by acting as prebiotics and stimulating the immune system. Although not yet used in infant formulas, β-glucans are known to have beneficial health effects, and are therefore of potential interest for supplementation. Methods and results: We investigated the in vitro fermentation of native and endo-1,3(4)-β-glucanase-treated oat β-glucan using pooled fecal inocula of 2-and 8-week-old infants. While native oat β-glucan was not utilized, both inocula specifically utilized oat β-glucan oligomers containing β(1→4)-linkages formed upon enzyme treatment. The fermentation rate was highest in the fecal microbiota of 2-week-old infants, and correlated with a high lactate production. Fermentation of media supplemented with native and enzyme-treated oat β-glucans increased the relative abundance of Enterococcus and attenuated proinflammatory cytokine production (IL-1β, IL-6, TNFα) in immature dendritic cells. This attenuating effect was more pronounced after enzyme treatment. This attenuation might result from the enhanced ability of fermented oat β-glucan to stimulate Dectin-1 receptors. Conclusion: Our findings demonstrate that endo-1,3(4)-β-glucanase treatment enhances the fermentability of oat β-glucan and attenuates pro-inflammatory responses. Hence, this study shows that especially enzyme-treated oat β-glucans have a high potential for supplementation of infant formula.</p

Effect of wheat bran derived prebiotic supplementation on gastrointestinal transit, gut microbiota, and metabolic health: a randomized controlled trial in healthy adults with a slow gut transit

Acute intake of the wheat bran extract Arabinoxylan-Oligosaccharide (AXOS) modulates the gut microbiota, improves stool characteristics and postprandial glycemia in healthy humans. Yet, little is known on how long-term AXOS intake influences gastrointestinal (GI) functioning, gut microbiota, and metabolic health. In this randomized, placebo-controlled, double-blind study, we evaluated the effects of AXOS intake on GI function and metabolic health in adults with slow GI transit without constipation. Forty-eight normoglycemic adults were included with whole-gut transit time (WGTT) of >35 h receiving either 15 g/day AXOS or placebo (maltodextrin) for 12-wks. The primary outcome was WGTT, and secondary outcomes included stool parameters, gut permeability, short-chain fatty acids (SCFA), microbiota composition, energy expenditure, substrate oxidation, glucose, insulin, lipids, gut hormones, and adipose tissue (AT) function. WGTT was unchanged, but stool consistency softened after AXOS. 12-wks of AXOS intake significantly changed the microbiota by increasing Bifidobacterium and decreasing microbial alpha-diversity. With a good classification accuracy, overall microbiota composition classified responders with decreased WGTT after AXOS. The incretin hormone Glucagon-like protein 1 was reduced after AXOS compared to placebo. Energy expenditure, plasma metabolites, AT parameters, SCFA, and gut permeability were unchanged. In conclusion, intake of wheat bran extract increases fecal Bifidobacterium and softens stool consistency without major effects on energy metabolism in healthy humans with a slow GI transit. We show that overall gut microbiota classified responders with decreased WGTT after AXOS highlighting that GI transit and change thereof were associated with gut microbiota independent of Bifidobacterium. NCT02491125.</p

Associations between Pro- and Anti-Inflammatory Gastro-Intestinal Microbiota, Diet, and Cognitive Functioning in Dutch Healthy Older Adults : The NU-AGE Study

Dietary modulation of the gastro-intestinal microbiota is a potential target in improving healthy ageing and age-related functional outcomes, including cognitive decline. We explored the association between diet, gastro-intestinal microbiota and cognition in Dutch healthy older adults of the 'New dietary strategies addressing the specific needs of the elderly population for healthy aging in Europe' (NU-AGE) study. The microbiota profile of 452 fecal samples from 226 subjects was determined using a 16S ribosomal RNA gene-targeted microarray. Dietary intake was assessed by 7-day food records. Cognitive functioning was measured with an extensive cognitive test battery. We observed a dietary and microbial pro- to anti-inflammatory gradient associated with diets richer in animal- or plant-based foods. Fresh fruits, nuts, seeds and peanuts, red and processed meat and grain products were most strongly associated to microbiota composition. Plant-rich diets containing fresh fruits, nuts, seeds and peanuts were positively correlated with alpha-diversity, various taxa from the Bacteroidetes phylum and anti-inflammatory species, including those related to Faecalibacterium prausnitzii and Eubacterium rectale and E. biforme. Animal product-rich diets associated with pro-inflammatory species, including those related to Ruminococcus gnavus and Collinsella spp.. Cognition was neither associated with microbiota composition nor alpha-diversity. In conclusion, diets richer in animal- and plant-based foods were related to a pro- and anti-inflammatory microbial profile, while cognition was associated with neither.Peer reviewe

Structure-Specific Fermentation of Galacto-Oligosaccharides, Isomalto-Oligosaccharides and Isomalto/Malto-Polysaccharides by Infant Fecal Microbiota and Impact on Dendritic Cell Cytokine Responses

SCOPE: Next to galacto-oligosaccharides (GOS), starch-derived isomalto-oligosaccharide preparation (IMO) and isomalto/malto-polysaccharides (IMMP) could potentially be used as prebiotics in infant formulas. However, it remains largely unknown how the specific molecular structures of these non-digestible carbohydrates (NDCs) impact fermentability and immune responses in infants. METHODS AND RESULTS: In vitro fermentation of GOS, IMO and IMMP using infant fecal inoculum of 2- and 8-week-old infants showed that only GOS and IMO were fermented by infant fecal microbiota. The degradation of GOS and IMO coincided with an increase in Bifidobacterium and production of acetate and lactate, which was more pronounced with GOS. Individual isomers with an (1↔1)-linkage or di-substituted reducing terminal glucose residue were more resistant to fermentation. GOS, IMO and IMMP fermentation digesta attenuated cytokine profiles in immature dendritic cells (DCs), but the extent was dependent on the infants age and NDC structure. CONCLUSION: The IMO preparation, containing reducing and non-reducing isomers, showed similar fermentation patterns as GOS in fecal microbiota of 2-week-old infants. Knowledge obtained on the substrate specificities of infant fecal microbiota and the subsequent regulatory effects of GOS, IMO and IMMP on DC responses might contribute to the design of tailored NDC mixtures for infants of different age groups. This article is protected by copyright. All rights reserved

Fecal microbiota transplantation as novel therapy in gastroenterology : A systematic review

AIM: To study the clinical efficacy and safety of Fecal microbiota transplantation (FMT). We systematically reviewed FMT used as clinical therapy. METHODS: We searched MEDLINE, EMBASE, the Cochrane Library and Conference proceedings from inception to July, 2013. Treatment effect of FMT was calculated as the percentage of patients who achieved clinical improvement per patient category, on an intention-to-treat basis. RESULTS: We included 45 studies; 34 on Clostridium difficile-infection (CDI), 7 on inflammatory bowel disease, 1 on metabolic syndrome, 1 on constipation, 1 on pouchitis and 1 on irritable bowel syndrome (IBS). In CDI 90% resolution of diarrhea in 33 case series (n = 867) was reported, and 94% resolution of diarrhea after repeated FMT in a randomized controlled trial (RCT) (n = 16). In ulcerative colitis (UC) remission rates of 0% to 68% were found (n = 106). In Crohn's disease (CD) (n = 6), no benefit was observed. In IBS, 70% improvement of symptoms was found (n = 13). 100% Reversal of symptoms was observed in constipation (n = 3). In pouchitis, none of the patients (n = 8) achieved remission. One RCT showed significant improvement of insulin sensitivity in metabolic syndrome (n = 10). Serious adverse events were rare. CONCLUSION: FMT is highly effective in CDI, and holds promise in UC. As for CD, chronic constipation, pouchitis and IBS data are too limited to draw conclusions. FMT increases insulin sensitivity in metabolic syndrome.Peer reviewe