Pseudomonas aeruginosa is intrinsically resistant to a variety of antimicrobials and can develop resistance during anti-pseudomonal chemotherapy both of which compromise treatment of infections caused by this organism. Resistance to multiple classes of antimicrobials (multidrug resistance) in particular is increasingly common in P. aeruginosa, with a number of reports of pan-resistant isolates treatable with a single agent, colistin. Acquired resistance in this organism is multifactorial and attributable to chromosomal mutations and the acquisition of resistance genes via horizontal gene transfer. Mutational changes impacting resistance include upregulation of multidrug efflux systems to promote antimicrobial expulsion, derepression of ampC, AmpC alterations that expand the enzyme's substrate specificity (i.e., extended-spectrum AmpC), alterations to outer membrane permeability to limit antimicrobial entry and alterations to antimicrobial targets. Acquired mechanisms contributing to resistance in P. aeruginosa include β-lactamases, notably the extended-spectrum β-lactamases and the carbapenemases that hydrolyze most β-lactams, aminoglycoside-modifying enzymes, and 16S rRNA methylases that provide high-level pan-aminoglycoside resistance. The organism's propensity to grow in vivo as antimicrobial-tolerant biofilms and the occurrence of hypermutator strains that yield antimicrobial resistant mutants at higher frequency also compromise anti-pseudomonal chemotherapy. With limited therapeutic options and increasing resistance will the untreatable P. aeruginosa infection soon be upon us?

An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant of Pseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5 mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, for example, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previously identified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretion of the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibility to tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for the ORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis of ORFC (oprK) revealed that its product is homologous to a class of outer membrane proteins involved in export. Similarly, the products of ORFA and ORFB exhibit homology to previously described bacterial export proteins located in the cytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug efflux contributes to multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multiple efflux) for ORFAB.

We have earlier described mexA-mexB-oprK, an operon involved in pyoverdine export in Pseudomonas aeruginosa, and suggested that the products of these genes also contribute to the active efflux of several antibiotics (K. Poole, K. Krebes, C. McNally, and S. Neshat, J. Bacteriol. 175:7363-7372, 1993). Recently the outer membrane component of this efflux system was shown to be OprM, rather than OprK (N. Gotoh and K. Poole, unpublished results). In the present study, the conclusion concerning the efflux activity of this system was confirmed and extended by the measurement of drug accumulation in intact cells. Thus, the steady-state accumulation levels of tetracycline and norfloxacin were increased in mexA and oprM null mutants. mexA and oprM null mutants also showed an increase in susceptibility to a wide variety of ␤-lactam antibiotics and an increase in the steady-state accumulation level of benzylpenicillin, indicating that the MexA-MexB-OprM pump also effluxes ␤-lactams. Furthermore, deenergization of the cytoplasmic membrane with a proton conductor always produced a strong increase in the accumulation level. Finally, a single-step mutant overproducing MexAB-OprM accumulated less tetracycline and chloramphenicol than the parent strain and was more resistant to a wide range of antimicrobial compounds, including ␤-lactams. These results support the notion that these proteins contribute to the intrinsic resistance of P. aeruginosa through the multidrug active efflux process.Pseudomonas aeruginosa shows significant degrees of intrinsic resistance to a wide variety of antimicrobial agents, including most ␤-lactams, tetracyclines, chloramphenicol, and fluoroquinolones. Although the outer membrane of this species has a very low nonspecific permeability to small, hydrophilic molecules (1, 26), this alone is insufficient to explain the degree of resistance observed (16), and an additional resistance mechanism must be postulated. With some ␤-lactams, hydrolysis of the drugs by the periplasmic ␤-lactamase can serve as this additional mechanism (see reference 18 for a discussion of this phenomenon in Escherichia coli). For other compounds that are not inactivated by wild-type cells, their active efflux out of the cell may be the most likely second contributing factor to resistance (17). Recently, a putative operon, mexA-mexB-oprK, which codes for the export of a siderophore, pyoverdine (20), was suggested to function also as a multidrug efflux pump because overexpression of this operon increased the resistance of P. aeruginosa to chloramphenicol, tetracycline, nalidixic acid, ciprofloxacin, and streptonigrin, and disruption of these genes made the mutants hypersusceptible to these agents (21). (The outer membrane component of this system, previously thought to be the OprK protein seen in the multidrug-resistant strain K385 [21], was shown recently [5], however, to be identical to the previously described protein OprM [12]. Thus, the operon contains genes mexA, mexB, and oprM rather than oprK.) An independent study also show...

Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.

Multiresistance in Gram-negative pathogens, particularly Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Acinetobacter spp. and the Enterobacteriaceae, is a significant problem in medicine today. While multiple mechanisms often contribute to multiresistance, a broadly distributed family of three-component multidrug efflux systems is an increasingly recognised determinant of both intrinsic and acquired multiresistance in these organisms. Homologues of these efflux systems are also readily identifiable in the genome sequences of a wide range of Gram-negative organisms, pathogens and non-pathogens alike, where they probably promote efflux-mediated resistance to multiple antimicrobials. Significantly, these systems often accommodate biocides, raising the spectre of biocide-mediated selection of multiresistance in Gram-negative pathogens. While there is some debate as to the natural function of these efflux systems, only some of which are inducible by their antimicrobial substrates, their contribution to resistance in a variety of pathogens nonetheless makes them reasonable targets for therapeutic intervention. Indeed, given the incredible chemical diversity of substrates accommodated by these efflux systems, it is likely that many novel or yet to be discovered antimicrobials will themselves be efflux substrates and, as such, efflux inhibitors may become an important component of Gram-negative antimicrobial therapy.