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

Enterobacter cloacae is a Gram-negative nosocomial pathogen of the ESKAPE priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, E. cloacae can also display phenotypic antimicrobial resistance, such as heteroresistance or persistence. Here we report that E. cloacae ATCC 13047 and six strains isolated from patients with blood infections display heteroresistance or persistence to aminoglycosides. E. cloacae heteroresistance is transient, accompanied with formation of petite colonies and increased MIC against 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 cpxRA two-component system abrogated aminoglycoside heteroresistance and petite colony formation, pointing to its indispensable role in phenotypic resistance. The introduction of a constitutively active allele of cpxA led to high aminoglycoside MICs, consistent with cell envelope stress driving these behaviours in E. cloacae. 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 the {Delta}cpxRA mutant. Moreover, copper exposure also elevated the gentamicin MICs of bloodstream isolates, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in E. cloacae strains. Altogether, we establish that E. cloacae relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic. ImportanceEnterobacter cloacae is a bacterium that belongs to the ESKAPE priority group and an increasing threat worldwide due its multidrug resistance. E. cloacae can also display phenotypic resistance to antibiotics, leading to treatment failure. We report that sensitive strains of E. cloacae, including six strains isolated from patients with bloodstream infections, show heteroresistance or persistence to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections, therefore understanding how resistance develops in 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. Further, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs in all tested strains of E. cloacae. Our study suggests phenotypic aminoglycoside resistance in E. cloacae could be broadly conserved and cautions against the undesirable effects of copper.