2'FL and LNnT Exert Antipathogenic Effects against C. difficile ATCC 9689 In Vitro, Coinciding with Increased Levels of Bifidobacteriaceae and/or Secondary Bile Acids.

Clostridioides difficile (formerly Clostridium difficile) infection (CDI) is one of the most common hospital-acquired infections, which is often triggered by a dysbiosed indigenous gut microbiota (e.g., upon antibiotic therapy). Symptoms can be as severe as life-threatening colitis. The current study assessed the antipathogenic potential of human milk oligosaccharides (HMOs), i.e., 2'-O-fucosyllactose (2'FL), lacto-N-neotetraose (LNnT), and a combination thereof (MIX), against C. difficile ATCC 9689 using in vitro gut models that allowed the evaluation of both direct and, upon microbiota modulation, indirect effects. During a first 48 h fecal batch study, dysbiosis and CDI were induced by dilution of the fecal inoculum. For each of the three donors tested, C. difficile levels strongly decreased (with >4 log CFU/mL) upon treatment with 2'FL, LNnT and MIX versus untreated blanks, coinciding with increased acetate/Bifidobacteriaceae levels. Interindividual differences among donors at an intermediate time point suggested that the antimicrobial effect was microbiota-mediated rather than being a direct effect of the HMOs. During a subsequent 11 week study with the PathogutTM model (specific application of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®)), dysbiosis and CDI were induced by clindamycin (CLI) treatment. Vancomycin (VNC) treatment cured CDI, but the further dysbiosis of the indigenous microbiota likely contributed to CDI recurrence. Upon co-supplementation with VNC, both 2'FL and MIX boosted microbial activity (acetate and to lesser extent propionate/butyrate). Moreover, 2'FL avoided CDI recurrence, potentially because of increased secondary bile acid production. Overall, while not elucidating the exact antipathogenic mechanisms-of-action, the current study highlights the potential of HMOs to combat CDI recurrence, help the gut microbial community recover after antibiotic treatment, and hence counteract the adverse effects of antibiotic therapies.

Human Milk Oligosaccharides Modulate Fecal Microbiota and Are Safe for Use in Children With Overweight: A Randomized Controlled Trial.

OBJECTIVES: Human milk oligosaccharides (HMOs) impact the intestinal microbiota by increasing beneficial bacteria in infants and adults, and are safe and well tolerated in these age groups. Effects on intestinal microbiota, safety, and digestive tolerance in children have not been, however, assessed. The aims of this trial were to evaluate if HMOs are able to specifically modulate the intestinal microbiota in children, and to assess safety and digestive tolerance. METHODS: In this randomized, double-blinded, placebo-controlled trial, 75 children with overweight (including obesity) ages 6 to 12 years were randomized to receive 2'-fucosyllactose (2'FL), a mix of 2'FL and lacto-N-neotetraose (Mix), or a glucose placebo orally administrated once per day for 8 weeks. RESULTS: The relative abundance of bifidobacteria increased significantly after 4 (P < 0.001) and 8 (P = 0.025) weeks of intervention in the 2'FL-group and after 4 weeks (P = 0.033) in the Mix-group, whereas no change was observed in the placebo group. Compared with placebo, the 2'FL-group had a significant increase in bifidobacteria abundance after 4 weeks (P < 0.001) and 8 weeks (P = 0.010) and the Mix-group showed a tendency to increased bifidobacteria abundance after 4 (P = 0.071) and 8 weeks (P = 0.071). Bifidobacterium adolescentis drove the bifidogenic effect in the 2 groups. Biochemical markers indicated no safety concerns, and the products did not induce digestive tolerance issues as assessed by Gastrointestinal Symptoms Rating Scale and Bristol Stool Form Scale. CONCLUSIONS: Both 2'FL and the Mix beneficially modulate intestinal microbiota by increasing bifidobacteria. Furthermore, supplementation with either 2'FL alone or a Mix is safe and well tolerated in children.

Effects of Human Milk Oligosaccharides on the Adult Gut Microbiota and Barrier Function.

Human milk oligosaccharides (HMOs) shape the gut microbiota in infants by selectively stimulating the growth of bifidobacteria. Here, we investigated the impact of HMOs on adult gut microbiota and gut barrier function using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), Caco2 cell lines, and human intestinal gut organoid-on-chips. We showed that fermentation of 2'-O-fucosyllactose (2'FL), lacto-N-neotetraose (LNnT), and combinations thereof (MIX) led to an increase of bifidobacteria, accompanied by an increase of short chain fatty acid (SCFA), in particular butyrate with 2'FL. A significant reduction in paracellular permeability of FITC-dextran probe was observed using Caco2 cell monolayers with fermented 2'FL and MIX, which was accompanied by an increase in claudin-8 gene expression as shown by qPCR, and a reduction in IL-6 as determined by multiplex ELISA. Using gut-on-chips generated from human organoids derived from proximal, transverse, and distal colon biopsies (Colon Intestine Chips), we showed that claudin-5 was significantly upregulated across all three gut-on-chips following treatment with fermented 2'FL under microfluidic conditions. Taken together, these data show that, in addition to their bifidogenic activity, HMOs have the capacity to modulate immune function and the gut barrier, supporting the potential of HMOs to provide health benefits in adults.

Human milk oligosaccharide supplementation in irritable bowel syndrome patients: A parallel, randomized, double-blind, placebo-controlled study.

OBJECTIVES: Human milk oligosaccharides safely and beneficially impact bifidobacteria abundance in healthy adults, while their effects in patients with irritable bowel syndrome (IBS) are unknown. Hence, we aimed to determine the dose of 4:1 mix of 2'-O-fucosyllactose and Lacto-N-neotetraose (2'FL/LNnT) that increases fecal bifidobacteria abundance without aggravating overall gastrointestinal symptoms in IBS patients in a randomized, double-blind, controlled study. Additionally, the impact of 2'FL/LNnT on the fecal bacterial profile was assessed. METHODS: Irritable bowel syndrome patients diagnosed according to the Rome IV criteria received placebo (glucose), or 5 g or 10 g 2'FL/LNnT for 4 weeks followed by a four-week follow-up period. Gastrointestinal Symptom Rating Scale-IBS was used to assess gastrointestinal symptom severity; fecal microbiota composition was evaluated by GA-map™ Dysbiosis Test. RESULTS: Of the included 60 patients, two (one placebo and one 10 g) discontinued prematurely. Fecal bifidobacteria abundance was increased at week 4, but not at week 8, in the 10 g group compared to the other groups. Severity of overall or individual gastrointestinal symptoms did not differ between the groups at week 4 or 8, and no symptom deterioration was seen in any of the groups. The 10 g dose influenced overall fecal microbiota composition, and responders-defined as bifidobacteria increase ≥50%-could be discriminated from non-responders based on fecal microbiota modulation. CONCLUSIONS: The 10 g dose of 2'FL/LNnT induced an increase in the beneficial Bifidobacterium spp. without aggravating gastrointestinal symptoms in patients with IBS. This approach may be worthwhile to modulate gut microbiota of IBS patients toward a healthier profile.

Human Milk Oligosaccharides Support Normal Bowel Function and Improve Symptoms of Irritable Bowel Syndrome: A Multicenter, Open-Label Trial.

INTRODUCTION: Treatment options for irritable bowel syndrome (IBS) are limited, causing many patients to remain symptomatic. This study assessed the potential of human milk oligosaccharides (HMOs) to normalize bowel habits. Secondary outcomes included IBS severity and health-related quality of life. METHODS: This multicenter, open-label trial recruited patients with IBS from 17 sites across the United States. Patients received daily orally administrated 5-g intervention of the HMOs 2'-fucosyllactose and lacto-N-neotetraose in a 4:1 mix. Bowel habits, IBS symptoms, and quality of life were assessed at baseline and every 4 weeks during the 12-week intervention. RESULTS: A total of 317 patients (70.7% women; mean age of 44.0 years, range 18-93 years) received the trial product, and 245 patients completed the trial according to protocol. Patients had a significant improvement from baseline to 12 weeks in total percentage of bowel movements with abnormal stool consistency (mean and [95% confidence interval]: 90.7 [88.9-92.9] vs 57.2% [53.9-60.5], P < 0.0001), overall IBS Symptom Severity Score (323 [314-332] vs 144 [133-155], P < 0.0001) and health-rela,ted quality of life (50.4 [48.0-52.8] vs 74.6 [72.3-76.9], P < 0.0001). Improvement was similar across IBS subtypes. Symptoms improved most in the first 4 weeks of intervention. The most common side effects were mild gastrointestinal symptoms such as flatulence, abdominal pain and discomfort, and distension. DISCUSSION: Supplementation with 2 selected HMOs improves IBS symptoms and quality of life without substantial side effects. These promising results suggest that this novel approach to IBS should be confirmed in a randomized, placebo-controlled trial.

Oral supplementation of healthy adults with 2'-O-fucosyllactose and lacto-N-neotetraose is well tolerated and shifts the intestinal microbiota.

The gut microbiota has been established as an important player influencing many aspects of human physiology. Breast milk, the first diet for an infant, contains human milk oligosaccharides (HMO) that shape the infant's gut microbiota by selectively stimulating the growth of specific bacteria, especially bifidobacteria. In addition to their bifidogenic activity, the ability of HMO to modulate immune function and the gut barrier makes them prime candidates to restore a beneficial microbiota in dysbiotic adults and provide health benefits. We conducted a parallel, double-blind, randomised, placebo-controlled, HMO-supplementation study in 100 healthy, adult volunteers, consuming chemically produced 2'-O-fucosyllactose (2'FL) and/or lacto-N-neotetraose (LNnT) at various daily doses and mixes or placebo for 2 weeks. All participants completed the study without premature discontinuation. Supplementation of 2'FL and LNnT at daily doses up to 20 g was shown to be safe and well tolerated, as assessed using the gastrointestinal symptoms rating scale. 16S rRNA sequencing analysis showed that HMO supplementation specifically modified the adult gut microbiota with the primary impact being substantial increases in relative abundance of Actinobacteria and Bifidobacterium in particular and a reduction in relative abundance of Firmicutes and Proteobacteria. This study provides the first set of data on safety, tolerance and impact of HMO on the adult gut microbiota. Collectively, the results from this study show that supplementing the diet with HMO is a valuable strategy to shape the human gut microbiota and specifically promote the growth of beneficial bifidobacteria.