Hijacking a small plasmid to confer high-level resistance to aztreonam-avibactam and ceftazidime-avibactam

Acquired β-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria which also commonly carry multicopy small plasmids. Here we report that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. We focused on aztreonam-avibactam (ATM-AVI) as it can be used to effectively counter almost all β-lactamases produced by bacteria and has been recommended against carbapenem-resistant Enterobacterales. We investigated a clinical strain (085003) of carbapenem-resistant Escherichia coli and obtained two mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L ATM-AVI as representatives of low- and high-level resistance, respectively, by induction. Through comparative genomics, we found that 085003R32 and 085003R512 had a single nucleotide mutation of β-lactamase gene bla_CMY-2, encoding a novel CMY with a Thr319Ile substitution, assigned CMY-2R. Using cloning and enzyme kinetics, we verified that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. We investigated mechanisms for the discrepant resistance between 085003R32 and 085003R512. We identified three tandem copies of bla_CMY-2R on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of bla_CMY-2R on the IncP1 plasmid but possessed anther bla_CMY-2R on an already present 4-kb small plasmid. IS1294-mediated mobilization onto this multicopy small plasmid significantly increased the copy number of bla_CMY-2R, rendering higher resistance. Our study demonstrates that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.