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

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

Antimicrobial agents and chemotherapy (2011-10-19)
Rebecca J Malott, Barbara R Steen-Kinnaird, Tracy D Lee, David P Speert
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
1-Naphthylamine, analytical standard
Pricing and availability is not currently available.
Sigma-Aldrich
N-Phenyl-1-naphthylamine, reagent grade, 98%
Pricing and availability is not currently available.
Sigma-Aldrich
1-Naphthylamine, 97%
Pricing and availability is not currently available.
Millipore
Nitrate Reagent A, suitable for microbiology
Pricing and availability is not currently available.