ThepmrCABOperon Mediates Polymyxin Resistance inAcinetobacter baumanniiATCC 17978 and Clinical Isolates through Phosphoethanolamine Modification of Lipid A

Abstract

ABSTRACTThe emergence of multidrug resistance amongAcinetobacter baumanniiis 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 thepmrCABoperon 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 thepmrBgene, encoding a sensor kinase, or in the response regulator PmrA. Knockout of thepmrBgene 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 clonedpmrABgenes from the mutants but not from the wild type. Reverse transcription-quantitative PCR (RT-qPCR) analysis also showed a correlation between the expression ofpmrCand 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 inSalmonella entericaserovar Typhimurium by the product of thepmrCgene, which is a homolog of thepmrCgene fromAcinetobacter. 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 thepmrCABoperon are responsible for resistance to polymyxins inA. baumannii.