Anaerogen gas pack

Total intestinal loads of VSL#3 bacteria were quantified in serial dilutions of fecal and large intestinal luminal samples streaked onto Columbia-Agar supplemented with 5% sheep blood and Columbia-CNA Agar supplemented with colistin and nalidixic acid (both Oxoid) in parallel and incubated under aerobic (with 5% CO₂), microaerophilic (in jars using CampGen gas packs; Oxoid) and obligate anaerobic (in jars using Anaerogen gas packs; Oxoid) conditions for at least 2 days. Bacterial species were identified according to their typical morphological appearances. The total VSL#3 bacterial loads of intestinal samples were approximated as the sum of identified colony forming units (CFU) derived from the respective culture conditions. The detection limit of viable bacteria was ≈100 CFU per g.
The complex intestinal microbiota composition in conventionally colonized SPF mice and mice subjected to FMT was assessed by quantitative 16S rRNA based real time PCR as described previously (Heimesaat et al., 2010 (link), 2014 (link); Rausch et al., 2013 (link); Thoene-Reineke et al., 2014 (link)).

The WT GBS strain used in this study is NEM316, the originally sequenced (RefSeq NC_004368.1) serotype III reference isolate [39 (link)]. The usual Todd Hewitt (TH, Difco Laboratories), Columbia supplemented with 10% horse blood (BioMérieux), and Granada medium (BioMérieux) were used for propagation and phenotypic tests. A chemically defined medium (CDM) containing inorganic salts, vitamins, amino acids, nucleobases, pyruvate and glucose (S5 Table) was adapted from reference [65 (link)]. Glycine betaine, potassium chloride, and sodium chloride (Sigma-Aldrich) are added when stated. Buffering at pH 7.3 was done by adding Hepes (50 mM). Liquid GBS cultures are done in static condition incubated in aerobiosis or anaerobiosis. Anaerobiosis is obtained in hermetic jars with AnaeroGen gas packs (Oxoid, ThermoFischer). Growth curves in aerobiosis were done in 96 wells microplates (150 μl) at 37°C with constant shaking and automatic recording of OD₆₀₀ every 20 minutes (BioTek Synergy). Erythromycin and kanamycin (Sigma-Aldrich) are used for plasmid selection at 10 and 500 μg/ml, respectively. Anhydrotetracycline (Sigma-Aldrich) is used for conditional expression from the P_tetO inducible promoter at 0–100 ng/ml [66 (link)]. Rifampicin (50 μg/ml) was used for the quantification of spontaneous resistant mutations.

C. jejuni strain 81–176 and L. johnsonii were quantitated in feces over time post reassociation or upon necropsy in luminal samples taken from the colon, in homogenates of whole tissue ex vivo biopsies derived from spleen and liver (approximately 1 cm³) and in cardiac blood (approximately 1 mL). Whereas cardiac blood was analyzed by direct plating, feces, luminal colon contents and organ homogenates were dissolved in sterile phosphate buffered saline (PBS; Gibco life technologies, Paisley, UK) and serial dilutions cultured on Karmali and Columbia agar supplemented with 5% sheep blood (Oxoid) for two days at 37 °C under microaerobic conditions using CampyGen gas packs (Oxoid) for C. jejuni detection as described earlier^{12 (link)}. L. johnsonii loads were determined on Columbia agar supplemented with 5% sheep blood, Columbia-CNA agar supplemented with colistin and nalidixic acid (both Oxoid), and MRS agar (Oxoid) in parallel and incubated under microaerobic (in jars using CampGen gas packs; Oxoid) and obligate anaerobic (in jars using Anaerogen gas packs; Oxoid) conditions for at least two days. Bacterial species were identified according to their typical morphological appearances and sequencing of the 16S rRNA genes. The detection limit of viable bacteria was ≈100 CFU per g.

Escherichia coli ATCC 11775, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, and Bacteroides fragilis ATCC 25285 were cultured and maintained in Luria-Bertani (LB) broth and agar (Sigma). Bifidobacterium longum ATCC 15707 and Lactobacillus brevis ATCC 14869 were cultured and maintained in Bifidus selective medium (BSM) (Sigma) and deMan, Rogosa, Sharpe (MRS) medium (Sigma), respectively, in broth and agar forms. B. fragilis and B. longum were maintained in anaerobic condition inside anaerobic jars (Oxoid) with Anaerogen gas packs (Oxoid). All cultures were incubated at 37 °C. Absorbance readings of bacterial broth cultures prior to experiments were done using Tecan Infinite F200 microplate reader.

A lyophilized stock of the intestinal representative bacteria was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The commensal bacteria Escherichia coli ATCC 11775, Enterococcus faecalis ATCC 29212, and Bacillus subtilis ATCC 6633 were cultured and maintained in Luria–Bertani (LB) broth and agar (Sigma). Bifidobacterium longum ATCC 15707 was grown and maintained in Bifidus selective medium (BSM) broth and agar (Sigma). Lactobacillus brevis ATCC 14869 was cultivated on deMan, Rogosa and Sharpe (MRS) medium (Sigma). The microorganisms causing opportunistic infections Bacteroides fragilis ATCC 25285 and Bacteroides vulgatus ATCC 8482 were grown in brain heart infusion (BHI) broth (Sigma), and the number of colony-forming units (CFU) of Bacteroides was counted on Trypticase soy agar with defibrinated sheep blood (BD). All the bacteria were cultured at 37 °C. Bacteroides fragilis, B. longum, and B. vulgatus were incubated in a jar equipped with Anaerogen gas packs (Oxoid).

Viable C. jejuni strain 81–176 were detected in feces or at time of necropsy in luminal samples taken from the colon, dissolved in sterile PBS and serial dilutions cultured on Karmali- and Columbia-Agar supplemented with 5% sheep blood (Oxoid, Wesel, Germany) for two days at 37 °C under microaerobic conditions using CampyGen gas packs (Oxoid) as described earlier [12 (link)]. Probiotic bacteria of the formulation VSL#3 were quantitated in serial dilutions streaked onto Columbia-Agar supplemented with 5% sheep blood and Columbia-CNA Agar supplemented with colistin and nalidixic acid (both Oxoid) in parallel and incubated under aerobic (with 5% CO₂), microaerophilic (in jars using CampGen gas packs; Oxoid) and obligate anaerobic (in jars using Anaerogen gas packs; Oxoid) conditions for at least 2 days. Bacterial species were identified according to their typical morphological appearances and confirmed by 16S rRNA based sequencing. The total probiotic bacterial loads in intestinal samples were assessed by the sum of identified CFU derived from the respective culture conditions. The detection limit of viable bacteria was ≈100 CFU per g.