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

Abstract

BackgroundClostridioides difficile is a Gram-positive anaerobic bacterium that can produce the toxins TcdA and/or TcdB and is considered an opportunistic pathogen. C. difficile is mainly transmitted as endospores, which germinate to produce the pathogenic vegetative cells under suitable conditions in the gut. To efficiently screen novel therapeutic- interventions against the proliferation of C. difficile within a complex microbial community, platforms are needed that facilitate parallel experimentation. In order to allow for screening of novel interventions a medium-to-high throughput in vitro system is desirable. To this end, we have developed the 96-well CDi-screen platform that employs an adapted simulated ileal effluent medium (CDi-SIEM) and allows for culturing of pathogenic C. difficile.MethodsC. difficile strain ATCC 43599 was inoculated in the form of vegetative cells and spores into the CDi-screen in the presence and absence of a cultured fecal microbiota and incubated for 48h. To demonstrate its utility, we investigated the effect of the human milk oligosaccharide 2’-Fucosyllactose (2’-FL) at 4 and 8 mg/mL on C. difficile outgrowth and toxin production in the CDi-screen. The test conditions were sampled after 24 and 48 hours. C. difficile -specific primers were used to monitor C. difficile growth via qPCR and barcoded 16S rRNA gene amplicon sequencing facilitated the in-depth analysis of gut microbial community dynamics.ResultsC. difficile ATCC 43599 proliferated in CDi-SIEM, both when inoculated as spores and as vegetative cells. The strain reached cell numbers expressed as C. difficile genome equivalents of up to 10 8 cells per mL after 24h of incubation. 2’-FL significantly inhibited the outgrowth of the ATTC 43599 strain within a complex human gut microbial community in the CDi-screen. In addition, a dose-dependent modulation of the gut microbial community composition by 2’-FL supplementation was detected, with a significant increase in the relative abundance of the genus Blautia in the presence of 2’-FL.ConclusionThe CDi-screen is suitable for studying C. difficile proliferation in a complex gut ecosystem and for screening for anti-pathogenic interventions that target C. difficile directly and/or indirectly through interactions with the gut microbiota. Different doses of compounds such as in this study the dose of the human milk oligosaccharide 2’-FL can be screened for efficacy in the inhibition of C. difficile proliferation.

Animal and In Vitro Models as Powerful Tools to Decipher the Effects of Enteric Pathogens on the Human Gut Microbiota

“…As a result of these changes in microbial composition, metabolic profiles of in vitro-cultured microbiota also changed, positively influencing the sporulation process of C. difficile [ 34 ]. The effect of specific dietary compounds on C. difficile proliferation was also evaluated in an in vitro model mimicking CDI, named CDi-Screen [ 35 ]. Vegetative cells and spores of C. difficile ATCC 43599 were separately co-cultured with the gut microbiota in the model in the presence of 2′-fucosyllactose (2′-FL), which was shown to significantly inhibit the overgrowth of C. difficile in vitro, reduce its abundance, and enhance the levels of Blautia in a dose-dependent manner [ 35 ].…”

A host-microbial metabolite interaction gut-on-a-chip model of the adult human intestine demonstrates beneficial effects upon inulin treatment of gut microbiome

“…Participants gave written informed consent. Fecal samples were collected and prepared as described [ 30 , 31 ] with some modifications. In brief, fecal samples were collected by the volunteers with the FecesCatcher (fecesvanger.nl).…”

2’-Fucosyllactose Inhibits Human Norovirus Replication in Human Intestinal Enteroids

“…In addition to serving as prebiotics for bacteria in the infant gut, other functions of HMOs include modulating epithelial and immune cell responses and acting as decoy receptors to reduce the attachment of pathogenic microbes to cell surface receptors (12). As such, HMOs have been shown to prevent pathogen adhesion to host epithelia for multiple enteric bacteria such as Campylobacter jejuni, Clostridioides difficile and Escherichia coli O157 as well as viruses such as rotavirus, coxsackievirus class A type 9 and SARS-CoV-2 (13)(14)(15)(16)(17)(18). Previous X-ray crystallography studies with three HuNoV genotypes (GI.1, GII.10 and GII.17) have shown that 2-fucosyllactose (2'FL), an α-1,2-fucosylated HMO, binds to the protruding domain of the HuNoV capsid protein VP1 in a similar pocket as HBGAs (19)(20)(21).…”