Fig. 3

B. subtilis CFS alters the pattern of genes expression and increases membrane permeability of S. aureus. a qRT-PCR analysis of the genes involved in adhesive molecules (Cna and ClfA), virulence factor Hla, quorum sensing (argA, argB and RNAIII) and biofilm formation (Ica and sarA) in S. aureus. S. aureus (1 × 10⁸ CFU/mL) was treated with PBS (control) or B. subtilis CFS for 3 h. N = 4/group, *P < 0.05, Mann-Whitney U test. b Representative images of SYTO9-PI staining to detect the effect of B. subtilis CFS on the membrane permeability of S. aureus. S. aureus (1 × 10⁸ CFU/mL) was treated with PBS (control) or B. subtilis CFS for 1 h, followed by staining with 10 µM of SYTO9 (membrane-permeable) and 3 µM of PI (membrane-impermeable). Both live and dead cells were stained with green, and dead ones stained red. Scale bar 20 μm. c The leakage of cellular ATP from S. aureus after treatment with B. subtilis CFS. Data are represented as means ± SD of 4 independent colonies. *P < 0.05 vs. control. Mann-Whitney U test. d Representative TEM images of PBS-treated (control) and B. subtilis CFS-treated S. aureus. Scale bar 200 nm. The white, black, yellow, and orange arrows indicate the normal cell, cells with extrusion of intracellular content, the disruption of cell wall and the displacement of cell membrane, respectively. e Representative images of Coomassie brilliant blue (CBB) staining for whole-cell and membrane proteins of PBS-treated (control) and B. subtilis CFS-treated S. aureus. Black arrows indicate decreased levels of proteins at 55, 70 and 100 kDa, and the blue arrow increased level of proteins. Experiments were repeated independently from 4 different colonies of S. aureus. f qRT-PCR analysis of the mRNA expression of mecA, the gene encoding PBP2a. S. aureus (1 × 10⁸ CFU/mL) was treated with PBS (control) or B. subtilis CFS for 3 h. N = 4/group, **P < 0.01, Mann-Whitney U test. Western blot analyses (g) and quantification (h)　of B. subtilis CFS on PBP2a protein expression after 3 h treatment. N = 3/group, *P < 0.05, Mann-Whitney U test