Alterations in Two-Component Regulatory Systems of <i>phoPQ</i> and <i>pmrAB</i> Are Associated with Polymyxin B Resistance in Clinical Isolates of <i>Pseudomonas aeruginosa</i>

<jats:title>ABSTRACT</jats:title> <jats:p> Polymyxins are often the only option to treat acquired multidrug-resistant <jats:italic>Pseudomonas aeruginosa</jats:italic> . Polymyxin susceptibility in <jats:italic>P. aeruginosa</jats:italic> PAO1 is associated with the lipopolysaccharide structure that is determined by <jats:italic>arnBCADTEF</jats:italic> and modulated by <jats:italic>phoPQ</jats:italic> and <jats:italic>pmrAB</jats:italic> . We examined five clonally unrelated clinical isolates of polymyxin B-resistant <jats:italic>P. aeruginosa</jats:italic> to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 μg/ml polymyxin B (MIC, 8 μg/ml), whereas <jats:italic>P. aeruginosa</jats:italic> PAO1 grew with 0.25 μg/ml polymyxin B (MIC, 0.5 μg/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 μg/ml) following the introduction of <jats:italic>phoPQ</jats:italic> (four isolates) and <jats:italic>pmrAB</jats:italic> (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking <jats:italic>phoPQ</jats:italic> or <jats:italic>pmrAB</jats:italic> that was transformed by <jats:italic>phoPQ</jats:italic> or <jats:italic>pmrAB</jats:italic> containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of <jats:italic>arnBCADTEF</jats:italic> (the MICs fell from 8 to 0.5 μg/ml). These results suggest that polymyxin B resistance among clinical isolates of <jats:italic>P. aeruginosa</jats:italic> is associated with alterations in two-component regulatory systems of <jats:italic>phoPQ</jats:italic> or <jats:italic>pmrAB</jats:italic> . </jats:p>