title: The pmrCAB operon mediates polymyxin resistance in Acinetobacter baumannii ATCC 17978 and clinical isolates through phosphoethanolamine modification of lipid A.
abstract: The emergence of multidrug resistance among Acinetobacter baumannii is leading to an increasing dependence on the use of polymyxins as last-hope antibiotics. Here, we utilized genetic and biochemical methods to define the involvement of the pmrCAB operon in polymyxin resistance in this organism. Sequence analysis of 16 polymyxin B-resistant strains, including 6 spontaneous mutants derived from strain ATCC 17978 and 10 clinical isolates from diverse sources, revealed that they had independent mutations in the pmrB gene, encoding a sensor kinase, or in the response regulator PmrA. Knockout of the pmrB gene in two mutants and two clinical isolates led to a decrease in the polymyxin B susceptibility of these strains, which could be restored with the cloned pmrAB genes from the mutants but not from the wild type. Reverse transcription-quantitative PCR (RT-qPCR) analysis also showed a correlation between the expression of pmrC and polymyxin B resistance. Characterization of lipid A species from the mutant strains, by thin-layer chromatography and mass spectrometry, indicated that the addition of phosphoethanolamine to lipid A correlated with resistance. This addition is performed in Salmonella enterica serovar Typhimurium by the product of the pmrC gene, which is a homolog of the pmrC gene from Acinetobacter. Knockout of this gene in the mutant R2 [pmrB(T235I)] reversed resistance as well as phosphoethanolamine modification of lipid A. These results demonstrate that specific alterations in the sequence of the pmrCAB operon are responsible for resistance to polymyxins in A. baumannii.
authors.affiliation: Centre for Microbial Diseases and Immunity Research, 232-2259 Lower Mall Research Station, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Arroyo
authors.forename: Carmen M
authors.initials: CM
authors.lastname: Herrera
authors.forename: Lucia
authors.initials: L
authors.lastname: Fernandez
authors.forename: Jessica V
authors.initials: JV
authors.lastname: Hankins
authors.forename: M Stephen
authors.initials: MS
authors.lastname: Trent
authors.forename: Robert E W
authors.initials: RE
authors.lastname: Hancock
mesh_terms: D040981:Acinetobacter baumannii / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D024901:Drug Resistance, Multiple, Bacterial; D004983:Ethanolamines / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009876:Operon; D011113:Polymyxins / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004983:Ethanolamines; D008050:Lipid A; C409039:PmrB protein, bacteria; D011113:Polymyxins; D014157:Transcription Factors; C081902:pmrA protein, Bacteria; C005448:phosphorylethanolamine