Artesunate enhances the antibacterial effect of β-lactam antibiotics against Escherichia coli by increasing antibiotic accumulation via inhibition of the multidrug efflux pump system AcrAB-TolC

Objectives: Occasionally, we found that artesunate enhanced the antibacterial effects of antibiotics in vitro. Therefore, the enhancement of various beta-lactam antibiotics by artesunate against Escherichia coli and the possible mechanism were investigated in the present study. Methods: Antibacterial effects were observed using the serial 2-fold dilution method and dynamic bacterial growth. Daunomycin accumulation within E. coli was observed using fluorospectrophotometry and laser confocal scanning microscopy. AcrAB-TolC, AmpC and TEM-1 mRNA expression was observed using a PCR method. Antisense oligonucleotides (as-ODNs) targeting AcrB were designed and used to block AcrB gene expression within E. coli ATCC 35218. Results: Although artesunate itself had no antibacterial ability, artesunate significantly increased the antibacterial effect of beta-lactam antibiotics against E. coli ATCC 35218 and an E. coli clinical strain. Artesunate increased daunomycin accumulation within E. coli ATCC 35218 in a dose-dependent manner and reduced the mRNA expression of AcrAB-TolC, an important multidrug efflux system for Gram-negative bacteria. The bacterial number was significantly reduced by as-ODN targeting AcrB, but did not further decrease after additional artesunate treatment. In contrast, artesunate lost its enhancement of beta-lactam antibiotics against E. coli AG100A, a strain lacking the gene encoding AcrAB, and artesunate did not increase daunomycin accumulation within E. coli AG100A. After the transformation of pET28a-AcrB into E. coli AG100A, artesunate regained enhancement of beta-lactam antibiotics. Furthermore, artesunate did not inhibit the expression of AmpC and TEM-1 mRNA. Conclusions: Artesunate enhances the antibacterial effect of various beta-lactam antibiotics against E. coli, which might be associated with the suppression of a major multidrug resistance system, AcrAB-TolC.