Expression of the MexXY efflux pump in amikacin-resistant isolates of Pseudomonas aeruginosa

Abstract

The MexZ-MexX-MexY multidrug efflux system in Pseudomonas aeruginosa was studied to determine its contribution to aminoglycoside resistance. Amikacin-resistant (AR) mutants were generated from P. aeruginosa strain PAO1, and clinical isolates of P. aeruginosa were collected from cystic fibrosis patients. The regulatory gene mexZ and the intergenic region (mexOZ) between mexZ and mexX were investigated for mutation by PCR and DNA sequence analysis. The results showed that 14 of 15 AR clinical isolates and one of ten laboratory mutants had at least one mutation in mexZ and/or mexOZ. To study the effect of mexZ and mexOZ mutations, the production of MexY mRNA was investigated quantitatively by real-time PCR. Seven of ten AR mutants (MIC 4-8 mg/L) produced 8-21-fold more MexY mRNA than PAO1. These isolates were sensitive to fluoroquinolones, carbapenems and ceftazidime. One AR mutant (MIC 64 mg/L) that produced > 200-fold more MexY mRNA than PAO1 was also resistant to fluoroquinolones, carbapenems and ceftazidime. Thirteen of 15 AR clinical isolates produced 3.4-727-fold more MexY mRNA. No evidence was found for the aminoglycoside-modifying enzymes 6'-N-acetyltransferase type Ib, 4'-O-nucleotidyltransferase type IIb or aminoglycoside 3'-phosphotransferase IIps in these strains. Nine AR mutants overproduced MexY without mutations in mexZ or mexOZ, suggesting that MexXY efflux is also regulated by gene(s) other than mexZ.

Efflux Unbalance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

“…These results were fully consistent with previously published data showing the absence of horizontally acquired aminoglycoside-modifying enzymes in most CF isolates of P. aeruginosa (29,45,70,83). As the efflux system MexXY-OprM is known to play a major role in emergence of aminoglycoside resistance in CF strains (31,83,84), we assessed its expression at the gene (mexY) and the protein (MexY) levels by reverse transcription RT-PCR and Western blotting, respectively. As expected, all the Tic hs strains were found to overexpress both the mexY gene (11.4-to 58.8-fold) (data not shown) and the MexY protein compared with aminoglycoside-susceptible strains PAO1, 615S, and 3020S (Fig.…”

“…Interestingly, studies in the 1990s demonstrated that the nalB gene encodes a negative regulator of MexAB-OprM (63), a polyspecific efflux system which contributes to the natural resistance of P. aeruginosa toward a wide range of antibiotics including ␤-lactams, tetracyclines, trimethoprim, fluoroquinolones, and novobiocin (36,40). In parallel, another efflux pump, MexXY, which is encoded by a distinct operon (mexXY) on the bacterial chromosome, was found to provide CF isolates with moderate resistance to amino-glycosides, fluoroquinolones, and the zwitterionic cephalosporin cefepime when stably overproduced upon various mutations (31,50,83,84).…”

Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

“…These data suggest a contribution by efflux pumps through their upregulation and/or permeability alterations in these strains. Antimicrobial compounds, including amikacin, ampicillin, cephalosporins, cefoxitin, chloramphenicol, ciprofloxacin, kanamycin, nalidixic acid, piperacillin, and tetracycline, which demonstrated a reduced effect on one or more of the biocide-tolerant Salmonella strains, are known efflux pump substrates (11,15,27,29,32) of the resistance nodulation and cell division (RND) family (32). In some instances, changes in antibiotic susceptibility profiles were sufficiently large that these could be defined according to CLSI guidelines, whereas others were more modest.…”

Antibacterial-ResistantPseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms

“…MexZ binds as a homodimer to an inverted repeat region, located in the mexZ-mexX intergenic region, which encompasses the putative mexXY promoter (154). Mutations in mexZ or the mexZ-mexX intergenic region have been associated with hyperexpression of mexXY (85,263). However, overexpression of mexXY has also been detected in mutants not harboring mutations within mexZ or the mexZ-mexX intergenic region (137,240,269).…”