Double deletion of cpxR and tolC significantly increases the susceptibility of Salmonella enterica serovar Typhimurium to colistin

Background The increasing use of colistin causes a serious breach in our last line of defence against MDR Gram-negative pathogens. Our previous study showed that CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin. Objectives To identify the mechanism of CpxAR and efflux pumps that synergistically enhance the susceptibility of S. Typhimurium to colistin. Methods A series of cpxR- and tolC-deleted mutants and a cpxR-complemented strain from a multidrug-susceptible standard strain of S. Typhimurium (JS) were generated in our previous study. Herein, we investigated the susceptibility of these strains to colistin through the broth microdilution method, time–kill curves and survival assays. Growth curves were measured by OD600 in LB broth, tryptone-soy broth (TSB) and M9-glucose (0.2%) minimal media. Finally, molecular mechanisms underlying the mode of action were elucidated by transcriptomic analysis. Results We found that in contrast to JS (0.8 mg/L), the MIC of colistin for JSΔtolC::kan showed a 16-fold decrease (0.05 mg/L). Notably, JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were associated with a 256-fold decrease (0.0031 mg/L) compared with JS. Growth curves identified that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR displayed a markedly lower growth rate and poorer adaptability. In addition, time–kill curves and survival assays showed that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were more susceptible to colistin. Lastly, double deletion of cpxR and tolC enhanced oxidative damage through promoting oxidative phosphorylation, the tricarboxylic acid (TCA) cycle and trimethylamine N-oxide (TMAO) respiration. Conclusions Our findings revealed that double deletion of cpxR and tolC significantly increases the susceptibility of S. Typhimurium to colistin.