Enspire 2300

ROS levels in S128P and WT^silent were measured using a reactive oxygen species assay kit (Beyotime, China) according to the manufacturer's protocol. This kit detects the ROS with the DCFH-DA (2,7-dichlorofluorescein diacetate) probe. DCFH-DA can pass through the cell membrane and would be hydrolyzed by intracellular esterase to produce DCFH (2,7-dichlorofluorescein). DCFH disrupts the membrane permeability and the intracellular ROS would oxidize DCFH to produce fluorescent DCF (dichlorofluorescein). The level of intracellular ROS was measured by detection of fluorescence via DCF. Briefly, after a 15-min exposure to 0.1 mM H₂O₂, 1 mL of bacterial cells (10⁹ CFU/mL) was treated with 30 μL 10 μM DCFH-DA and incubated at 28°C for 20 min. After the extracellular DCFH-DA was removed, the bacterial cells were collected by centrifugation at 4,000 g for 5 min, washed three times and re-suspended in fresh medium. Cells (200 μL) were analyzed using a multimode reader (ENSPIRE 2300, Promega, USA) with excitation and emission wavelengths of 488 and 525 nm, respectively. Eight biological replicates were analyzed in this experiment.

During specific time intervals, the intra- and extracellular ATP levels were determined by a previously described bioluminescence-based method (Suzuki et al., 2005 (link)) using a microplate reader (PE & EnSpire 2300) and a CellTiter-Glo 2.0 Kit (Promega, USA).

A damaged cell membrane results in the leakage of intracellular ATP and UV-absorbing materials (nucleic acid substances and proteins) from CD cells. Thus, the changes in extracellular ATP and UV-absorbing material levels accurately reflect the degree of damage to CD cells. 1 mL of a CD cell suspension (10⁸ CFU/mL) was mixed with 1 mL of different doses of YH68-CFCS before anaerobically co-cultured at 37°C. Sampling 200 μL from the co-cultured mixed bacterial solution at 3 h, and the CFCS of the mixed bacterial solution was collected after centrifugation (4°C, 12,000 r/min, and 10 min) and filtration (0.22 μm). 100 μL of the CFCS was used to determine the extracellular ATP levels by a previously described bioluminescence-based method (Suzuki et al., 2005 (link)) using a microplate reader (PE and EnSpire 2300) and a CellTiter-Glo 2.0 Kit (Promega, United States). The rest 100 μL of the CFCS was used to determine the extracellular UV-absorbing material levels according to a method reported by Liu et al., 2017 (link), with some modifications. The absorbance of the CFCS was recorded at OD₂₆₀ and OD₂₈₀ using a UV spectrophotometer (Lamda 950, China). All samples were tested in triplicate and this assay was repeated at least three times independently.