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

Abstract

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.

Infant Formula With a Specific Blend of Five Human Milk Oligosaccharides Drives the Gut Microbiota Development and Improves Gut Maturation Markers: A Randomized Controlled Trial

“…Acetate might also have contributed directly to the lower relative abundance of C. difficile as results from an in vivo study indicate that acetate promotes innate host responses against C. difficile through coordinated action on neutrophils and group 3 innate lymphoid cells ( 47 ). A direct effect of the HMOs preventing epithelial adhesion of C. difficile by acting as decoy receptors might have been possible too; however, pre-clinical work suggests that the effect is rather indirect via gut microbiota modulation ( 48 ). Our findings about toxigenic C. difficile are of importance because they indicate that the five-HMO blend can reduce a risk factor for infectious diarrhea in formula-fed infants.…”

2’-Fucosyllactose inhibits proliferation of Clostridioides difficile ATCC 43599 in the CDi-screen, an in vitro model simulating Clostridioides difficile infection

“…The inclusion of technical replicates in the CDi-screen results in statistically relevant data output and displays an advantage over other model systems that do not include technical replicates in the experimental design, likely due to high costs, large working volumes, and low experimental throughput (e.g. a recent study by Vigsnaes et al., 2021 ( Vigsnaes et al., 2021 )). Though in this study we used pooled human fecal material to mimic a complex microbial community as is common in these type of approaches ( Aguirre et al., 2014 ), we note that future application of the CDi-screen model would also allow to investigate differences in the response to therapeutic interventions that are mediated by differences in microbiome composition between hosts.…”

“…Therefore, it is pivotal to develop models that facilitate the study of C. difficile biology and anti- C. difficile interventions in vitro . A few C. difficile in vitro models exist; some models operate at a relatively large volume e.g., the Pathogut™ model (specific application of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME ® ) ( Crowther et al., 2016 ; Vigsnaes et al., 2021 ).…”

“…The anti-pathogenic effects of human milk oligosaccharides (HMOs) against C. difficile ATCC 9689 were reported based on Pathogut experiments. The effects coincided with increased levels of Bifidobacteriaceae and/or secondary bile acids ( Vigsnaes et al., 2021 ).…”

Battling Enteropathogenic Clostridia: Phage Therapy for Clostridioides difficile and Clostridium perfringens

“…These human milk oligosaccharides or their mixture decreased the presence of Cd in human gut microbiota versus untreated conditions. This decrease coincided with increased acetate and Bifidobacteriaceae levels, thereby suggesting a microbiota-modulated anti-Cd effect, rather than a direct effect of the human milk oligosaccharides ( Vigsnaes et al, 2021 ). These findings suggest that CDI can possibly be prevented by modulating microbiota composition and activity.…”