Aminoglycosides are widely used to treat infections of Pseudomonas aeruginosa. Genes encoding aminoglycoside-modifying enzymes (AMEs), acquired by horizontal gene transfer, are commonly associated with aminoglycoside resistance, but their effects have not been quantified. The aim of this research was to determine the extent to which AMEs increase the antibiotic tolerance of P. aeruginosa. Bioinformatics analysis identified AME-encoding genes in 48 out of 619 clinical isolates of P. aeruginosa, with ant(2′)-Ia and aac(6′)-Ib3, which are associated with tobramcyin and gentamicin resistance, being the most common. These genes and aph(3′)-VIa (amikacin resistance) were deleted from antibiotic-resistant strains. Antibiotic minimum inhibitory concentrations (MICs) were reduced by up to 64-fold, making the mutated bacteria antibiotic-sensitive in several cases. Introduction of the same genes into four antibiotic-susceptible P. aeruginosa strains increased the MIC by up to 128-fold, making the bacteria antibiotic-resistant in all cases. The cloned genes also increased the MIC in mutants lacking the MexXY-OprM efflux pump, which is an important contributor to aminoglycoside resistance, demonstrating that AMEs and this efflux pump act independently in determining levels of aminoglycoside tolerance. Quantification of the effects of AMEs on antibiotic susceptibility demonstrates the large effect that these enzymes have on antibiotic resistance.
Introduction. Aminoglycoside antibiotics are widely used to treat infections of
Pseudomonas aeruginosa
. The MexXY-OprM efflux pump is an important contributor to aminoglycoside tolerance in
P. aeruginosa
reference strains and expression of the mexXY genes is repressed by the MexZ repressor protein. Direct investigation of the role of this efflux pump in clinical isolates is relatively limited.
Hypothesis. The contribution of MexXY-OprM to
P. aeruginosa
aminoglycoside resistance is isolate-specific.
Aim. To quantify the role of MexXY-OprM and its repressor, MexZ, in clinical isolates of P. aeruginosa.
Methodology. The mexXY genes were deleted from ten clinical isolates of
P. aeruginosa
, and the mexZ gene from nine isolates. Antimicrobial susceptibility testing was carried out for commonly used antipseudomonal drugs on the engineered mutants and the isogenic wild-type isolates. RT-qPCR was used to measure expression of the mexX gene.
Results. All but one of the mexXY mutants were more susceptible to the clinically used aminoglycosides tobramycin, gentamicin and amikacin but the degree to which susceptibility increased varied greatly between isolates. The mexXY mutants were also more susceptible to a fluoroquinolone, ciprofloxacin. In three isolates with functional MexZ, deletion of mexZ increased expression of mexXY and aminoglycoside tolerance. Conversely, deleting mexZ from six clinical isolates with mexZ sequence variants had little or no effect on expression of mexXY or on aminoglycoside susceptibility, consistent with the variants abolishing MexZ function. Genome analysis showed that over 50 % of 619 clinical isolates had sequence variants predicted to reduce the affinity of MexZ for DNA, likely increasing mexXY expression and hence efflux of aminoglycosides.
Conclusion. Our findings show that the interplay between MexXY, MexZ and the level of mexXY expression plays an important role in aminoglycoside resistance in clinical isolates of
P. aeruginosa
but the magnitude of the contribution of this efflux pump to resistance is isolate-specific.
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.
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