Efflux pump inhibitor chlorpromazine effectively increases the susceptibility of <i>Escherichia coli</i> 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.研究目的：本研究旨在评估大肠杆菌ATCC8739对Brevinin-2CE（B2CE）的应答反应，以此探索预防抗菌肽（antimicrobial peptide, AMP）耐药菌生成的可行策略。 方法：采用转录组测序与逆转录聚合酶链式反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）检测基因表达水平；借助CRISPR-Cas9技术敲除靶基因；通过最小抑菌浓度（minimum inhibitory concentration, MIC）测定评估菌株耐药性。 结果：经B2CE刺激后，acrZ与sugE的表达水平显著上调。ATCC8739ΔacrZ与ATCC8739ΔsugE的敏感性分别提升2倍与4倍。在B2CE与氯丙嗪（chlorpromazine, CPZ）联合处理条件下，大肠杆菌ATCC8739的存活率显著降低。其他抗菌肽与氯丙嗪联用同样展现出抗菌活性。 结论：本研究结果表明，抗菌肽与外排泵抑制剂（efflux pump inhibitor, EPI）联用有望成为对抗耐药菌的潜在策略。 亚致死浓度的B2CE可引发大肠杆菌内大量基因的表达水平发生显著改变。 敲除acrZ与sugE基因证实，这两种外排泵负责将B2CE泵出菌体，从而削弱其对大肠杆菌的抑制作用。 B2CE与氯丙嗪联用可使大肠杆菌的抗菌效果提升5倍，该结果验证了外排泵抑制剂联用可在更低的肽类浓度下实现有效抗菌，进而降低细菌对B2CE产生耐药性的风险。 B2Ka与P2CE与氯丙嗪联用同样可增强对大肠杆菌的抗菌效果，提示两栖动物来源抗菌肽与外排泵抑制剂联用是未来临床应用的可行策略。