Bacteroides fragilis Reactivation and Transplantation

We obtained Bacteroides fragilis NCTC 9343 from the National Collection of Type Cultures (London, UK). The bacterium was reactivated following the manufacturer’s protocol. B. fragilis was grown in Brain Heart Infusion (BHI) broth (Sigma Aldrich, St. Louis, MO, USA) and used for transplantation in the antibiotics-treated mice. The culture was incubated anaerobically at 37 °C for 24 h [34 (link)]. A 5 × 10⁷ CFU/mL culture suspension of B. fragilis was centrifuged at 3000× g for 15 min. Bacterial pellets were resuspended in phosphate-buffered saline and washed twice. Viability was determined by plating an aliquot of serially diluted bacterial suspension on blood agar (Sigma Aldrich, St. Louis, MO, USA). The mice were gavaged with 200 µL of B. fragilis suspension a day after stopping antibiotics (Figure 1C).