Mutations in Gene fusA1 as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa

Abstract

Resistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1 and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.

Keywords: EF-G1A; Pseudomonas aeruginosa; aminoglycosides; mechanisms of resistance.

FIG 1

Aminoglycoside resistance-associated mutations in gene fusA1. Mutations identified in spontaneous in vitro mutants (in boldface type), clinical strains (underlined), and specifically engineered in vitro mutants (in italic type) are mapped on the gene sequence of strain PAO1.

FIG 2

Three-dimensional structure of EF-G protein from strain PAO1, as predicted by RaptorX (http://raptorx.uchicago.edu/). EF-G domains were predicted by using Pfam database. The amino acid substitutions responsible for aminoglycoside resistance are indicated by blue spots. The blue and orange circles represent the fusidic acid and argyrin B binding sites, respectively.

FIG 3

Growth curves of strain PAO1 (circles, solid line), mutants PAOR13 (squares, dashed line), and PAOR15 (triangles, dotted line) at 30°C (A), 37°C (B), and 44°C (C).