Drivers of Clostridioides difficile hypervirulent ribotype 027 spore germination, vegetative cell growth and toxin production in vitro

Objectives: Clostridioides difficile infection (CDI) is a considerable healthcare and economic burden worldwide. Faecal microbial transplant remains the most effective treatment for CDI, but is not at the present time the recommended standard of care. We hereby investigate which factors derived from a healthy gut microbiome might constitute the colonisation resistance barrier (CRB) in the gut, inhibiting CDI. Method: CRB drivers pH, short chain fatty acid (SCFA), and oxidation-reduction potential (ORP) were investigated in vitro using C. difficile NAP1/BI/027. Readouts for inhibitory mechanisms included germination, growth, toxin production and virulence gene expression. pH ranges (3 – 7.6), SCFA concentrations (25 – 200mM) and ORP (-300 - +200mV) were manipulated in brain heart infusion broth cultures under anaerobic conditions to assess the inhibitory action of these mechanisms. Results: <pH 5.3 completely inhibited C. difficile growth to OD of 0.019 vs. 1.19 for control pH 7.5. Toxin production was reduced to 25 units vs 3125 units for pH 7.6 (1 in 5 dilutions). Virulence gene expression reduced by 150 fold compared with pH 7.6 (p<0.05). Germination and proliferation of spores below pH 6.13 yielded an average OD of 0.006 vs. 0.99 for control. SCFA were potent regulators of toxin production at 25mM and above (p<0.05). Acetate significantly inhibited toxin production to 25 units independent of OD (0.8733) vs. control (OD 0.6 and toxin titer 3125) (p<0.05). ORP did not impact C. difficile growth. Conclusion: This study highlights the critical role that pH has in the CRB, regulating CDI in vitro and that SCFA can regulate C. difficile function independent of pH

The effects of dietary supplementation with inulin and inulin-propionate ester on hepatic steatosis in adults with non-alcoholic fatty liver disease

The short chain fatty acid (SCFA) propionate, produced through fermentation of dietary fibre by the gut microbiota, has been shown to alter hepatic metabolic processes that reduce lipid storage. We aimed to investigate the impact of raising colonic propionate production on hepatic steatosis in adults with non-alcoholic fatty liver disease (NAFLD). Eighteen adults were randomised to receive 20g/day of an inulin-propionate ester (IPE), designed to deliver propionate to the colon, or an inulin-control for 42-days in a parallel design. The change in intrahepatocellular lipid (IHCL) following the supplementation period was not different between groups (P=0.082), however IHCL significantly increased within the inulin-control group (20.9±2.9 to 26.8±3.9%; P=0.012; n=9), which was not observed within the IPE group (22.6±6.9 to 23.5±6.8%; P=0.635; n=9). The predominant SCFA from colonic fermentation of inulin is acetate, which in a background of NAFLD and a hepatic metabolic profile that promotes fat accretion, may provide surplus lipogenic substrate to the liver. The increased colonic delivery of propionate from IPE appears to attenuate this acetate- mediated increase in IHC

A novel dietary strategy to increase colonic propionate production in humans and improve appetite regulation and bodyweight management

A number of animal and human investigations have demonstrated that dietary supplementation with non-digestible carbohydrates (NDC) improves appetite regulation and bodyweight. These positive metabolic effects have been linked to the elevated production of short chain fatty acids (SCFAs) through fermentation of NDC by the colonic microbiota. Of the principle SCFAs, propionate has the highest affinity for free fatty acid receptor 2 (FFAR2), which is expressed on colonic L-cells and upon stimulation secretes anorectic hormones. Augmenting colonic propionate is therefore an attractive target to improve appetite regulation and, as part of the BBSRC DRINC initiative, we developed a novel dietary molecule, inulin-propionate ester that we demonstrate increases colonic propionate levels in humans. Our further studies with inulin-propionate ester in human volunteers suggest that dietary interventions that enhance colonic propionate levels may be a strategy to improve appetite regulation and weight management at the population level