EXPRESSION OF PSEUDOMONAS-AERUGINOSA MULTIDRUG EFFLUX PUMPS MEXA-MEXB-OPRM AND MEXC-MEXD-OPRJ IN A MULTIDRUG-SENSITIVE ESCHERICHIA-COLI STRAIN

The mexCD-oprJ and mexAB-oprM operons encode components of two distinc t multidrug efflux pumps in Pseudomonas aeruginosa. To assess the cont ribution of individual components to antibiotic resistance and substra te specificity, these operons and their component genes were cloned an d expressed in Escherichia coli. Western immunoblotting confirmed expr ession of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (Ac rAB), although only the Delta acrAB strain, KZM120, demonstrated incre ased resistance to antibiotics in the presence of the P. aeruginosa ef flux genes. E. coli KZM120 expressing MexAB-OprM showed increased resi stance to quinolones, chloramphenicol, erythromycin, azithromycin, sod ium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significan tly, several beta-lactams, which is reminiscent of the operation of th is pump in P. aeruginosa. This confirmed previous suggestions that Mex AB-OprM provides a direct contribution to beta-lactam resistance via t he efflux of this group of antibiotics. An increase in antibiotic resi stance, however, was not observed when MexAB or OprM alone was express ed in KZM120. Thus, despite the fact that beta-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these a gents. E. coli KZM120 expressing MexCD-OprJ also showed increased resi stance to quinolones, chloramphenicol, macrolides, SDS, and crystal vi olet, though not to most beta-lactams or novobiocin, again somewhat re miniscent of the antibiotic resistance profile of MexCD-OprJ-expressin g strains of P. aeruginosa. Surprisingly, E. coli KZM120 expressing Me xCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antib iotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in co njunction with TolC, the previously identified outer membrane componen t of the AcrAB-TolC efflux system. These data confirm that a tripartit e efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.