Pseudomonas aeruginosa exhibits high intrinsic resistance to penem antibiotics such as faropenem, ritipenem, AMA3176, sulopenem, Sch29482, and Sch34343. To investigate the mechanisms contributing to penem resistance, we used the laboratory strain PAO1 to construct a series of isogenic mutants with an impaired multidrug efflux system MexAB-OprM and/or impaired chromosomal AmpC ␤-lactamase. The outer membrane barrier of PAO1 was partially eliminated by inducing the expression of the plasmid-encoded Escherichia coli major porin OmpF. Susceptibility tests using the mutants and the OmpF expression plasmid showed that MexAB-OprM and the outer membrane barrier, but not AmpC ␤-lactamase, are the main mechanisms involved in the high intrinsic penem resistance of PAO1. However, reducing the high intrinsic penem resistance of PAO1 to the same level as that of penem-susceptible gram-negative bacteria such as E. coli required the loss of either both MexAB-OprM and AmpC ␤-lactamase or both MexAB-OprM and the outer membrane barrier. Competition experiments for penicillin-binding proteins (PBPs) revealed that the affinity of PBP 1b and PBP 2 for faropenem were about 1.8-and 1.5-fold lower, than the respective affinity for imipenem. Loss of the outer membrane barrier, MexAB, and AmpC ␤-lactamase increased the susceptibility of PAO1 to almost all penems tested compared to the susceptibility of the AmpC-deficient PAO1 mutants to imipenem. Thus, it is suggested that the high intrinsic penem resistance of P. aeruginosa is generated from the interplay among the outer membrane barrier, the active efflux system, and AmpC ␤-lactamase but not from the lower affinity of PBPs for penems.Penem antibiotics such as faropenem (formerly SUN5555 or

Expression of the multidrug efflux system MexC-MexD-OprJ innfxB mutants of Pseudomonas aeruginosacontributes to resistance to fluoroquinolones and the “fourth-generation” cephems (cefpirome and cefozopran), but not to most β-lactams, including the ordinary cephems (ceftazidime and cefoperazone). nfxB mutants also express a second multidrug efflux system, MexA-MexB-OprM, due to incomplete transcriptional repression of this operon by the mexR gene product. To characterize the contribution of the MexC-MexD-OprJ system to drug resistance in P. aeruginosa, a site-specific deletion method was employed to remove the mexA-mexB-oprM region from the chromosome of wild-type and nfxB strains ofP. aeruginosa. Characterization of mutants lacking themexA-mexB-oprM region clearly indicated that the MexC-MexD-OprJ efflux system is involved in resistance to the ordinary cephems as well as fluoroquinolones and the fourth-generation cephems but not to carbenicillin and aztreonam. Rabbit polyclonal antisera and murine monoclonal antibody against the components of the MexA-MexB-OprM system were prepared and used to demonstrate the reduced production of this efflux system in the nfxB mutants. Consistent with this, transcription of the mexA-mexB-oprM operon decreased in an nfxB mutant. This reduction appears to explain the hypersusceptibility of the nfxB mutant to β-lactams, including ordinary cephems.

No abstract

The high intrinsic penem resistance of Pseudomonas aeruginosa is due to the interplay among the outer membrane barrier, the active efflux system MexAB-OprM, and AmpC ␤-lactamase. We studied the roles of two other efflux systems, MexCD-OprJ and MexXY-OprM, in penem resistance by overexpressing each system in an AmpC-and MexAB-OprM-deficient background and found that MexAB-OprM is the most important among the three efflux systems for extrusion of penems from the cell interior.Pseudomonas aeruginosa is a clinically significant pathogen exhibiting intrinsic and acquired resistance to various antimicrobial agents. This resistance is attributable to the limited permeability of the outer membrane and the extrusion of a wide variety of antibiotics from the cell interior by a tripartite multidrug efflux system, which is composed of membrane fusion protein-type periplasmic, resistance-nodulation-cell division-type inner-membrane, and outer-membrane efflux proteins (10). Among these efflux systems, 9,19) and 11,14) contribute to both intrinsic resistance and acquired resistance, while and MexEF-OprN (8) contribute only to acquired resistance in P. aeruginosa. Recently, Masuda et al. (12) reported the substrate specificities of MexAB-OprM, MexXY-OprM, and MexCD-OprJ. Although most antimicrobial agents are substrates of all three efflux systems, these systems have slight but significant differences in substrate specificities to ␤-lactams. MexAB-OprM extrudes the broadest variety of ␤-lactams, including penicillins, cephems, and meropenem-type carbapenems. MexXY-OprM and MexCD-OprJ extrude most penicillins but not carbenicillin, sulbenicillin, various cephems, and many carbapenems. Penem antibiotics display potent activities against a variety of gram-positive and gram-negative bacteria but not against P. aeruginosa (2,3,4,13,15,16,17,21,24). Studies with mutants that overproduce or lack MexAB-OprM demonstrated that this efflux system extrudes penem antibiotics (18). However, it is unclear whether MexXY-OprM and MexCD-OprJ extrude penem antibiotics.We compared the susceptibilities of a series of previously described isogenic AmpC-lacking mutants, each of which constitutively overexpressed an individual efflux pump (12, 18) ( Table 1). We used AmpC-lacking P. aeruginosa mutants because the presence of chromosomal AmpC ␤-lactamase makes it difficult to interpret data on the MICs of ␤-lactams, including penems, due to the interplay between ␤-lactamase and the efflux system(s). The MICs of various penems, norfloxacin, and tetracycline for the mutants were determined by the twofold agar dilution method (23) with L agar with an inoculum of 10 4 cells. These results are shown in Table 2. Although the susceptibilities of the mutant KG5002, which lacked MexABOprM, MexCD-OprJ, and MexXY-OprM, to all penems tested were reduced by the overexpression of MexAB-OprM, MexCD-OprJ, or MexXY-OprM, as were those to norfloxacin and tetracycline, the degree of reduction resulting from the overexpression of MexAB-OprM (128-to 4,096-fold reduction ...

No abstract