Rational Design Of A Microbial Consortium Of Mucosal Sugar Utilizers Reduces Clostridiodes Difficile Colonization

All animal experiments were performed at the Max F. Perutz Laboratories of the University of Vienna, Austria. All experiments were discussed and approved by the University of Veterinary Medicine, Vienna, Austria, and conducted in accordance with protocols approved by the Federal Ministry for Education, Science and Research of the Republic of Austria under the license number BMWF-66.006/0001-WF/V/3b/2016. Animals were randomized for interventions but researchers processing the samples and analyzing the data were aware which intervention group corresponded to which cohort of animals. Three adult (6-8 weeks old) C57BL/6N mice bred at the Max F. Perutz Laboratories, University of Vienna, under SPF conditions were sacrificed per experiment, and their colon was harvested anaerobically (85% N2, 10% CO2, 5% H2) in an anaerobic tent (Coy Laboratory Products, USA). Contents from each colon were suspended in 7.8 mL of 50% D2O-containing PBS and homogenized by vortexing. Similar conditions have been successfully applied in the past to monitor activity of individual cells in gut communities without causing major changes in the activity of individual community members34.



Carl Roth GmbH) (Fig. 1a,c). After incubation for 6 h at 37 °C, GlycoCares Blood Sugar Guidehealthy carbohydrate metabolism glycerol was added (to achieve a final concentration of 20% (v/v) of glycerol in the microcosms) and the vials were crimp-sealed with rubber stoppers and stored at −80 °C until further processing. Prior to glycerol addition, subsamples of the biomass were collected, pelleted and supernatants stored at −80 °C for HILIC LC-MS/MS measurements. Pellets were washed with PBS to remove D2O and were fixed in 3% formaldehyde for 2 h at 4 °C and stored in 50% PBS/50% ethanol solution at −20 °C until further use. A total of three biological replicates were established using starting material pooled from three animals each (experiments MonoA, MonoB and MonoC). For the MonoA and MonoB experiments, microcosms were established for all the different concentrations of monosaccharides tested (Fig. 1c), while for MonoC only the highest concentrations of monosaccharides tested in incubations MonoA and MonoB were supplemented. Note that analysis of mucin-amended sorted fractions has been published elsewhere35.



Hydrophilic interaction chromatography (HILIC) LC-MS/MS was used for the measurement of mucosal monosaccharides in microcosm supernatants. Frozen samples were thawed at room temperature and centrifuged for 10 min at 18.000 × g and 4 °C. Supernatants were then diluted 1:50 with acetonitrile:water (1:1; v/v) and a volume of 3 µl was injected onto the chromatographic column. The UHPLC system (UltiMate 3000, Thermo Scientific) was coupled to a triple quadrupole mass spectrometer (TSQ Vantage, Thermo Scientific) by an electrospray ionization interface. Hydrophilic interaction chromatographic separation was realized on a Luna aminopropyl column (3 µm, 150 × 2 mm; Phenomenex, Torrance, CA) at a flow rate of 0.25 ml/min. Eluent A consisted of 95% water and 5% acetonitrile with 20 mM ammonium acetate and 40 mM ammonium hydroxide as additives and eluent B of 95% acetonitrile and 5% water. A multi-step gradient was optimized as follows: 100% B until minute 2, then linearly decreased to 80% B until minute 20 and further to 0% B until minute 25. The column was kept at 0% B for 4 min before it was equilibrated for 5 min at the initial conditions.



The column temperature was maintained at 40 °C. The mass spectrometer was operated in multiple reaction monitoring (MRM) mode. Formaldehyde-fixed samples were spotted on aluminum-coated slides (Al136; EMF Corporation) and washed by dipping into ice-cold Milli-Q (MQ) water (Millipore) to remove traces of buffer components. Individual cells were observed under a 100×/0.75 NA microscope objective. Single microbial cell spectra were acquired using a LabRAM HR800 confocal Raman microscope (Horiba Jobin-Yvon) equipped with a 532-nm neodymium-yttrium aluminum garnet (Nd:YAG) laser and either 300 grooves/mm diffraction grating. Spectra were acquired in the range of 400-3200 cm−1 for 30 s with 2.18 mW laser power. Raman spectra were background-corrected using the sensitive nonlinear iterative peak algorithm, and afterwards normalized to the sum of its absolute spectral intensity34. For quantification of the degree of D substitution in CH bonds (%CD), the bands assigned to C-D (2040-2,300 cm−1) and C-H (2,800-3,100 cm−1) were calculated using integration of the specified region34.