Abstract
Pseudomonas aeruginosa is an opportunistic pathogen capable of causing a wide range of infections. Tobramycin, amikacin, and gentamicin are the most widely used aminoglycoside antibiotics for the treatment and management of P. aeruginosa infections. A number of mechanisms may contribute to the development of resistance to aminoglycosides, including increased activity of the MexXY-OprM efflux pump, acquired mutations, and modifications and inactivation of aminoglycosides through enzymatic action.
The MexXY-OprM pump is controlled by a repressor MexZ, which is one of the most frequently mutated genes in P. aeruginosa isolated from cystic fibrosis patients. There has been considerable research conducted on the role of the MexXY-OprM efflux pump in the P. aeruginosa reference isolate PAO1, but the exact role of the efflux pump and its repressor MexZ in clinical isolates of P. aeruginosa has not been thoroughly investigated. Resistance to aminoglycosides is multifactorial, with mutations in multiple genes influencing the resistance phenotype. It is not well understood how individual and multiple combinations of mutations contribute to the amount of aminoglycoside that P. aeruginosa can tolerate. Genes encoding aminoglycoside-modifying enzymes (AMEs) are often frequently correlated with aminoglycoside resistance, although their contribution to the amount of aminoglycoside that can be tolerated remains to be understood. Understanding the contribution of each of these mechanisms to resistance in reference and clinical isolates of P. aeruginosa could assist with the prediction of resistance phenotypes based on genotypes, something that could aid with effective therapeutic regimens for this pathogen. The overall aim of this research was to understand the genetic basis of aminoglycoside resistance by quantifying the role of the MexXY-OprM pump and its repressor MexZ, quantifying the effects of individual and combination mutations, and quantifying the effect of AMEs on antibiotic resistance in reference and clinical isolates of P. aeruginosa.
The mexXY and mexZ genes were deleted from clinical isolates of P. aeruginosa. All but one of the resulting mutants were more susceptible to tobramycin, gentamicin and amikacin confirming the MexXY efflux pump as a key contributor of aminoglycoside resistance in clinical isolates. Deleting the mexZ gene in clinical isolates containing MexZ sequence variants did not result in any change in the expression of the mexXY or in the susceptibility to aminoglycosides, whereas deleting mexZ in isolates without MexZ sequence variants had a greater impact on resistance and mexXY expression. Therefore, MexZ variants are associated with an increased level of aminoglycoside resistance and mexXY expression in clinical isolates. These results show that the interaction between mexZ and mexXY, as well as the extent of mexXY expression, contributes significantly to reduced aminoglycoside sensitivity in P. aeruginosa clinical isolates, although the degree of its contribution to resistance varies by isolate.
To quantify the contributions of mutations in other genes, frequent clinically identified genetic variations from a cohort of 619 clinical strains were screened. Sequence variants in mexY (G287S), mexZ(∆), amgS (V121G) and fusA1(R680C) were engineered in reference strain PAO1 in single and multiple combinations. Minimum Inhibitory Concentration (MIC) testing was carried out on the engineered mutants. Engineered mutants had increased aminoglycoside MICs and mexXY expression, with triple mutants having the greatest effect on phenotype and genotype. These findings show that analysing the genetic basis of individual and combination mutations in genes associated with aminoglycoside resistance can advance the understanding of how aminoglycoside resistance can develop in clinical isolates of P. aeruginosa.
This research further aimed to understand the aminoglycoside resistance of P. aeruginosa contributed by AMEs. AME-encoding genes in 48 of 619 clinical isolates of P. aeruginosa were identified through a bioinformatic approach and the most prevalent ant(2′)-Ia and aac(6′)-Ib3 along with aph(3′)-VIa were cloned and expressed in P. aeruginosa strains to study their effects on resistance phenotype. The cloned genes conferred resistance to aminoglycosides and elevated aminoglycoside MICs when introduced into mutants lacking the MexXY-OprM efflux pump, demonstrating that AMEs and this efflux pump operate independently in determining aminoglycoside resistance levels.
Overall, this research improves the genetic understanding of aminoglycoside resistance by investigating the intrinsic and acquired resistance mechanisms in P. aeruginosa. Performing a comprehensive analysis of mexXY expression, screening for variants in mexZ, amgS, and fusA1, and detecting AMEs in clinical isolates will help to predict resistance phenotype from genotype. Analysing these aminoglycoside resistance markers from patient samples prior to treatment will reduce treatment delays, control antibiotic overuse, stop recurrent infections, and decrease mortality rates associated with P. aeruginosa infections.