Mechanistic insights into mixed bile salt-weak base disinfectants against wild-type and multidrug-resistant...

Staphylococcus aureus biofilms on stainless-steel surfaces show high tolerance to disinfectants, particularly in multidrug-resistant (MDR) strains, posing risks in food and pharmaceutical processing. Repurposing Generally Recognized as Safe (GRAS) compounds for low-dose biofilm control is therefore attractive. This study evaluated the antibacterial and antibiofilm efficacy and key mechanisms of mixed bile salt-weak base disinfectants against wild-type and MDR S. aureus biofilms. In brief, sodium cholate (CHO) and sodium chenodeoxycholate (CHE) were combined with ammonium hydroxide (AMH) or sodium bicarbonate (BIC) and tested against SA. ATCC and a multidrug-resistant SA. CCARM strains. Antibacterial activity and interactions were determined by minimum inhibitory concentration and checkerboard assays. Biofilms on sandblasted stainless-steel discs were assessed by viable cell counts, biofilm formation index, and extracellular DNA and polysaccharide levels. Transmission electron microscopy and quantitative real-time PCR of selected genes were used to examine structural damage and stress-response modulation. All preparations showed antibacterial activity, with higher MICs in the MDR strain. CHO + AMH displayed clear synergy against both strains, whereas CHE + BIC ranged from synergistic to additive. On stainless steel, CHO + AMH produced the greatest and most sustained reduction in biofilm-associated cells and the lowest biofilm formation indices, together with marked depletion of matrix components. TEM revealed extensive envelope disruption and cell damage under CHO + AMH compared with single agents. Combination treatments altered stress-response and efflux gene expression in patterns consistent with severe cellular injury and limited adaptive capacity. Accordingly, mixed bile salt-weak base disinfectants, particularly CHO + AMH, enhance antibacterial and antibiofilm activity against wild-type and MDR S. aureus by targeting cell envelopes, biofilm matrix, and stress-adaptation pathways. These GRAS-based combinations represent promising low-dose disinfectant candidates for controlling S. aureus biofilms on stainless-steel surfaces in processing environments.