title: Identification of hopanoid biosynthesis genes involved in polymyxin resistance in Burkholderia multivorans.
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.
authors: Centre for Understanding and Preventing Infection in Children, Department of Pediatrics, University of British Columbia, Vancouver, British Columbia, Canada. rmalott@cfri.ubc.ca
authors: Rebecca J
authors: RJ
authors: Malott
authors: Barbara R
authors: BR
authors: Steen-Kinnaird
authors: Tracy D
authors: TD
authors: Lee
authors: David P
authors: DP
authors: Speert
mesh_terms: D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D019121:Burkholderia Infections / Q000188:drug therapy / Q000382:microbiology; D042602:Burkholderia cepacia complex / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000235:genetics*; D003091:Colistin / Q000378:metabolism / Q000494:pharmacology; D004251:DNA Transposable Elements; D024881:Drug Resistance, Bacterial; D015698:Genomic Library; D008826:Microbial Sensitivity Tests; D005810:Multigene Family; D009154:Mutation; D016366:Open Reading Frames; D011112:Polymyxin B / Q000378:metabolism / Q000494:pharmacology*; D013261:Sterols / Q000096:biosynthesis*
chemical_list: D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D013261:Sterols; C005444:N-phenyl-1-naphthylamine; D015057:1-Naphthylamine; D011112:Polymyxin B; D003091:Colistin