Identifying glycan consumers in human gut microbiota samples using metabolic labeling coupled with fluorescence-activated cell sorting.

The composition and metabolism of the human gut microbiota are strongly influenced by dietary complex glycans, which cause downstream effects on the physiology and health of hosts. Despite recent advances in our understanding of glycan metabolism by human gut bacteria, we still need methods to link glycans to their consuming bacteria. Here, we use a functional assay to identify and isolate gut bacteria from healthy human volunteers that take up different glycans. The method combines metabolic labeling using fluorescent oligosaccharides with fluorescence-activated cell sorting (FACS), followed by amplicon sequencing or culturomics. Our results demonstrate metabolic labeling in various taxa, such as Prevotella copri, Collinsella aerofaciens and Blautia wexlerae. In vitro validation confirms the ability of most, but not all, labeled species to consume the glycan of interest for growth. In parallel, we show that glycan consumers spanning three major phyla can be isolated from cultures of sorted labeled cells. By linking bacteria to the glycans they consume, this approach increases our basic understanding of glycan metabolism by gut bacteria. Going forward, it could be used to provide insight into the mechanism of prebiotic approaches, where glycans are used to manipulate the gut microbiota composition.

Characterizing the Effect of Amylase Inhibitors on Maltodextrin Metabolism by Gut Bacteria Using Fluorescent Glycan Labeling.

Diet-derived polysaccharides are an important carbon source for gut bacteria and shape the human gut microbiome. Acarbose, a compound used clinically to treat type 2 diabetes, is known to inhibit the growth of some bacteria on starches based on its activity as an inhibitor of α-glucosidases and α-amylases. In contrast to acarbose, montbretin A, a new drug candidate for the treatment of type 2 diabetes, has been reported to be more specific for the inhibition of α-amylase, notably human pancreatic α-amylase. However, the effects of both molecules on glycan metabolism across a larger diversity of human gut bacteria remain to be characterized. Here, we used ex vivo metabolic labeling of a human microbiota sample with fluorescent maltodextrin to identify gut bacteria affected by amylase inhibitors. Metabolic labeling was performed in the presence and absence of amylase inhibitors, and the fluorescently labeled bacteria were identified by fluorescence-activated cell sorting coupled with 16S rDNA amplicon sequencing. We validated the labeling results in cultured isolates and identified four gut bacteria species whose metabolism of maltodextrin is inhibited by acarbose. In contrast, montbretin A slowed the growth of only one species, supporting the fact that it is more selective. Metabolic labeling is a valuable tool to characterize glycan metabolism in microbiota samples and could help understand the untargeted impact of drugs on the human gut microbiota.

A Natural Polyphenol Exerts Antitumor Activity and Circumvents Anti-PD-1 Resistance through Effects on the Gut Microbiota.

Several approaches to manipulate the gut microbiome for improving the activity of cancer immune-checkpoint inhibitors (ICI) are currently under evaluation. Here, we show that oral supplementation with the polyphenol-rich berry camu-camu (CC; Myrciaria dubia) in mice shifted gut microbial composition, which translated into antitumor activity and a stronger anti-PD-1 response. We identified castalagin, an ellagitannin, as the active compound in CC. Oral administration of castalagin enriched for bacteria associated with efficient immunotherapeutic responses (Ruminococcaceae and Alistipes) and improved the CD8+/FOXP3+CD4+ ratio within the tumor microenvironment. Moreover, castalagin induced metabolic changes, resulting in an increase in taurine-conjugated bile acids. Oral supplementation of castalagin following fecal microbiota transplantation from ICI-refractory patients into mice supported anti-PD-1 activity. Finally, we found that castalagin binds to Ruminococcus bromii and promoted an anticancer response. Altogether, our results identify castalagin as a polyphenol that acts as a prebiotic to circumvent anti-PD-1 resistance. SIGNIFICANCE: The polyphenol castalagin isolated from a berry has an antitumor effect through direct interactions with commensal bacteria, thus reprogramming the tumor microenvironment. In addition, in preclinical ICI-resistant models, castalagin reestablishes the efficacy of anti-PD-1. Together, these results provide a strong biological rationale to test castalagin as part of a clinical trial. This article is highlighted in the In This Issue feature, p. 873.

Fluorescence activated cell sorting and fermentation analysis to study rumen microbiome responses to administered live microbials and yeast cell wall derived prebiotics.

Rapid dietary changes, such as switching from high-forage to high-grain diets, can modify the rumen microbiome and initiate gastrointestinal distress, such as bloating. In such cases, feed additives, including prebiotics and live microbials, can be used to mitigate these negative consequences. Bio-Mos® is a carbohydrate-based prebiotic derived from yeast cells that is reported to increase livestock performance. Here, the responses of rumen bacterial cells to Bio-Mos® were quantified, sorted by flow cytometry using fluorescently-labeled yeast mannan, and taxonomically characterized using fluorescence in situ hybridization and 16S rRNA sequencing. Further, to evaluate the effects of bovine-adapted Bacteroides thetaiotaomicron administration as a live microbial with and without Bio-Mos® supplementation, we analyzed microbial fermentation products, changes to carbohydrate profiles, and shifts in microbial composition of an in vitro rumen community. Bio-Mos® was shown to be an effective prebiotic that significantly altered microbial diversity, composition, and fermentation; while addition of B. thetaiotaomicron had no effect on community composition and resulted in fewer significant changes to microbial fermentation. When combined with Bio-Mos®, there were notable, although not significant, changes to major bacterial taxa, along with increased significant changes in fermentation end products. These data suggest a synergistic effect is elicited by combining Bio-Mos® and B. thetaiotaomicron. This protocol provides a new in vitro methodology that could be extended to evaluate prebiotics and probiotics in more complex artificial rumen systems and live animals.