Simultaneous overexpression of multidrug efflux pumps in Pseudomonas aeruginosa non-cystic fibrosis clinical isolates

Abstract

The purpose of this study was to examine expression and regulation of 6 multidrug efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY, MexJK, and MexVW, in 13 non-cystic fibrosis (CF) clinical isolates of Pseudomonas aeruginosa. These isolates displayed a high level of resistance to many clinically important antibiotics. Some isolates simultaneously overexpressed up to 4 different Mex systems, as determined by quantitative real-time reverse transcription PCR. None of the isolates overexpressed MexCD-OprJ, and only 1 isolate overproduced MexJK. All the isolates overexpressed MexXY, while overexpression of MexEF-OprN and MexVW was common. DNA sequencing analysis of regulatory genes showed that no clear correlation could be established among (i) the presence of mutations, (ii) the type of mutations, (iii) the expression level of the Mex systems, and (iv) resistance to antibiotic substrates. The results suggest that the concomitant overexpression of some Mex systems may superimpose their antimicrobial drug efflux capabilities, contributing to the multidrug resistance phenotype in the P. aeruginosa non-CF clinical isolates. The existence of uncharacterized regulators for the Mex systems was signified.

MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of Pseudomonas aeruginosa from chronic infections

“…While western blots can provide more definitive data on the expression of efflux pumps, lack of appropriate antibodies for analysis of a large subset of proteins often hampers the use of this technique. Therefore, qRT-PCR is widely used to measure the expression of efflux pumps (Eaves et al 2004;Kumar et al 2008;Fernando et al 2013;Poonsuk et al 2014). All 4 pumps we studied have been shown to be overexpressed in clinical isolates displaying a MDR phenotype (Llanes et al 2004;Hocquet et al 2007;Jeannot et al 2008;Poonsuk et al 2014).…”

Genome-wide antibiotic resistance and virulence profiling of Pseudomonas Aeruginosa isolated from clinical samples in Lebanon

“…PCR assays were performed using the following primers: oprI F (5'-ATGAACAACGTTCTGAAATTCTCTGCT-3') and oprI R (5'-CTTGCGGCTGGCTTTTTCCAG-3') amplifying a 249 bp fragment of the oprI gene, oprL F (5'-ATGGAAATGCTGAAATTCGGC-3') and oprL R (5'-CTTCTTCAGCTCGACGCGACG) amplifying a 504 bp fragment of the oprL gene, oprD F (5'-CGCCGACAAGAAGAACTAGC-3') and oprD R (5'-GTCGATTACAGGATCGACAG-3') amplifying a 1412 bp of the oprD gene (Adabi et al, 2015;Poonsuk et al, 2014). PCR mixture contained 2 µl of template DNA, 20…”

Genome-wide association study of Pseudomonas aeruginosa adaptations to the cystic fibrosis lung

“…Beta-lactams, carbapenems and fluoroquinolones are some of the drug classes affected by these changes [29,30]. Mutations in gyrA, gyrB, parC or parE, which encode for drug target enzymes DNA gyrase and topoisomerase IV, can cause resistance to fluoroquinolones [31].…”

“…Mutlidrug efflux pumps (Mex proteins) confer antibiotic resistance by pumping out drugs and other materials from the cell [124]. The role of efflux pumps in antibiotic resistance in CF PA strains has been widely studied [30,125,126]. mexZ encodes for a well characterized protein that confers aminoglycoside and fluoroquinolone resistance [127,128,129].…”