Efflux pump inhibitor chlorpromazine effectively increases the susceptibility of Escherichia coli to antimicrobial peptide Brevinin-2CE

Aim: The response of E. coli ATCC8739 to Brevinin-2CE (B2CE) was evaluated as a strategy to prevent the development of antimicrobial peptide (AMP)-resistant bacteria. Methods: Gene expression levels were detected by transcriptome sequencing and RT-PCR. Target genes were knocked out using CRISPR-Cas9. MIC was measured to evaluate strain resistance. Results: Expression of acrZ and sugE were increased with B2CE stimulation. ATCC8739ΔacrZ and ATCC8739ΔsugE showed twofold and fourfold increased sensitivity, respectively. The survival rate of ATCC8739 was reduced in the presence of B2CE/chlorpromazine (CPZ). Combinations of other AMPs with CPZ also showed antibacterial effects. Conclusion: The results indicate that combinations of AMPs/efflux pump inhibitors (EPIs) may be a potential approach to combat resistant bacteria. The sub-lethal concentration of B2CE resulted significant changes in the expression levels of numerous genes in E. coli. The deletion of acrZ and sugE proved that these efflux pumps are responsible for pumping B2CE out of the cells, weakening its inhibitory effect on E. coli. The combination of B2CE and CPZ increased the antibacterial effect on E. coli by fivefold, which verified the combined use of EPI achieves effective antibacterial effects at lower peptide concentrations, thus reducing the risk of bacterial resistance to B2CE. The B2Ka and P2CE combined with CPZ also increased the antibacterial effects on E. coli, which suggested that the combined use of amphibian AMPs and EPIs is a feasible future clinical application strategy.