A part of each stool sample was submitted to a density gradient in order to separate the microbiota from the rest of the fecal material, according to the method of Courtois and colleagues with some modifications25 (link). Two grams of feces were homogenized in 18 mL of sterile NaCl 0.9% (w/v), in a laboratory paddle blender (Stomacher Lab Blender 400, Seward Ltd. UK) for 1 min. A solution of Nycodenz® 80% (w/v) (PROGEN Biotechnik GmbH, Heidelberg, Denmark) was prepared in ultrapure water, and sterilized at 121 °C for 15 min. A volume of 10.5 mL of the diluted, homogenized fecal sample was placed on top of 3.5 mL of the Nycodenz® solution, and centrifuged for 40 min at 4 °C (10,000 × g, TST41.14 rotor, Kontron, Milan, Italy). The upper phase, containing soluble debris, was discarded after the centrifugation step, and the layers corresponding to the microbiota extracted with 10.5 mL of PBS (Fig. 1 ) were collected. Cellular suspensions were kept on ice for 5 minutes, in order to allow non-soluble debris to precipitate, were then washed twice, and stored in aliquots of 1 mL, at −80 °C, until DNA extraction was performed. In all the cases, DNA directly from homogenized stool samples, or from the corresponding separated microbiota fractions was extracted using the QIAamp DNA Stool Mini kit (Qiagen Ltd., Strasse, Germany), as described in a previous work26 (link).
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