The Role of Multidrug Efflux Pumps in the Antibiotic Resistance of Pseudomonas aeruginosa and Other Gram-negative Bacteria

Jeffrey R. Aeschlimann, PharmD

Disclosures

Pharmacotherapy. 2003;23(7) 

In This Article

Multidrug Efflux Pump-Based Resistance in Gram-Negative Bacteria

Gram-negative efflux pumps were first identified and characterized in strains of Escherichia coli and P. aeruginosa.[5,8] Many similar efflux pumps have since been discovered in nearly all clinically relevant gram-negative bacteria ( Table 1 ).[5,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31] Because the efflux pump systems of these bacteria share substantial homology with the P. aeruginosa systems, further research will most likely reveal similar multidrug resistance characteristics.

The P. aeruginosa genome contains at least 10 distinct tripartite (3-protein) efflux pump system gene operons.[32] All of the efflux pump systems are energy dependent (i.e., proton motive force-driven) and consist of three components: a resistance-nodulation-division (RND) exporter protein (located in the cytoplasmic membrane), a gated outer membrane protein (located in the outer membrane), and a membrane fusion protein (MFP) that links the RND exporter protein with the gated outer membrane protein. A schematic of the proposed structure and function of these efflux pump systems is depicted in Figure 1.

Proposed structure and function of the MexAB-OprM and related efflux pumps of P. aeruginosa. Antibiotics can be captured from the periplasmic space, cytoplasmic membrane, and/or cytoplasmic space by MexB, D, F, or Y (RND exporter proteins). MexA, C, E, or X (MFP proteins) serve as conduits between the cytoplasmic and outer membranes. OprM, J, or N (gated outer membrane porin proteins) serve as the final step in removal of the antibiotic from the cell.

Four multidrug efflux pumps have been well characterized in P. aeruginosa: MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN, and MexX-MexY-OprM.[7,23,33,34] The MexA-MexB-OprM efflux pump is produced at low levels by the mexA-mexB-oprM gene operon in all strains of P. aeruginosa. However, this gene operon becomes overexpressed in so-called nalB mutants as a result of mutations in the mexR repressor gene.[35] The resulting efflux pump overproduction increases the minimum inhibitory concentrations (MICs) for a number of unrelated antibiotics, such as aztreonam and nalidixic acid, by 8-fold or more; a more complete list is provided in Table 2 .[4,5,6,7,23,33,34,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50] The MexA-MexB-OprM system has the broadest-spectrum activity of the efflux pumps described to date. Genetic deletion of this efflux pump results in impressive (≥ 128-fold) decreases in MICs for antibiotics that are not considered to have clinically significant antipseudomonal activity (e.g., amoxicillin, cefuroxime, cefotaxime, nafcillin, tetracycline). In some cases the MICs are reduced below current breakpoint values for resistance.[34]

The MexC-MexD-OprJ and MexE-MexF-OprN efflux pumps are detected and overproduced only in so-called nfxB and nfxC mutants. Both have a somewhat narrower substrate specificity than the MexA-MexB-OprM efflux pump. The MexC-MexD-OprJ efflux pump can expel cefepime, fluoroquinolones, macrolides, and tetracycline, whereas MexE-MexF-OprN can expel fluoroquinolones, imipenem, trimethoprim, and some -lactamase inhibitors ( Table 2 ).[7,23,34,43,45] Although regulation of the gene operons encoding these two efflux pumps has not been fully characterized, production of MexC-MexD-OprJ appears to increase in the presence of sub-inhibitory concentrations of pump substrates.[46]

The MexX-MexY-OprM pump system is a particularly intriguing multidrug resistance efflux system. It has a much narrower substrate profile than MexA-MexB-OprM ( Table 2 ), but it can remove many clinically useful antipseudomonal antibiotics (e.g., aminoglycosides, cefepime, ciprofloxacin, and levofloxacin).[34,25,26,27,46,47,48] Like MexC-MexD-OprJ, its production can be increased by the presence of low concentrations of its substrates. In fact, overproduction of the MexX-MexY-OprM efflux pump system appears to be responsible for the "adaptive resistance" that P. aeruginosa develops to aminoglycosides (i.e., the antibacterial activity of the aminoglycoside decreases with subsequent exposures of the bacteria to the antibiotic). The MexX-MexY-OprM system also plays an integral role in the long-recognized antagonism of aminoglycoside activity by divalent cations, such as calcium and magnesium, and it has been proposed as a compensatory resistance mechanism to fluoroquinolones.[25,47]

Overproduction of one or more of these multidrug efflux pumps appears to be a critical step toward development of high-level multidrug resistance to fluoroquinolones and other antipseudomonal antibiotics.[4,6] Although all antibiotics appear capable of selecting mutants that overproduce efflux pumps, the fluoroquinolone antibiotics appear especially adept at mutant selection.[4] Older quinolones (e.g., nalidixic acid and norfloxacin) select mutants that primarily overproduce MexA-MexB-OprM, whereas newer fluoroquinolones (e.g., ciprofloxacin, levofloxacin, and trovafloxacin) select mutants that overproduce MexC-MexD-OprJ and/or MexE-MexF-OprN. These so-called mutant selection preferences have been documented in both in vitro and in vivo investigations.[4,49]

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