Aminoglycoside heteroresistance in is driven by the cell envelope stress response.

is a Gram-negative nosocomial pathogen of the ESKAPE (, and spp.) priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, can also display forms of antibiotic refractoriness, such as heteroresistance and tolerance. Here, we report that displays transient heteroresistance to aminoglycosides, which is accompanied with the formation of small colony variants (SCVs) with increased minimum inhibitor concentration (MIC) of gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the two-component system abrogated aminoglycoside heteroresistance and SCV formation, pointing to its indispensable role in these processes. The introduction of a constitutively active allele of led to high aminoglycoside MICs consistent with cell envelope stress response driving these behaviors in . Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild-type but not in the Δ mutant. Moreover, copper exposure also elevated the gentamicin MICs of clinical isolates from bloodstream infections, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in strains. Altogether, we establish that relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic.IMPORTANCE is a bacterium that belongs to the WHO high-priority group and an increasing threat worldwide due its multi-drug resistance. can also display heteroresistance, which has been linked to treatment failure. We report that shows heteroresistance to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections; therefore, understanding how resistance develops among sensitive strains is important. We show that aminoglycoside resistance is driven by the activation of the cell envelope stress response and transcriptional reprogramming via the CpxRA two-component system. Furthermore, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs of the type strain and clinical strains isolated from bloodstream infections. Our study suggests aminoglycoside recalcitrance in could be broadly conserved and cautions against the undesirable effects of copper.