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 (p12). We show that this plasmid drives transient {beta}-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, without the involvement of traditional large resistance plasmids.