Aminoglycoside heteroresistance inEnterobacter cloacaeis driven by the cell envelope stress response
Authors: Choi Ana J. , Bennison Daniel J. , Kulkarni Esha , Sun Haoyu , Li Hanqi , Bradshaw Jonathan , Yeap Hui Wen , Lim Nicholas , Mishra Vishwas , Crespo-Puig Anna , Davies Frances , Sriskandan Shiranee , Shenoy Avinash R. 
Published: October 2023

Enterobacter cloacaeis a Gram-negative nosocomial pathogen of the ESKAPE priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However,E. cloacaecan also display phenotypic antimicrobial resistance, such as heteroresistance or persistence. Here we report thatE. cloacaeATCC 13047 and six strains isolated from patients with blood infections display heteroresistance or persistence to aminoglycosides.E. cloacaeheteroresistance 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 thecpxRAtwo-component system abrogated aminoglycoside heteroresistance and petite colony formation, pointing to its indispensable role in phenotypic resistance. The introduction of a constitutively active allele ofcpxAled to high aminoglycoside MICs, consistent with cell envelope stress driving these behaviours inE. 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 ΔcpxRAmutant. Moreover, copper exposure also elevated the gentamicin MICs of bloodstream isolates, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved inE. cloacaestrains. Altogether, we establish thatE. cloacaerelies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic.

Importance

Enterobacter cloacaeis a bacterium that belongs to the ESKAPE priority group and an increasing threat worldwide due its multidrug resistance.E. cloacaecan also display phenotypic resistance to antibiotics, leading to treatment failure. We report that sensitive strains ofE. 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 ofE. cloacae. Our study suggests phenotypic aminoglycoside resistance inE. cloacaecould be broadly conserved and cautions against the undesirable effects of copper.