Identification of hopanoid biosynthesis genes involved in polymyxin resistance in Burkholderia multivorans

Antimicrob Agents Chemother. 2012 Jan;56(1):464-71. doi: 10.1128/AAC.00602-11. Epub 2011 Oct 17.

Abstract

A major challenge to clinical therapy of Burkholderia cepacia complex (Bcc) pulmonary infections is their innate resistance to a broad range of antimicrobials, including polycationic agents such as aminoglycosides, polymyxins, and cationic peptides. To identify genetic loci associated with this phenotype, a transposon mutant library was constructed in B. multivorans ATCC 17616 and screened for increased susceptibility to polymyxin B. Compared to the parent strain, mutant 26D7 exhibited 8- and 16-fold increases in susceptibility to polymyxin B and colistin, respectively. Genetic analysis of mutant 26D7 indicated that the transposon inserted into open reading frame (ORF) Bmul_2133, part of a putative hopanoid biosynthesis gene cluster. A strain with a mutation in another ORF in this cluster, Bmul_2134, was constructed and named RMI19. Mutant RMI19 also had increased polymyxin susceptibility. Hopanoids are analogues of eukaryotic sterols involved in membrane stability and barrier function. Strains with mutations in Bmul_2133 and Bmul_2134 showed increased permeability to 1-N-phenylnaphthylamine in the presence of increasing concentrations of polymyxin, suggesting that the putative hopanoid biosynthesis genes are involved in stabilizing outer membrane permeability, contributing to polymyxin resistance. Results from a dansyl-polymyxin binding assay demonstrated that polymyxin B does not bind well to the parent or mutant strains, suggesting that Bmul_2133 and Bmul_2134 contribute to polymyxin B resistance by a mechanism that is independent of lipopolysaccharide (LPS) binding. Through this work, we propose a role for hopanoid biosynthesis as part of the multiple antimicrobial resistance phenotype in Bcc bacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Naphthylamine / analogs & derivatives
  • 1-Naphthylamine / metabolism
  • Animals
  • Anti-Bacterial Agents / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Outer Membrane Proteins / metabolism*
  • Burkholderia Infections / drug therapy
  • Burkholderia Infections / microbiology
  • Burkholderia cepacia complex / genetics
  • Burkholderia cepacia complex / metabolism*
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Cell Membrane Permeability / genetics*
  • Colistin / metabolism
  • Colistin / pharmacology
  • DNA Transposable Elements
  • Drug Resistance, Bacterial
  • Genomic Library
  • Microbial Sensitivity Tests
  • Multigene Family
  • Mutation
  • Open Reading Frames
  • Polymyxin B / metabolism
  • Polymyxin B / pharmacology*
  • Sterols / biosynthesis*

Substances

  • Anti-Bacterial Agents
  • Bacterial Outer Membrane Proteins
  • DNA Transposable Elements
  • Sterols
  • N-phenyl-1-naphthylamine
  • 1-Naphthylamine
  • Polymyxin B
  • Colistin