Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis

Inmaculada García-Romero, Mugdha Srivastava, Julia Monjarás-Feria, Samuel O. Korankye, Lewis MacDonald, Nichollas E. Scott, Miguel A. Valvano*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

Objectives
A concern with the ESKAPE pathogen, Enterobacter bugandensis, and other species of the Enterobacter cloacae complex, is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins.

Methods
Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis.

Results
In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain, W3110. The efflux pump, AcrABTolC, and the two component regulators, PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modelling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps.

Conclusion
Our results support a model where drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.

Original languageEnglish
Pages (from-to)108-121
Number of pages14
JournalJournal of Global Antimicrobial Resistance
Volume37
Early online date23 Apr 2024
DOIs
Publication statusPublished - Jun 2024

Keywords

  • Cationic antimicrobial peptides
  • Enterobacter cloacae complex
  • Intrinsic antibiotic resistance
  • KexD
  • Neonatal sepsis
  • Outer membrane permeability

ASJC Scopus subject areas

  • Microbiology
  • Immunology and Allergy
  • Immunology
  • Microbiology (medical)

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