SUMMARY The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

Drug efflux pumps play a key role in drug resistance and also serve other functions in bacteria. There has been a growing list of multidrug and drug-specific efflux pumps characterized from bacteria of human, animal, plant and environmental origins. These pumps are mostly encoded on the chromosome although they can also be plasmid-encoded. A previous article (Li X-Z and Nikaido H, Drugs, 2004; 64[2]: 159–204) had provided a comprehensive review regarding efflux-mediated drug resistance in bacteria. In the past five years, significant progress has been achieved in further understanding of drug resistance-related efflux transporters and this review focuses on the latest studies in this field since 2003. This has been demonstrated in multiple aspects that include but are not limited to: further molecular and biochemical characterization of the known drug efflux pumps and identification of novel drug efflux pumps; structural elucidation of the transport mechanisms of drug transporters; regulatory mechanisms of drug efflux pumps; determining the role of the drug efflux pumps in other functions such as stress responses, virulence and cell communication; and development of efflux pump inhibitors. Overall, the multifaceted implications of drug efflux transporters warrant novel strategies to combat multidrug resistance in bacteria.

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...

OprJ, overproduced in nfxB multidrug-resistant strains of Pseudomonas aeruginosa, and OprK, overproduced in the multidrug-resistant strain K385, were demonstrated to be immunologically cross-reactive using an OprJ-specific monoclonal antibody. Treatment of the purified proteins with trypsin or chymotrypsin yielded virtually indistinguishable digestion patterns, and the N-terminal sequence of two trypsin fragments was identical for both proteins, indicating that OprJ and OprK share identity. The N-terminal amino acid sequences were used to facilitate cloning of the oprJ gene on a 5kbp Kpnl fragment and a 10 kbp BamHl fragment. Nucleotide sequencing of portions of these fragments revealed that oprJ was the terminal gene in a putative three-gene operon, mexC-mexD-oprJ. The predicted mexC-mexD-oprJ gene products exhibit homology to the MexA-MexB-OprM components of the multidrug-resistance efflux pump of P. aeruginosa (43-46% identity). Consistent with an implied role for mexC-mexD-oprJ in drug efflux, the mexC-mexD-oprJ-hyperexpressing strain K385 showed reduced accumulation of a variety of antibiotics as compared with its parent strain, and this drug 'exclusion' was abrogated by energy inhibitors. The mexC and oprJ products are putative lipoproteins of a molecular mass of 40,707 and 51,742 Da, respectively, while mexD was predicted to encode a protein of 111 936 Da. Sequencing upstream of mexC revealed the presence of the nfxB gene transcribed divergently from the efflux genes. Overproduction of OprJ and the attendant multiple-antibiotic resistance of strain K385 was shown to result from a point mutation in nfxB, resulting in a H87-->R change in the predicted NfxB polypeptide. OprJ overproduction and multidrug resistance in K385 was reversed by the cloned nfxB gene, suggesting that nfxB encodes a repressor of mexC-mexD-oprJ expression. Consistent with this, the cloned nfxB gene repressed synthesis of a mexC-lacZ fusion in Escherichia coli. nfxB also repressed expression of a nfxB-lacZ fusion, indicating that NfxB negatively regulates its own expression. These data indicate that the multidrug resistance of nfxB strains is due to overexpression of an efflux operon, mexC-mexD-oprJ, encoding components of a second efflux pump in P. aeruginosa.

Antibiotic resistance and associated genes are ubiquitous and ancient, with most genes that encode resistance in human pathogens having originated in bacteria from the natural environment (eg, β-lactamases and fluoroquinolones resistance genes, such as qnr). The rapid evolution and spread of "new" antibiotic resistance genes has been enhanced by modern human activity and its influence on the environmental resistome. This highlights the importance of including the role of the environmental vectors, such as bacterial genetic diversity within soil and water, in resistance risk management. We need to take more steps to decrease the spread of resistance genes in environmental bacteria into human pathogens, to decrease the spread of resistant bacteria to people and animals via foodstuffs, wastes and water, and to minimize the levels of antibiotics and antibiotic-resistant bacteria introduced into the environment. Reducing this risk must include improved management of waste containing antibiotic residues and antibiotic-resistant microorganisms.

Most strains of Pseudomonas aeruginosa are significantly more resistant, even in the absence of R plasmids, to many antimicrobial agents, including P-lactams, tetracycline, chloramphenicol, and fluoroquinolones, than most other gram-negative rods. This broad-range resistance has so far been assumed to be mainly due to the low permeability of the P. aeruginosa outer membrane. The intrinsic-resistance phenotype becomes further enhanced in "intrinsically carbenicillin-resistant" isolates, which were often assumed to produce outer membranes of even lower permeability. It has been shown, however, that this hypothesis cannot explain the f-lactam resistance of these isolates (D. M. Livermore and K. W. M. Davy, Antimicrob. Agents Chemother. 35:916-921, 1991). In this study, we examined the uptake of tetracycline, chloramphenicol, and norfloxacin by intact cells using strains showing widely different levels of intrinsic resistance. Their accumulation and the response to the addition of a proton conductor showed that even relatively susceptible strains of P. aeruginosa actively pump out these compounds from the cell and that the efflux activity becomes much stronger in strains showing higher levels of intrinsic resistance. We conclude that the efilux mechanism(s) are likely to contribute significantly to the intrinsic resistance of P. aeruginosa isolates to tetracycline, chloramphenicol, and fluoroquinolones, as does the low permeability of the outer membrane. This conclusion is supported by the observation that the hypersusceptibility to various agents of the mutant K799/61 (W. Zimmermann, Antimicrob. Agents Chemother. 18:94-100, 1980) was apparently caused by the lack of active eftlux. Although the hypersusceptibility of this mutant has hitherto been assumed to be solely due to its higher outer membrane permeability, its outer membrane was shown to have a coefficient of permeability to cephaloridine that was not significantly different from that of the parent, resistant strain K799/WT. The strains with elevated intrinsic resistance overproduced two cytoplasmic membrane proteins and one outer membrane protein; at least two of these proteins appeared different from the proteins overproduced in the recently described mutant with a derepressed multidrug efilux system, MexA-MexB-OprK (K. Poole, K. Krebes, C. McNally, and S. Neshat, J. Bacteriol. 175:7363-7372, 1993).It is well known that most strains of Pseudomonas aeruginosa show significant degrees of intrinsic resistance to a wide variety of antimicrobial agents, including most ,-lactams, tetracyclines, chloramphenicol, and fluoroquinolones. The outer membrane of this species shows a very low nonspecific permeability to small, hydrophilic molecules (1, 46), and this has generally been thought to be the main cause of the resistance of this organism.Nevertheless, the low outer membrane permeability cannot be the entire explanation of the intrinsic resistance. First, theoretical analysis shows that even the low-permeability outer membrane of this species should allow a suf...

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Silver-resistant mutants were selected by stepwise exposure of silver-susceptible clinical strains of Escherichia coli, two of which did not contain any plasmids, to either silver nitrate or silver sulfadiazine. These mutants showed complete cross-resistance to both compounds. They showed low-level cross-resistance to cephalosporins and HgCl 2 but not to other heavy metals. The Ag-resistant mutants had decreased outer membrane (OM) permeability to cephalosporins, and all five resistant mutants tested were deficient in major porins, either OmpF or OmpF plus OmpC. However, the well-studied OmpF-and/or OmpC-deficient mutants of laboratory strains K-12 and B/r were not resistant to either silver compound. Resistant strains accumulated up to fourfold less 110m AgNO 3 than the parental strains. The treatment of cells with carbonyl cyanide m-chlorophenylhydrazone increased Ag accumulation in Ag-susceptible and -resistant strains, suggesting that even the wild-type Ag-susceptible strains had an endogenous Ag efflux activity, which occurred at higher levels in Ag-resistant mutants. The addition of glucose as an energy source to starved cells activated the efflux of Ag. The results suggest that active efflux, presumably coded by a chromosomal gene(s), may play a major role in silver resistance, which is likely to be enhanced synergistically by decreases in OM permeability.Silver (Ag) is a biologically nonessential metal which is used widely in photographic emulsions. The toxicity of Ag has also led to clinical applications, including the topical treatment of bacterial infections with silver nitrate (AgNO 3 ) and silver sulfadiazine (AgSD). The action of AgNO 3 and AgSD is assumed to be dependent on the Ag ϩ ions, which strongly inhibit bacterial growth through poisoning of respiratory enzymes and electron transport components and through interference with DNA functions (3, 18).Ag-resistant bacterial strains have been isolated from both clinical and environmental sources. Examples include strains of Acinetobacter baumannii (4), Escherichia coli (7), Enterobacter cloacae (7), Klebsiella pneumoniae (7), Pseudomonas aeruginosa (19), Pseudomonas stutzeri (5), and Salmonella typhimurium (15). In some cases the resistance was shown to be encoded by plasmids (4,5,15,27,31).Mechanisms of resistance to other metals such as arsenic, cadmium, copper, and mercury have been elucidated at the molecular level (for reviews, see references 3, 27, and 28). Resistance is sometimes due to enzymatic detoxification as in the case of mercury and organomercurials (28). But in a majority of cases energy-dependent ion efflux seems to be responsible, as exemplified by bacterial resistance to many oxyanions (such as arsenite, antimonite, and chromate) as well as to the cations zinc, cobalt, cadmium, and nickel (3, 27, 28). Solioz and Odermatt (30) have shown recently that a P-type copper efflux ATPase from a gram-positive bacterium, Enterococcus hirae, can also pump out, perhaps fortuitously, Ag ϩ . Little is known, however, about the mechanism of Ag...