The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump

ABSTRACT Sanguinarine-containing phytobiotic is becoming increasingly popularly utilized as antibiotic alternatives in animal production. However, their potential risks to the human health and environment remain poorly understood. The present study aims to evaluate the occurrence of drug resistance in Escherichia coli after treatment with sanguinarine and investigate the molecular basis associated with sanguinarine resistance. Repetitive exposure of E. coli to subinhibitory dose of this phytochemical selected for mutants within genes encoding AcrR or MarR, transcriptional regulators of the multidrug tripartite efflux pump AcrAB-TolC. Disruption of the gene acrB caused a 16-fold MIC decrease for sanguinarine against E. coli strains, while the susceptibility of acrB-deficient mutants to sanguinarine was restored in complemented strains with ectopically expressed acrB. Accumulation assays suggested that the high susceptibility of acrB-deficient mutants was a consequence of a decrease in drug efflux. Consistent with this, the antibacterial activity of sanguinarine against wild-type (WT) strains of E. coli was significantly strengthened by combining it with 1-(1-naphthylmethyl)-piperazine (NMP), a classical efflux pump inhibitor. In addition, the combination of in silico prediction, competitive inhibition assay, and functional analysis of AcrB variants led to the identification of sanguinarine as a CH3-preferring substrate of AcrB, which could partly explain the selectively enhanced antibacterial effect of NMP on sanguinarine. IMPORTANCE The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.