Adhesion assay for B. infantis

Adhesion assays with B. infantis were performed as previously described [18 (link),19 (link)]. HT-29 cells were washed twice with PBS, and 250 µL of the bacteria and media suspensions were added to the wells, corresponding to approximately 40 bacterial cells per human cell. Bacterial cells were incubated with the HT-29 cells for 2 h at 37 °C under anaerobic conditions using an Anaerocult A system (Merck). The HT-29 cells were then washed five times with PBS to remove non-adherent bacteria. HT-29 cells were then lysed with 250 µL of 1% TritonTM X-100 (Merck) for 5 min at 37 °C. The lysates were serially diluted and enumerated by spot-plating on MRS plates to enumerate bacterial colony-forming units (CFU). The adhesion of the bacteria was determined as the percentage of original inoculum which attached, thus accounting for variations in the starting inoculum. Percentage adhesion = (CFU/mL of recovered adherent bacteria/CFU/mL of inoculum) × 100. Experiments were performed in triplicate on three separate occasions.