Simultaneous inhibition of MarR by salicylate and efflux pumps by curcumin sensitizes colistin resistant clinical isolates of Enterobacteriaceae

Abstract

Carbapenem resistant Enterobacteriaceae (CRE) infection has been widely treated with last resort antibiotics like colistin. Resistance to colistin has further jeopardized the situation. We have previously reported a combination of MarR inhibitor – salicylate (Sal) and an efflux pump inhibitor (BC1) that successfully restored colistin (Col) sensitivity in multidrug and colistin resistant clinical isolate of E. coli U3790. Since synthetic compounds usually fail during drug development initiatives, we attempted to replace synthetic efflux pump inhibitor (BC1) with plant metabolite as efflux pump inhibitor to restore colistin sensitivity in CRE. Screening 13 plant metabolites, we narrowed on curcumin (CUR) to effectively inhibit efflux in both colistin resistant E. coli U3790 and K. pneumoniae BC936. Combination of Col + CUR showed a remarkable reversal in colistin MIC by 128 fold and 32 fold in E. coli U3790 and K. pneumoniae BC936 respectively. Studies with knock out mutant strains of AcrAB-TolC pump components show that curcumin's efflux inhibition is partly mediated by acrB. Thus, curcumin reduced colistin MIC well below the CLSI breakpoint (<2 μg/ml). Curcumin also exhibited synergy with colistin against most of the clinical isolates of Enterobacteriaceae tested. Efficiency of Col + Sal + CUR was evident in time kill curve analysis, which displayed a 6 log and a 4 log decline in CFU/ml by 24 h in U3790 and BC936 strains respectively. In vivo intramuscular fish infection studies showed that the triad combination reduced the bacterial bioburden of E. coli U3790 by 2.6 log and that of K. pneumoniae BC936 by 1.6 log. Hence, our study shows the efficacy of inhibiting MarR by salicylate and inhibiting efflux pump with curcumin restores colistin sensitivity in colistin resistant Enterobacteriaceae in vitro and in vivo.