Authors and affiliations: Jari Verbunt* [1,2], Johan Jocken [2], Lars Vliex [2], John Penders [1], Ellen Blaak [2], Paul Savelkoul [1] and Frank Stassen [1]

*Presenting author, 1: Department of Medical Microbiology, infectious diseases & Infection prevention, Maastricht University Medical Center+, 2: Department of Human Biology, School of Nutrition and Translational Research in Metabolism (NUTRIM), Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands. Email: jari.verbunt@maastrichtuniversity.nl

Background The microbiome influences human health and substrate metabolism. Gut-bacteria produce factors that exit the intestine to signal host organs such as the liver, adipose tissue, and skeletal muscle. In animal studies, bacterial membrane vesicles (bMVs) have been found to modulate tissue substrate and energy metabolism. Antibiotic-induced disruptions of microbiota yield aberrant vesicle populations that could be modulated through fiber supplementation. Here we investigate characteristics of human gut-derived bMVs following microbiota disruption with vancomycin, and restoration with 2’-fucosyllactose (2FL) fibre supplementation.

Methods Fecal samples from 37 study participants with overweight/obesity (BMI 25-40 kg/m^2) and normal glucose tolerance were collected at (i) baseline, (ii) following a seven day course of vancomycin, and (iii) after 8 weeks of 2FL supplementation or placebo. bMVs were purified using (ultra)centrifugation and size exclusion chromatography. Nanoparticle tracking analysis was performed to determine bMV size and concentrations. 16S rDNA sequencing will be performed to identify bacterial origins bMVs. In vitro stimulation of human macrophages and HEK-cells allow functional characterization of vesicles (ongoing).

Results Post-vancomycin, mean bMV diameters decrease (183.6±4.977nm to 138.6±2.886nm, p<0.0001). Concurrently, median concentrations increased (1.7×10^10±2.2×10^9 bMVs/mL to 5.0×10^10±4.6×10^9 bMVs/mL, p<0.0001). This suggests that post-vancomycin, gut-microbiota produce smaller and more bMVs.

Discussion/Conclusion Here we show for the first time how vancomycin disrupts the gut-microbial bMV repertoire in humans. Vancomycin-induced outgrowth of Gram-negative bacteria is accompanied by an increased number of smaller bMVs. As these bacteria and their vesicles contain LPS, their increased abundance and translocation from the intestine could contribute to metabolic endotoxemia. Therefore, bMVs add complexity to microbe-host interactions in the pathophysiology of metabolic diseases such as diabetes type 2.