Gene–Gene Interactions Reduce Aminoglycoside Susceptibility of Pseudomonas aeruginosa through Efflux Pump-Dependent and -Independent Mechanisms

Abstract

Pseudomonas aeruginosa causes a wide range of acute and chronic infections. Aminoglycosides are a cornerstone of treatment, but isolates are often resistant. The purpose of this research was to better understand the genetic basis of aminoglycoside resistance in P. aeruginosa. Bioinformatic approaches identified mutations in resistance-associated genes in the clinical isolates of P. aeruginosa. The common mutations were then engineered into the genome of P. aeruginosa reference strain PAO1. Mutations in the elongation factor gene fusA1 caused the biggest reduction in aminoglycoside susceptibility, with mutations in the two-component regulator gene amgS and the efflux pump regulator gene mexZ having less impact. This susceptibility was further reduced by combinations of mutations. Mutations in fusA1, amgS and mexZ all increased the expression of the mexXY efflux pump that is strongly associated with aminoglycoside resistance. Furthermore, the fusA1 amgS mexZ triple mutant had the highest efflux pump gene expression. Engineering fusA1 and amgS mutants lacking this efflux pump showed that fusA1 and amgS also reduce aminoglycoside susceptibility through additional mechanisms. The fusA1 and amgS mutations reduced bacterial growth, showing that these mutations have a fitness cost. Our findings demonstrate the complex interplay between mutations, efflux pump expression and other mechanisms for reducing the susceptibility of P. aeruginosa to aminoglycosides.