Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges for controlling this type of horizontal transfer. Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance. Although such inhibitors are rare, they typically exhibit relatively high toxicity and low efficacy in vivo and their mechanisms of action are inadequately understood. Here, we studied the effects of dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, on conjugation. DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene ( mcr-1) by more than 160-fold in vitro in Escherichia coli, and more than two-fold (IncI2 plasmid) in vivo in a mouse model. It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene bla NDM-5 by more than two-fold in vitro. Detection of intracellular adenosine triphosphate (ATP) and proton motive force (PMF), in combination with transcriptomic and metabolomic analyses, revealed that DHA impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid (TCA) cycle pathway, thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure, indicating that the transfer apparatus for conjugation may be inhibited. Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.
质粒介导的接合转移是细菌耐药基因水平传播的重要途径。研究报道越来越多的抗生素与非抗生素药物可加速耐药基因的水平传播，因此如何控制耐药基因水平传播显得尤为迫切。通过开发能延缓或阻断耐药质粒的接合转移抑制剂是控制细菌耐药性传播的有效策略之一。但目前接合转移抑制剂较少，且它们通常在体内具有相对较高的毒性和较低的疗效，并且现有研究缺乏对其作用机制的深入探究。因此，该研究探索了用于治疗疟疾的青蒿素衍生物双氢青蒿素对质粒接合转移的效果及机制。结果表明双氢青蒿素可显著降低携带多黏菌素耐药基因 mcr-1阳性IncI2质粒（180倍）和IncX4质粒（160倍）的接合转移频率。小鼠体内接合转移试验表明双氢青蒿素可降低2倍的 mcr-1-IncI2质粒接合转移频率。此外，双氢青蒿素还在体外降低携带碳青霉烯耐药基因 bla NDM-5阳性IncX3质粒的接合转移转移频率超过2倍。通过测定双氢青蒿素处理后的细菌胞内ATP含量和质子动力势，并结合转录组和代谢组学分析发现，双氢青蒿素可能通过抑制三羧酸循环通路来破坏电子传递链的功能，进而扰乱质子动力势，导致细菌ATP产生受到限制，降低了对接合转移系统的能量供应，并进一步下调了与接合转移和菌毛产生相关基因转录水平，最终使接合转移系统效率受损。综上，该研究为控制细菌耐药性的传播提供了新的见解，并拓宽了双氢青蒿素的潜在用途。.
Keywords: Conjugation inhibitors; Dihydroartemisinin; Plasmid; TCA cycle; blaNDM-5; mcr-1.