Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides

Abstract

A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501 insertion mutagenesis of Pseudomonas aeruginosa PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene, mexZ, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P. aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in Escherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415-417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P. aeruginosa to aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the mexXY operon in P. aeruginosa had no detectable effect on susceptibility to these agents.

FIG. 1

Physical map of the mexZXY operon in P. aeruginosa. Inserts of recombinant plasmids pJR120 and pAGH97 are indicated. The site of insertion of transposon Tn501 in mutant 11B is indicated with an arrowhead.

FIG. 2

Accumulation of [³H]dihydrostreptomycin by intact cells of P. aeruginosa PAO1 (⧫) and mutant 11B (▴). The MICs of dihydrostreptomycin for PAO1 and mutant 11B were 16 and 4 μg ml⁻¹, respectively. The cells were grown in LB, harvested, washed (50 mM phosphate buffer, 100 mM LiCl), and resuspended in 50 mM of phosphate buffer (pH 7) containing 1 mM MgSO₄ and 0.2% (wt/vol) glucose as a source of carbon. After 5 min at 37°C, a mixture of dihydrostreptomycin and [³H]dihydrostreptomycin (20 Ci mmol⁻¹) was added to a final concentration of 8 μg ml⁻¹; and the samples were taken and filtered, and the radioactivity was counted. The data are means for three independent experiments.

FIG. 3

Accumulation of [³H]tetracycline by intact cells of P. aeruginosa PAM1275 (●, ○) and PAM1275(pAGH97) (⧫, ◊). The MICs of tetracycline for PAM1275 and PAM1275(pAGH97) were 2 and 16 μg ml⁻¹, respectively. The cells were prepared as described in the legend to Fig. 2. After 5 min at 37°C, a mixture of tetracycline and [³H]tetracycline (0.55 Ci mmol⁻¹) was added to a final concentration of 2 μg ml⁻¹; and the samples were taken and filtered, and the radioactivity was counted. After 7.5 min of incubation, a 500 μM final concentration of CCCP was added to one-half of the reaction mixture. Accumulation in CCCP-treated samples is represented by open symbols, and that in samples that did not receive CCCP is indicated by closed symbols. The data are means for three independent experiments. No modification of intracellular accumulation of antibiotic was observed when strains were transformed with pAK1900.