In vivo emergence of multidrug-resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA-MexB-OprM

Abstract

During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal beta-lactam antibiotics were isolated from hospitalized patients. In vivo emergence of beta-lactam resistance was associated with the overexpression of AmpC beta-lactamase in 10 patients. In the other 11 patients, the posttherapy isolates produced only low, basal levels of beta-lactamase and had increased levels of resistance to a variety of non-beta-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of beta-lactamase production and resistance of their pretherapy counterparts. These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains. Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates. To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR. Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics. These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.

FIG. 1

Western immunoblots of outer membrane proteins (10 μg per lane) developed with a rabbit polyclonal antibody raised against OprM. Posttherapy resistant isolates 273, 92, 128, and A2 (lanes 1, 3, 5, and 7, respectively) and their respective pretherapy susceptible counterparts (isolates 287, 70, 141, and A1; lanes 2, 4, 6, and 8, respectively) were tested. Lane 9, strain 4098E overexpressing the active efflux system MexA-MexB-OprM. The arrow indicates the position of OprM (ca. 50 MDa).