In vitro Assay of Bacterial Adhesion Onto the Human Intestinal Caco-2 Cell Line

An in vitro bacterial adhesion assay was performed as described by Letourneau et al. with some modifications (Letourneau et al., 2011). To prepare monolayers of Caco-2 cells for the in vitro bacterial adhesion assay, one milliliter of cell suspension (2×10⁵ cells/ml) was seeded in three sets of duplicate wells (one for each treatment) of a 24-well plate, and the plate was incubated in a cell culture incubator until the cells were fully confluent. The cells were then washed with phosphate-buffered saline (PBS), and the culture medium was replaced with 900μl of antibiotic-free DMEM supplemented with 10% FBS; then, 100μl of B. subtilis natto supernatant or M9B medium was added.

Overnight cultures of E. faecalis cells were centrifuged, washed twice with PBS containing 2mm EDTA, and resuspended in DMEM with 10% FBS. A volume of bacterial culture corresponding to 10⁶ E. faecalis cells was used to inoculate Caco-2 cells. The same volume of E. faecalis culture was also added to a medium mixture (90% DMEM containing 10% FBS; 10% M9B medium) without Caco-2 cells to determine the total number of bacterial cells in the inoculum. The E. faecalis and Caco-2 cells were then cocultured at 37°C with 5% CO₂ for 3h. After 3h of incubation, the culture medium was removed, and the infected Caco-2 cells were washed 3 times with PBS. All cells were then detached with 0.05% trypsin-EDTA for 20min at 37°C. Then, serial dilutions of these samples were plated on selective Todd Hewitt broth agar medium containing 30g/L Bacto Todd Hewitt Broth (Neogen Cor., United States), 15g/L agar and 50μg/ml rifampicin, and the adherent E. faecalis cells were counted.