Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents for Combination Therapy

Active efflux of toxic compounds out of cells is a general mechanism that bacteria have developed to protect themselves against the adverse effects of their environments. Antibiotics that are used in clinical settings are among these toxic compounds, and extrusion of antibiotics from bacterial cells significantly decreases their clinical utility. Antibiotics are expelled from the cells by membrane transporter proteins, the so-called drug-efflux pumps. Of particular interest are efflux pumps capable of extruding out of the cell a large variety of structurally unrelated compounds with different antibacterial modes of action (13, 15, 30–32). Most of the genes encoding these multidrug resistance (MDR) pumps are normal constituents of bacterial chromosomes. Some of these genes have a relatively high level of constitutive expression and confer so-called intrinsic resistance to antibiotics. Expression of other genes that confer an efflux capability is not detected in wild-type cells, but such genes can become expressed after the acquisition of regulatory mutations. In gram-negative bacteria, most of the efflux pumps that contribute to both intrinsic and acquired resistance to clinically useful antibiotics are three-component structures that traverse both inner membranes and outer membranes. Each tripartite pump contains a transporter located in the cytoplasmic membrane, an outer membrane channel, and a periplasmic linker protein, which is thought to bring the other two components into contact (54, 55). This structural organization allows extrusion of substrates directly into the external medium, bypassing the periplasm. Direct efflux as a mechanism of drug extrusion is required since these rather slow tripartite MDR pumps (46) rely heavily on the help of the outer membrane, which serves as a permeability barrier for both hydrophobic and hydrophilic antibiotics (33). Several classes of MDR pumps have been identified on the basis of sequence comparisons (42). Most of the inner membrane components of clinically relevant tripartite efflux pumps from gram-negative bacteria belong to a single class of transporters called resistance-nodulation-division (RND) efflux pumps (5). Pseudomonas aeruginosa is an important opportunistic pathogen which is highly resistant to antibiotic therapy. Fluoroquinolones, β-lactams, and aminoglycosides are among the primary agents available for treatment of infections caused by this pathogen. Four multicomponent MDR RND efflux pumps have been identified in P. aeruginosa, namely, MexAB-OprM (39), MexCD-OprJ (38), MexEF-OprN (12), and MexXY-OprM (1). These pumps have somewhat overlapping spectra of antibiotic substrates. For example, all four pumps confer various degrees of resistance to fluoroquinolones, and mutants that overexpress three of these pumps have been isolated among fluoroquinolone-resistant bacteria in clinical settings: nalB mutants that overproduce the MexAB-OprM pump (3), nfxB mutants that overproduce MexCD-OprJ (53), and nfxC mutants that overproduce the MexEF-OprN efflux pump (6). So far, overexpression of MexXY-OprM has not been reported as a cause of fluoroquinolone resistance in P. aeruginosa; however, this pump has recently been implicated in low-level resistance to aminoglycosides (1, 50). Of these four pumps, only MexAB-OprM is expressed at a level sufficient to confer intrinsic MDR in wild-type cells. Deletion of the mexA, mexB, or oprM gene renders P. aeruginosa more susceptible to multiple antibiotics, including fluoroquinolones (8, 39, 51). Importantly, overexpression of the MexCD-OprJ or the MexEF-OprN efflux pumps restores resistance to fluoroquinolones in strains lacking the MexAB-OprM efflux pump (9, 14, 18). Besides efflux, resistance to fluoroquinolones is also conferred by target mutations. These mutations mainly occur in quinolone resistance-determining regions (QRDRs) (11, 36, 52) in DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE) in many organisms including P. aeruginosa (26). Importantly, it has been demonstrated recently in P. aeruginosa (18) and Escherichia coli (35) that when both target-based and efflux-mediated resistance mechanisms are present in the same cell, they contribute independently to fluoroquinolone resistance. As a result, deletion of the MexAB-OprM efflux pump from a strain in which this pump is overexpressed resulted in a 64-fold reduction in the MIC of levofloxacin, regardless of the presence of additional resistance mechanisms. It was also demonstrated that deletion of all described pumps significantly reduces the frequency of emergence of fluoroquinolone-resistant mutant strains (18). On the basis of these genetic data, it appears that inhibition of efflux pumps in P. aeruginosa may significantly improve the clinical performance of fluoroquinolones. Inhibition of efflux pumps is expected to (i) decrease the level of intrinsic resistance, (ii) significantly reverse acquired resistance, and (iii) decrease the frequency of emergence of P. aeruginosa mutants highly resistant to fluoroquinolones. The same considerations can also be applied to gram-positive bacteria. Indeed, when reserpine (which was long ago identified as an inhibitor of the mammalian MDR pump, P-glycoprotein, and which was later found to inhibit MDRs from gram-positive bacteria) was added to the selection media, it suppressed the emergence of Staphylococcus aureus and Streptococcus pneumonia mutants resistant to ciprofloxacin (19, 20). Recently, several new inhibitors of the NorA pump from S. aureus which potentiated the activity of ciprofloxacin against both wild-type and NorA-overexpressing strains have been reported (21). These compounds decreased the MICs of ciprofloxacin up to fourfold (at concentrations ranging from 0.2 to 1.5 μg/ml) for strains of S. aureus overexpressing the NorA pump. Some of these compounds also inhibited efflux-mediated resistance in S. pneumoniae. Importantly, all compounds dramatically suppressed the frequency of emergence of ciprofloxacin-resistant strains when selection was performed with 1 μg of ciprofloxacin per ml (21). The potential benefits of broad-spectrum efflux pump inhibitors (EPIs) prompted us to screen our synthetic compound and natural product libraries to search for inhibitors of the Mex pumps from P. aeruginosa. Recently, we have described one such inhibitor, MC-207,110, and presented a description of a portion of our efforts to optimize the biological and physiochemical properties of this lead compound (41). Here we present an additional characterization of MC-207,110: evidence of its mode of action as an inhibitor of efflux pump activity, its potentiating effect of the activity of levofloxacin against both laboratory strains with multiple target mutations and recent clinical isolates of P. aeruginosa, and its ability to decrease the emergence of P. aeruginosa strains with clinically relevant resistance to levofloxacin.