To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the ␤-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously.Several tripartite efflux systems coded on the chromosome play important roles in the intrinsic and acquired resistance in Pseudomonas aeruginosa. Each system consists of a cytoplasmic membrane component of the resistance-nodulation-division family presumed to function as a transporter, an outer membrane component presumed to form channels, and a membrane fusion protein presumed to link the two membrane proteins for reviews, see references (13, 14, and 15). The MexAMexB-OprM efflux system (7, 16) contributes to both intrinsic and acquired resistance in P. aeruginosa, while the MexCMexD-OprJ (17) and MexE-MexF-OprN (6) efflux systems contribute only to the acquired resistance in this bacterium. Studies with mutants that overproduce or lack MexAB-OprM demonstrated that this efflux system extrudes quinolones, macrolides, tetracycline, chloramphenicol, novobiocin, and most ␤-lactams but not imipenem (7,8,22). Studies with mutants overproducing MexCD-OprJ demonstrated that this efflux system extrudes quinolones, erythromycin, tetracycline, chloramphenicol, and expanded-spectrum cephems such as cefpirome (9, 10). Furthermore, characterization of mutants lacking the mexA-mexB-oprM region demonstrated that the MexCD-OprJ efflux system extrudes ceftazidime and cefoperazone as well as cefpirome, while it does not extrude carbenicillin and aztreonam (3). Recently, Aires et al. (1) and our group (12) showed that MexX-MexY extrudes aminoglycosides, tetracycline, and erythromycin in cooperation with spontaneously expressed OprM, thereby contributing to the intrinsic resistance to these agents in P. aeruginosa. Our group also showed that no expression of MexXY is observed in wild-type strains but that the expression can be induced by subinhibitory concentrations of its substrates such as tetracycline a...

To test the possibility that MexX-MexY, a new set of efflux system components, is associated with OprM and contributes to intrinsic resistance in Pseudomonas aeruginosa, we constructed a series of isogenic mutants lacking mexXY and/or mexAB and/or oprM from a laboratory strain PAO1, and examined their susceptibilities to ofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Loss of either MexXY or OprM from the MexAB-deficient mutant increased susceptibility to all agents tested, whereas loss of MexXY from the MexAB-OprM-deficient mutant caused no change in susceptibility. Introduction of an OprM expression plasmid decreased the susceptibility of the mexAB-oprM-deficient-/mexXY-maintaining mutant, yet caused no change in the susceptibility of a mexAB-oprM-and mexXY-deficient double mutant. Immunoblot analysis using anti-MexX polyclonal rabbit serum generated against synthetic oligopeptides detected expression of MexX in the PAO1 cells grown in medium containing tetracycline, erythromycin, or gentamicin, although expression of MexX was undetectable in the cells incubated in medium without any agent. These results suggest that MexXY induced by these agents is functionally associated with spontaneously expressed OprM and contributes to the intrinsic resistance to these agents.A variety of multicomponent efflux systems are coded on the chromosomes of gram-negative bacteria and contribute to intrinsic and acquired resistance against antimicrobial agents, disinfectants, organic solvents, and heavy metals (17)(18)(19)(20)24). Pseudomonas aeruginosa is a clinically significant pathogen exhibiting highly intrinsic resistance to various antimicrobial agents. One of the mechanisms contributing to its intrinsic resistance is a spontaneous expression of the efflux system MexA-MexB-OprM encoded on a mexA-mexB-oprM operon of the chromosome of P. aeruginosa (10). This system energydependently extrudes many antimicrobial agents from the cell interior in cooperation with the periplasmic, inner membrane, and outer membrane components organized through the two membranes. While the disruption of each component gene of the mexA-mexB-oprM operon increases the susceptibility to many antimicrobial agents, the disruption of oprM increases the susceptibility to a greater extent than the disruption of mexA or mexB (5,21,30,31). Due to the presence of a weak promoter in the mexB gene upstream of oprM gene, the polar effect from the disruptions of mexA and mexB does not entirely suppress the expression of the oprM gene (31). Thus, OprM can contribute to the intrinsic resistance by cooperation with unknown periplasmic and inner membrane components. Recently, mexG-mexH (GenBank accession no. AF073776 To test the possibility that MexXY is associated with OprM and contributes to intrinsic resistance, we constructed a series of isogenic mutants lacking mexXY and/or mexAB and/or oprM from laboratory strain PAO1 and compared their susceptibilities to antimicrobial agents. We also showed that the expression of MexXY is induced by expo...

Three types of multiple-drug-resistant mutants which were phenotypically similar to previously described nalB, nfxB, and nfxC mutants were isolated from Pseudomonas aeruginosa PAO1 and two clinical isolates. Type 1 (nalB-type) mutants showed cross-resistance to meropenem, cephems, and quinolones. They overproduced an outer membrane protein with an apparent molecular mass of 50 kDa (OprM). Type 2 (nfxB-type) mutants showed cross-resistance to quinolones and new cephems, i.e., cefpirome and cefozopran, concomitant with overproduction of an outer membrane protein with an apparent molecular mass of 54 kDa (OprJ). Type 3 (nfxC-type) mutants showed cross-resistance to carbapenems and quinolones. They produced decreased amounts of OprD and increased amounts of a 50-kDa protein (OprN), which was almost the same molecular weight as that of OprM, but it was distinguishable from OprM by its heat modifiability on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of salicylate, the parent strains showed an increased level of resistance to carbapenems and quinolones and produced decreased amounts of OprD and increased amounts of OprN. Salicylate caused the repression of OprJ production and the loss of resistance to cefpirome and cefozopran in two of the three OprJ-overproducing mutants, although salicylate slightly increased the level of resistance in the parent strains. The changes in susceptibilities were transient in the presence of salicylate. These data suggest that at least three different outer membrane proteins, OprM, OprJ, and OprN, are associated with multiple drug resistance in P. aeruginosa.Pseudomonas aeruginosa is a clinically significant opportunistic pathogen that infects compromised hosts in hospitals. This organism has an outer membrane with a low level of permeability and is thereby intrinsically resistant to a wide variety of commonly used antibiotics (1,22). Only a few antimicrobial agents, such as carbapenems and quinolones, show potent antibacterial activity against this species. In recent years, a number of clinical P. aeruginosa isolates have been reported to be resistant to some of these antibiotics, especially quinolones. Several mutations conferring quinolone resistance have been identified and mapped on chromosomes in P. aeruginosa. The gyrA (nfxA, nalA, or cfxA) mutation causes an alteration in the subunit A of DNA gyrase (5,7,16,17). The nalB (cfxB) (12,16,17), nfxB (5), and nfxC (3) mutations cause a decrease in the level of accumulation of norfloxacin, and strains with these mutations show cross-resistance to structurally unrelated antimicrobial agents. The nalB mutant shows cross-resistance to quinolones, cephems, carbenicillin, meropenem, tetracycline, chloramphenicol, and novobiocin and overproduces an outer membrane protein with an apparent molecular mass of 49 kDa (OprM). The nfxB mutant shows cross-resistance to quinolones and hypersusceptibility to ␤-lactams and aminoglycosides and overproduces an outer membrane protein with an apparent molecular mass of 54 kDa....

The Pseudomonas aeruginosa nfxB mutant lacking mexAB-oprM showed hypersusceptibility to 9 out of 24 ␤-lactams tested. This hypersusceptibility was found for the nfxB mutant lacking mexAB-oprM-mexXY (N108) but not for the nfxB mutant lacking both mexAB-oprM-mexXY and ampC. The level of the AmpC ␤-lactamase induction was reduced in N108. Thus, the reduced AmpC induction must be the cause of the hypersusceptibility.A series of multicomponent efflux systems, each made up of three components, play important roles in the intrinsic and acquired resistance of gram-negative bacteria (20,21,22). Four of these efflux systems, MexAB-OprM (11, 24), MexCDOprJ (25), MexEF-OprN (9), and MexXY-OprM (1,19,26), have been characterized for Pseudomonas aeruginosa. MexABOprM and MexXY-OprM contribute to both intrinsic and acquired resistance, whereas MexCD-OprJ and MexEF-OprN contribute to only acquired resistance. MexAB-OprM is slightly expressed in wild-type strains, and a nalB mutation causes an overexpression of the efflux system. The expression of MexCD-OprJ and MexEF-OprN is strictly suppressed in wild-type strains, and mutations in nfxB and nfxC cause overexpression of MexCD-OprJ and MexEF-OprN, respectively. The expression of MexXY, which is not detectable in the wild-type strain, is induced by several antimicrobial agents such as tetracycline, erythromycin, and gentamicin (17). MexXY is associated with OprM and contributes to the intrinsic resistance to these agents. While nfxB mutants show resistance to quinolones, tetracycline, erythromycin, chloramphenicol, and expanded-spectrum cephems such as cefpirome, they show hypersusceptibility to penicillins, carbapenems, and aminoglycosides (15). A characterization of mutants lacking the mexABoprM region demonstrated that the hypersusceptibility to ␤-lactams such as carbenicillin and aztreonam is caused by the reduced expression of MexAB-OprM in the nfxB mutants (4).To investigate whether the hypersusceptibility of the nfxB mutants to ␤-lactams is generally attributable to this mechanism, in this study, we compared the susceptibilities of the isogenic mutants, i.e., the MexCD-OprJ-producing KG2259 (⌬MexAB-OprM of COR6 [4]) and the non-MexCD-OprJproducing KG2239 (⌬MexAB-OprM of PAO1 [4]), to the 24 ␤-lactams. The results indicated that these ␤-lactams can be classified into three groups. The first group, which consists of piperacillin, cloxacillin, nafcillin, cefpirome, cefepime, cefozopran, and cefoselis, showed 8-to 64-fold-lower activities against KG2259 than against KG2239. The second group, which consists of penicillin G, cefuroxime, cefmetazole, ceftazidime, cefsulodin, meropenem, S-4661 (7), and aztreonam, showed almost the same activities against KG2239 and KG2259. The third group, which consists of sulbenicillin, cefpodoxime, ceftriaxone, moxalactam, flomoxef, imipenem, panipenem, biapenem, and R-95867 (an active form of a new oral carbapenem, CS-834 [3]), showed 4-to 32-fold-higher activities when MexCD-OprJ was expressed and MexAB-OprM was not. These results suggest tha...

We investigated the role of chromosomal β-lactamase and the MexAB-OprM efflux system in intrinsic resistance to β-lactams inPseudomonas aeruginosa. Determination of the susceptibilities of a series of isogenic mutants with impaired production of the β-lactamase and the efflux system to 16 β-lactams including penicillins, cephems, oxacephems, carbapenems, and a monobactam demonstrated that the intrinsic resistance of P. aeruginosa to most of the β-lactams is due to the interplay of both factors.