Polymyxin B resistance in clinical isolates of Pseudomonas aeruginosa is associated with alterations in two-component regulatory systems of phoPQ and pmrAB

Polymyxins are often the only option to treat acquired multidrug resistant P. aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with lipopolysaccharide structure determined by arnBCADTEF and modulated by phoPQ and pmrAB. I examined five clonally unrelated clinical isolates of polymyxin B resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 mug/mL of polymyxin B (MIC 8 mug/mL) whereas a reference strain P. aeruginosa PAO1 grew with 0.25 mug/mT (MIC 0.5 mug/mL). The resistant isolates were converted to susceptible ones (MIC fell from 8 to 0.5 mug/mL) following introduction of phoPQ (4 isolates) and pmrAB (1 isolate) that had been cloned from the strain PAO1. DNA sequence analysis of the five isolates, revealed a single nucleotide substitution in three isolates that substituted a single amino acid in PhoQ, a 17 nucleotide deletion in one isolate that truncated protein PhoQ and dual nucleotide substations in one isolate that substituted two amino acids in PmrB. Involvement of these amino acid substitutions or truncated-protein of PhoQ and PmrB in polymyxin B resistance was confirmed using the strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or truncated-protein. The resistant clinical isolates were sensitized by inactivation of arnBCADTEF (MIC fell from 8 to 0.5 mug/mL). These results suggest that polymyxin B resistance among clinical isolate of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.