Preparation of Bacterial Co-Cultures

High toxigenic ({"type":"entrez-protein","attrs":{"text":"VPI10463","term_id":"1642177071","term_text":"VPI10463"}}VPI10463, HTCD) and nontoxigenic ({"type":"entrez-protein","attrs":{"text":"VPI11186","term_id":"1645792007","term_text":"VPI11186"}}VPI11186) C. difficile strains (purchased from ATCC) were cultured in brain heart infusion (BHI) broth (BD, BBL BHI) at 37 °C overnight in an anaerobic chamber. For supernatant culture, the overnight cultured cells (O.D. 600 = 0.89 ± 1%) were centrifuged at 3000 g for 5 min. The residual cells in the supernatants were removed by a 0.2 μm pore size filter (Millipore). The supernatants were freshly prepared before each experiment. Pelleted HTCD or NTCD cells were resuspended in fresh BHI. The HTCD suspension (200 μL) was inoculated in 800 μL of the respective supernatants (BHI, HTCD supernatant or NTCD supernatant, O.D. 600 = 0.331 ± 1%). For transwell co-culture, 200 μL of the HTCD suspension and 1.2 mL of BHI were inoculated in the wells within a 12-well plate for co-culture with 200 μL of NTCD or HTCD suspensions (co-culture control) and 600 μL of BHI inoculated in the transwell insert. The cells inoculated in supernatants or in the transwell co-culture setup were incubated at 37 °C overnight (16–18 h) in an anaerobic chamber. Prior to the DEP or ROT, the medium was replaced with 8.8% sucrose water, with medium conductivity (Mettler Toledo FE20) of 5 ± 5% mS/m as adjusted by the BHI medium. Using the colony-forming unit (CFU) assay, we confirmed (Supporting Information Table S1) that C. difficile can survive for up to 3 h under the aerobic conditions within this altered medium (typical time frame for DEP and ROT analysis is <5 min).