Synergistic antimicrobial activity of alpha-linolenic acid in combination with tetracycline or florfenicol against multidrug-resistant Salmonella typhimurium.

Salmonella is a major foodborne pathogen that can be transmitted from livestock and poultry to humans through the food chain. Due to the widespread use of antibiotics, antibiotic resistance Salmonella has become an important factor threatening food safety. Combining antibiotic and non-antibiotic agents is a promising approach to address the widespread emergence of antibiotic-resistant pathogens. In this study, we investigated the antibiotic resistance profile and molecular characterization of different serotypes of Salmonella isolated from large-scale egg farms using drug susceptibility testing and whole genome sequencing. The synergistic effect of alpha-linolenic acid (ALA) with antibiotics was evaluated using the checkerboard test and time-kill curve. The molecular mechanism of α-linolenic acid synergism was explored using biochemical assays, pull-down assays, and molecular docking. In vivo efficacy of ALA in combination with florfenicol (FFC) or tetracycline (TET) against multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar typhimurium was also investigated using a mouse model. We found that ALA reduced the minimum inhibitory concentration (MIC) of tetracycline and florfenicol in all strains tested. When ALA (512 mg/L) was combined with florfenicol (32 mg/L) or tetracycline (16 mg/L), we observed disruption of cell membrane integrity, increased outer membrane permeability, lowered cell membrane potential, and inhibition of proton-drive-dependent efflux pumps. The synergistic treatment also inhibited biofilm production and promoted oxidative damage. These changes together led to an increase in bacterial antibiotic susceptibility. The improved efficacy of ALA combination treatment with antibiotics was validated in the mouse model. Molecular docking results indicate that ALA can bind to membrane proteins via hydrogen bonding. Our findings demonstrated that combined treatment using ALA and antibiotics is effective in preventing infections involving MDR bacteria. Our results are of great significance for the scientific and effective prevention and control of antibiotic resistance Salmonella, as well as ensuring food safety.