Plasmid-chromosome transcriptional crosstalk in multidrug resistant clinical enterobacteria

Conjugative plasmids are the main vehicle for the horizontal spread of antimicrobial resistance (AMR). Although AMR plasmids provide advantages to their hosts under antibiotic pressure, they can also disrupt the cell’s regulatory network, impacting the fitness of their hosts. Despite the importance of plasmid-bacteria interactions on the evolution of AMR, the effects of plasmid carriage on host physiology has remained underexplored, and most studies have focused on model bacteria and plasmids that lack clinical relevance. Here, we analyzed the transcriptional response of 11 clinical enterobacterial strains (2 Escherichia coli, 1 Citrobacter freundii and 8 Klebsiella spp.) and the laboratory-adapted E. coli MG1655 to carriage of pOXA-48, one of the most widely spread carbapenem-resistance plasmids. Our analyses revealed that pOXA-48 produces variable responses on their hosts, but commonly affects processes related to metabolism, transport, response to stimulus, cellular organization and motility. More notably, the presence of pOXA-48 caused an increase in the expression of a small chromosomal operon of unknown function in Klebsiella spp. and C. freundii, which is not present in E. coli. Phylogenetic analysis suggested that this operon has been horizontally mobilized across different Proteobacteria species. We demonstrate that a pOXA-48-encoded LysR transcriptional regulator controls the expression of the operon in Klebsiella spp. and C. freundii. In summary, our results highlight a crosstalk between pOXA-48 and the chromosome of its natural hosts. First, we performed differential expression (DE) analysis to analyze the transcriptional responses of clinical enterobacterial strains to carriage of the carbapenem-resistance plasmid pOXA-48. We sequenced three biological replicates per strain and condition (carrying and not carrying pOXA-48), except for strains CF13, TC_KPN10, PF_KPN10 and MG1655 that we sequenced two replicates due insufficient RNA Integrity number (RIN). Additionally, we performed a second RNA-Seq and DE analysis for strains KPN15 and CF13 carrying pOXA-48 or pOXA-48ΔlysR (three biological replicates per strain and condition) to characterize the plasmid-chromosome transcriptional crosstalk.