, Grass Lake, MI, USA) with an atmosphere of 85% N2, 10% CO2 and 5% H2 (PanGas AG, Dagmersellen, Switzerland). Fecal aliquots were prepared for immediate culture, while further aliquots were stored at ?80��C prior to selleck chem inhibitor DNA extraction for qPCR and pyrosequencing. Culture and strain isolation Fresh fecal aliquots of 0.5�C1 g were used to prepare 10% (wet wt/vol) suspensions in pre-reduced anaerobic peptone water (Oxoid AG, Pratteln, Switzerland, supplemented with 0.5 g/L L-cysteine-HCl, Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) and further serial 10-fold dilutions (vol/vol) of which 100 ��L of appropriate dilutions were plated in duplicate on two non-selective and seven selective agar media. Media targeting anaerobic gut-associated bacterial populations, bacteroides mineral salts agar for Bacteroides spp.

(using 5 g/L D-glucose as carbon source, VWR International, Dietikon, Switzerland) [32], Beerens agar for Bifidobacterium spp. [33], reinforced clostridial agar for members of the Clostridia [34] and Wilkins-Chalgren anaerobe agar for total anaerobes (Oxoid; supplemented with 0.5 g/L L-cysteine-HCl, Sigma-Aldrich), were incubated in an anaerobic chamber. On the other hand, media targeting facultative anaerobic populations, MacConkey agar no2 for Enterobacteriaceae/Enterococcus spp. (Oxoid), mannitol salt agar for Staphylococcus spp. (Oxoid) and nutrient agar for total facultative anaerobes (Oxoid) were incubated aerobically; except for Lactobacillus anaerobic de Man, Rogosa and Sharpe agar with vancomycin and bromocresol green (LAMVAB) targeting Lactobacillus spp.

[35] and azide blood agar for gram-positive cocci/Streptococcus spp. (Oxoid), which were incubated in anaerobic jars. Plates were incubated for up to 14 days at 37��C and population levels were reported as log cfu/g feces. Based on different morphologies, a set of colonies was isolated per sample and medium, streaked for purity and cultured in liquid media, Wilkins-Chalgren anaerobe broth for presumptive anaerobes (Oxoid; supplemented with 0.5 g/L L-cysteine-HCl, Sigma-Aldrich), tryptone soy broth for facultative anaerobes (Oxoid) and de Man, Rogosa and Sharpe broth for presumptive Lactobacillus spp. (Labo-Life S��rl, Pully, Switzerland; supplemented with 0.5 g/L L-cysteine-HCl, Sigma-Aldrich).

Brefeldin_A Purity was verified microscopically and finally viable isolates were maintained at ?80��C in a final concentration of 20% (vol/vol) glycerol, while centrifuged cells were stored at ?20��C until DNA extraction and subsequent Sanger sequencing. DNA extraction DNA was extracted from pure culture cell pellets using a Wizard Genomic DNA purification kit (Promega AG, D��bendorf, Switzerland), and total DNA was extracted from 0.1�C0.3 g of feces using a FastDNA SPIN Kit for Soil (MP Biomedicals, Illkirch, France) according to the manufacturers’ instructions.