Distinct plasmid- and host-encoded mechanisms drive small plasmid copy number-mediated heteroresistance in Escherichia coli

Antibiotic heteroresistance, the presence of a rare resistant subpopulation within an otherwise susceptible bacterial population, poses a significant clinical challenge. Understanding its genetic mechanisms is critical for early detection and treatment efficacy. Here, we investigate the contribution of small plasmids to heteroresistance using a clinical bloodstream Escherichia coli isolate carrying a 12 kb ColE1-type plasmid. We show that this plasmid drives transient β-lactam heteroresistance through massive increases in plasmid copy number. Two distinct genetic mechanisms drive this amplification: mutations in the plasmid RNAI/RNAII that deregulate replication control, and a chromosomal recD mutation that induces multimerization and a shift toward rolling-circle replication. Notably, this recD -mediated amplification is restricted to small ColE1 and F-plasmids. This study highlights the crucial role of small plasmids in resistance evolution, demonstrating that they can cause this phenotype via alternative genetic pathways.