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

Abstract

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.

Keywords: AcrB; Escherichia coli; antibiotic alternatives; antimicrobial resistance; efflux pump; sanguinarine.

Fig 1

Effect of mutation in acrR or marR on the expression of the acrA, and acrB, and marA. The expression of the acrA, acrB, regulatory gene marA, as well as the control gene of 16 s rRNA were measured by RT–qPCR in the WT E. coli ATCC 35218 (35218) and its derived strains (T1, T2, T5, and T6). The results were presented as average   ± SEM (n  =  3) and shown normalized to the expression of each gene in the WT strain. Statistically significant differences between the WT and mutant strains are shown as **P  <  0.01, ***P  <  0.001, and ****P  <  0.0001.

Fig 2

Intracellular accumulation of sanguinarine in E. coli with or without WT acrB gene. A, normalized fluorescence of intracellular sanguinarine in E. coli DH5α, ΔacrB, CΔacrB, and ΔacrB–pBBR. B, normalized fluorescence of intracellular sanguinarine in E. coli ATCC 35218, 35218m, C35218m, 35218m-pBBR. The fluorescence intensity was measured at 1 h after sanguinarine treatment and normalized with OD₆₀₀ values in order to eliminate the effect of cell growth. Data are presented as mean ± SD of four independent experiments. Amber color cycle, 1.6%(v/v) DMSO; Cyan cycle, 4 µg/mL SAN; Magenta color cycle, 8 µg/mL SAN; Orange red color cycle, 16 µg/mL SAN. Statistical significance was determined by a two-way ANOVA (**P < 0.01, ***P < 0.001, ****P < 0.0001). SAN, sanguinarine.

Fig 3

Conformations of the most stable binding modes of sanguinarine (shown as sticks: carbon, green; oxygen, red; nitrogen, blue) within the distal binding pocket (DBP) and channel 3 (CH3) of AcrB (pale cyan, shown as cartoon or surface). The images were created by PyMol. The yellow (A) and yellow orange (C) box indicated the binding site in DBP and CH3, respectively. B and D were zoomed images to show the residues (shown as surface and sticks) of AcrB have hydrophobic contacts with sanguinarine.

Fig 4

The competitive inhibition of sanguinarine on ethidium bromide efflux. Fluorescence intensity of ethidium bromide in E. coli ATCC 35218 (A) 35218m (B) E. coli DH5α (C) and ΔacrB (D) treated with different concentration sanguinarine (1/4 × MIC,1/2 × MIC, 1 × MIC). The vertical axis displays the percentage of fluorescence relative to the accumulation of EB treated alone in E. coli cells, indicating the degree of cellular uptake. The experiment was repeated three times with similar results, and one representative result is shown. SAN, sanguinarine; EB, ethidium bromide.