Dihydroartemisinin inhibits plasmid transfer in drug-resistant <i>Escherichia coli</i> via limiting energy supply

Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARGs dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges to control 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 have inadequately understood mechanisms of action. Here we studied the effects of dihydroartemisinin, an artemisinin derivative used to treat malaria, on conjugation. Dihydroartemisinin inhibited conjugation of IncI2 and IncX4 plasmids carrying mobile colistin resistance gene (mcr-1) by over 160-fold in vitro in Escherichia coli, and by two-fold (IncI2 plasmid) in vivo in a mouse model. It also suppressed the transfer of IncX3 plasmid carrying carbapenem resistance gene blaNDM-5 by over two-fold in vitro. Detection of intracellular ATP and proton motive force (PMF), in combination with transcriptome and metabolomics analyses, revealed that dihydroartemisinin impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid cycle pathway, thereby disrupting PMF and limiting intracellular ATP available for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly downregulated during dihydroartemisinin exposure, indicating that the transfer apparatus for conjugation might be inhibited. Our findings provide new insights for controlling the spread of antibiotic resistance and broaden the potential uses of dihydroartemisinin.