Mechanistic insights into plasmid transfer inhibition in Enterobacterales by nucleoside analogues

Antimicrobial resistance (AMR) poses a major global health threat, with carbapenem-resistant and extended-spectrum β-lactamase (ESBL)-producing Enterobacterales causing widespread infections and deaths. Much of this resistance spreads through conjugative plasmids, autonomously replicating mobile genetic elements that transfer between bacteria and carry multiple AMR genes. Targeting plasmid conjugation could therefore help curb the spread of AMR. In this study, we tested whether clinically approved nucleoside analogues (NAs) inhibited the transfer of the GFP-tagged ESBL-encoding IncK plasmid pCTgfp in Escherichia coli and the carbapenemase-encoding IncF plasmid pKpQILgfp in Klebsiella pneumoniae using flow cytometry. Alongside the known inhibitor azidothymidine (AZT), didanosine, stavudine, and trifluridine reduced plasmid conjugation in both species without affecting growth. Conversely, famciclovir and zalcitabine promoted pCTgfp conjugation in E. coli, while aciclovir and valaciclovir enhanced pKpQILgfp conjugation in K. pneumoniae. Mechanistic studies showed that plasmid conjugation-promoting NAs altered intracellular ATP levels. RNA sequencing revealed that AZT downregulated the expression of genes linked to motility in E. coli. Genetic inactivation of motility in E. coli mirrored the decrease in pCTgfp conjugation, like AZT. In K. pneumoniae, AZT upregulated genes linked to DNA damage and the SOS response, but downregulated methionine biosynthesis and metabolism genes. The exogenous addition of zinc acetate to inhibit RecA or the end product of methionine metabolism, S-adenosyl-methionine, restored pKpQILgfp conjugation in K. pneumoniae. Overall, our results indicated that existing NAs, including AZT, represent structural scaffolds for the development of potent conjugation inhibitors and highlight motility, DNA repair, and methionine metabolism as potential key factors in plasmid conjugation.