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.

Genomics of Aminoglycoside Resistance in Pseudomonas aeruginosa Bloodstream Infections at a United States Academic Hospital

“…Naturally occurring mutations in the fusA1 gene have been linked to enhanced resistance to aminoglycosides ( 40 , 41 ). We therefore applied the same approach described for mexZ to fusA1.…”

Pseudomonas Aeruginosa Colonising Patients with Cystic Fibrosis: Antibiotic Resistance and Growth Conditions Determined by in vivo and in vitro Gene Expression

“…The fact that no statistically significant differences were observed in mexY expression between strains fully susceptible or resistant to one, two or three antibiotic classes, could indicate that other factors influence the expression of antibiotic resistance and/or of MexXY-OprM. Actually, it has recently been demonstrated that mutations in genes other than mexZ (fusA1 and amgRS) have a higher impact on reducing aminoglycoside susceptibility and that combination of mutations in the three genes have a stronger effect 23 . For three patients the type of mexZ mutation correlated with the resistance profile, although the association was inverse to what was expected: strains harbouring wild type mexZ were resistant to 2 classes (not including aminoglycosides) or multiresistant (including aminoglycosides) while double mutants were fully susceptible.…”

“…This observation was already made twenty years ago but in that case no mutations in mexZ regulator were found 26 , although the authors conclude that MexXY overexpression in aminoglycoside resistant strains occurs via mutation in one or more genes that were not identified at the time of the study. We now know that mutations in fusA1 and amgRS have a greater impact than mexZ in reducing aminoglycoside susceptibility 23 . Few studies have so far analysed gene expression of P. aeruginosa in vivo in patients with CF, either by measuring gene expression directly into clinical samples [42][43][44] or under laboratory conditions mimicking human conditions or animal models 45,46 .…”

“…On the other hand, narG was not always induced (data not shown). The MexXY-OprM efflux pump was chosen because its overexpression is the most common mechanism of aminoglycoside resistance in CF isolates 23 and it can also contribute to ß-lactam and fluroquinolone resistance, three main antibiotics in the treatment of CF patients, although these resistances may be isolated from patients without CF 25,26 . Real time PCR (Rotor-Gene 3000 -Corbett TM , Australia) was used to quantify cDNA with 3 µl of cDNA as template and the following reaction components: 7.5 µl of QuantiTect SYBR Green TM (Qiagen), 0.9 µl of each template and 2.7 µl of water.…”

“…P. aeruginosa can also acquire resistance to aminoglycosides by two main mechanisms: active elimination of the molecule by efflux pumps and enzymatic hydrolysis 21 . Overexpression of the inducible efflux pump MexXY-OprM is considered the most frequent mechanisms of acquired aminoglycoside resistance in CF patients 22 and is due to mutations in different genes involved in the regulation of expression, notably mexZ repressor, fusA1 that encodes for an elongation factor G and amgRS that encodes for a two-component regulatory system 23 . We aimed to investigate the growth conditions of P. aeruginosa and the expression of some determinants of resistance by measuring genetic expression directly into the sputa (in vivo) and in strains isolated from these sputa (in vitro) in patients with CF.…”