Effects of l -arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis ( S . Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). l -Arabinose, a common inducer for gene expression, modulated S . Choleraesuis efflux pump activity in a dose-dependent manner. At low l -arabinose concentrations, increasing l -arabinose led to a corresponding increase in fluorophore efflux, while at higher l -arabinose concentrations, increasing l -arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (Δ tolC ), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude l -arabinose. Further, due to the rapid export of l -arabinose, an efficient method for recombination-mediated gene knockout, the l -arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of l -arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that l -arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of l -arabinose should facilitate the optimization of l -arabinose induction in strains with high efflux activity.