Fecal DNA was extracted from the weighted stool samples as previously described37 (link). More precisely, the feces samples were weighed and then resuspended for 10 min at room temperature in 250 μl of 4 M guanidine thiocyanate in 0.1 M Tris (pH 7.5) (Sigma) and 40 μl of 10% N-lauroyl sarcosine (Sigma). After the addition of 500 μl of 5% N-lauroyl sarcosine in 0.1 M phosphate buffer (pH 8.0), the 2-ml tubes were incubated at 70 °C for 1 h. One volume (750 ml) of a mixture of 0.1- and 0.6-mm-diameter silica beads (Sigma) (previously sterilized by autoclaving) was added, and the tube was shaken at 6.5 Meter/second three times for 30 s each in a FastPrep (MP Biomedicals) apparatus. Polyvinylpolypyrrolidone (15 mg) was added to the tube, which was then vortexed and centrifuged for 5 min at 20,000g. After recovery of the supernatant, the pellets were washed with 500 μl of TENP (50 mM Tris (pH 8), 20 mM EDTA (pH 8), 100 mM NaCl, 1% polyvinylpolypyrrolidone) and centrifuged for 5 min at 20,000g, and the new supernatant was added to the first supernatant. The washing step was repeated two times. The pooled supernatant (about 2 ml) was briefly centrifuged to remove particles and then split into two 2-ml tubes. Nucleic acids were precipitated by the addition of 1 volume of isopropanol for 10 min at room temperature and centrifugation for 10 min at 20,000g. Pellets were resuspended and pooled in 450 μl of 100 mM phosphate buffer, pH 8, and 50 ml of 5 M potassium acetate. The tube was placed on ice overnight and centrifuged at 20,000g for 30 min. The supernatant was then transferred to a new tube containing 20 μl of RNase (1 mg/ml) and incubated at 37 °C for 30 min. Nucleic acids were precipitated by the addition of 50 μl of 3 M sodium acetate and 1 ml of absolute ethanol. The tube was incubated for 10 min at room temperature, and the nucleic acids were recovered by centrifugation at 20,000g for 15 min. The DNA pellet was finally washed with 70% ethanol, dried, and resuspended in 100 μl of Tris–EDTA (TE) buffer. DNA suspensions were stored at –20 °C for real-time qPCR analysis of the 16S rDNA or ITS2 sequences. DNA was then subjected to qPCR by using a Takyon SYBR Green PCR kit (Eurogentec) for quantification of all fungal sequences or by using TaqMan Gene Expression Assays (Life Technologies) for quantification of all bacterial sequences. The probes and primers for the bacterial 16S rDNA genes and primers for the fungal 18S rDNA genes were used as described previously19 (link),37 (link). The threshold cycle for each sample was determined for each gene normalized to the CT value of the all-bacteria 16S ribosomal RNA gene. Data were calculated using the 2−ΔΔCt method.