Quantifying Gut Microbiome Depletion

Day 13 and day 24 of the antibiotic treatment mice were fixed to defecate directly into a pre-weighted 2 ml capped microtube (Sarstedt, Nümbrect, Germany) prefilled with 1 ml sterile ice-cold phosphate-buffered saline (PBS). Tubes with fecal pellets were kept on ice, weighed and the weight of the pellets calculated (median 46 mg, range 17–120). Fecal pellets were resuspended in the 1 ml PBS by vortexing and by bashing with a sterile bacteriological loop. The fecal suspension was then plated on blood agar, anaerobic blood agar (hemin – vitamin K agar), and yeast agar (Sabouraud agar) in doubles with 100 µl suspension on each plate. Blood agar and Sabouraud agar plates were incubated aerobically at 37°C with 5% CO₂ for 72 hours, while anaerobic blood agar plates were incubated at 37°C in anaerobic conditions for 96 hours. At the end of incubation the numbers of colonies on the plates were counted and the number of bacteria per mg of feces calculated. Evaluation of cultivated agar plates was performed by an experienced bacteriologist (P.G.) The detection limit of the assay was defined as 1 cfu/mg feces. Only mice successfully depleted (<1 cfu/mg feces) were included in phenotypic and gene expression analyses.
As a positive control for the depletion verification assay, and to enumerate cultivable microbes with the fecal collection procedure, fecal pellets from untreated mice were collected with the above described procedure. Serial dilutions made in sterile PBS and suitable dilutions were plated on selective media for intestinal Gram negative rods, enterococci, anaerobic Gram negative rods (Bacteroides spp), Clostridium spp, Lactobacillus spp and Bifidobacterium spp. The aerobic agar plates were incubated in 37°C with 5% CO₂ for 48 hours while anaerobic agar plates were incubated in 37°C for 48 hours. After incubation the numbers of colonies on the plates were counted and the number of bacteria per mg of feces was calculated.