Bacterial Viability/Cytotoxicity Assay.

Staining of living and dead bacteria was performed following the manufacturer’s protocol (Viability/Cytotoxicity Assay Kit for Bacteria Live and Dead Cells, Biotium). The 5-d-cultured Lcr bacterial cells and overnight-cultured Bs, Xcv, E. coli, and At bacterial cells were used for the assay. The cultures were centrifuged (7,000 g, 10 min, 22 °C) to pellet, resuspended, and washed with 0.85% NaCl solution three times. The bacterial cells were suspended in 0.85% NaCl solution, adjusted to OD₆₀₀ 1.0, and diluted 100-fold for staining. A 100× MaSAMP stock (1 M, 100 μM, and 10 μM in dimethylsulfoxide [DMSO]) was prepared to dilute the bacterial suspension to create the final MaSAMP concentrations of 10 μM, 1 μM, and 100 nM. At the end of treatment, the stained bacterial cell suspensions were concentrated 100-fold and observed with Leica SP5 confocal microscopy. Alternatively, the fluorescence intensity of stained bacterial cell suspension was measured with the Promega GloMax Discover Microplate Reader.