Bacterial resistance to antibiotics: Active efflux and reduced uptake

Advanced Drug Delivery Reviews 57 (2005) 1486 – 1513 www.elsevier.com/locate/addr

Bacterial resistance to antibiotics: Active efflux and reduced uptake Ayush Kumar, Herbert P. Schweizer* Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA Received 7 December 2004; accepted 11 April 2005 Available online 4 June 2005

Abstract Antibiotic resistance of bacterial pathogens is a fast emerging global crisis and an understanding of the underlying resistance mechanisms is paramount for design and development of new therapeutic strategies. Permeability barriers for and active efflux of drug molecules are two resistance mechanisms that have been implicated in various infectious outbreaks of antibioticresistant pathogens, suggesting that these mechanisms may be good targets for new drugs. The synergism of reduced uptake and efflux is most evident in the multiplicative action of the outer membrane permeability barrier and active efflux, which results in high-level intrinsic and/or acquired resistance in many clinically important Gram-negative bacteria. This review summarizes the current knowledge of these two important resistance mechanisms and potential strategies to overcome them. Recent advances in understanding the physical structures, function and regulation of efflux systems will facilitate exploitation of pumps as new drug targets. D 2005 Elsevier B.V. All rights reserved. Keywords: Outer membrane permeability; Porins; Active efflux; RND pumps; Regulation

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bacterial drug efflux systems . . . . . . . . . . . . . . . . . . . 2.1. Major facilitator superfamily (MFS) . . . . . . . . . . . 2.2. ATP-binding cassette (ABC) superfamily . . . . . . . . . 2.3. Small multidrug resistance (SMR) family . . . . . . . . . 2.4. Resistance-nodulation-cell division (RND) superfamily . . 2.5. Multidrug and toxic compound extrusion (MATE) family 2.6. Efflux pumps in Gram-positive bacteria. . . . . . . . . . 2.7. Efflux pumps in Gram-negative bacteria . . . . . . . . . 2.8. Structure and function of RND efflux pumps . . . . . . .

* Corresponding author. Tel.: +1 970 491 3536; fax: +1 970 491 1815. E-mail address: [email protected] (H.P. Schweizer). 0169-409X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2005.04.004

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

1487 1487 1488 1489 1489 1489 1489 1490 1490 1495

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

2.9. Natural functions of RND pumps . . . . . . . . . 2.10. Regulation of RND pump gene expression. . . . . 3. Overcoming efflux . . . . . . . . . . . . . . . . . . . . . 4. Outer membrane permeability . . . . . . . . . . . . . . . 4.1. LPS and antibiotic resistance . . . . . . . . . . . . 4.2. Porins and antibiotic resistance . . . . . . . . . . . 4.3. Overcoming restricted outer membrane permeability 5. Summary and conclusions . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The discovery and use of antibiotics has been one of the major scientific achievements of the 20th century. During the early period of antibiotic usage, bacterial infections were considered tamed. Antibiotics were being used to cure potentially lethal infections. Infected cuts and wounds were no longer lifethreatening, and various bacterial diseases, such as syphilis and cholera, were considered on their way to eradication. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains [1–3]. Resistance is spreading rapidly, particularly in hospitals, where various bacteria can come in close contact with one another, spreading the resistance traits in the process. Since bacteria share resistance genes, nosocomial antibiotic resistance can spread to surrounding communities. The various antibiotic resistance mechanisms include alteration/modification of the target site, degradation of the antibiotic molecule and reduction of effective intracellular antibiotic concentration as a result of decreased permeability and energy-dependent (or active) efflux. Resistance genes are either carried on the chromosomes of wild-type bacteria or on elements of extachromosomal, sometimes extraneous origins, such a resistance (R) plasmids and transposons. In the mid-1970s, Pglycoprotein was the first example of an efflux pump implicated in the drug resistance of mammalian cancer cells [4] and antibiotic efflux as a resistance mechanism was first recognized for tetracycline in the late 1970s [5–8]. Since that time, efflux-mediated resistance to a wide range of antibacterial agents, including antibiotics, biocides and solvents, has been reported in many bacteria. Although some are

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

1487

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

1496 1497 1499 1500 1500 1501 1502 1502 1502 1502

drug-specific, many efflux systems accommodate multiple drugs and thus contribute significantly to bacterial intrinsic and acquired multidrug resistance (MDR). It is thus not surprising that over the past decade efflux-mediated drug resistance has been extensively studied as documented by an impressive body of literature, including many minireviews (cited throughout this review), reviews [9–12] and a book [13]. Although drug efflux pumps are found in Gram-negative and Gram-positive bacteria, effluxmediated resistance in Gram-negative bacteria is a more complex problem due to the molecular architecture of the cell envelope. As a consequence, drug resistance in many cases is attributable to synergy between reduced drug intake (mainly due to low outer membrane permeability) [14] and active drug export (via efflux pumps).

2. Bacterial drug efflux systems Drug efflux systems pump out a broad range of chemically and structurally unrelated compounds from bacteria in an energy-dependent manner, without drug alteration or degradation. Analysis of various available bacterial genome sequences has shown that known and putative drug efflux transporters constitute from 6% to 18% of all transporters found in any given bacterial cell [15]. Bacterial drug efflux transporters are currently classified into five families (Fig. 1): (1) the major facilitator superfamily (MFS) [16,17]; (2) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily [18]; (3) the small multidrug resistance (SMR) family [19]; (4) the resistance-nodulation-cell division (RND) superfamily [20]; and (5) the multidrug and

1488

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513 Drug

Outer Membrane

TolC

Drug H+ Drug

Drug

H+

AcrA

Na+ H+

Drug

AcrB NorA

Drug

EmrE H+

Drug

LmrA

NorM H+

Drug

Na+

H+

Drug

Drug ATP

MFS

SMR

MATE

RND

Plasma Membrane Cytoplasm ADP + Pi

ABC

Fig. 1. Schematic illustration of the main types of bacterial drug efflux pumps. Illustrated are Staphylococcus aureus NorA, a member of the major facilitator superfamily (MFS); Escherichia coli EmrE, a member of the small multidrug resistance (SMR) superfamily; Vibrio parahaemolyticus NorM, a member of the multidrug and toxic compound extrusion (MATE) superfamily; E. coli AcrAB–TolC, a member of the resistance-nodulation-cell division (RND) superfamily; and Lactococcus lactis LmrA, a member of the ATP-binding cassette (ABC) superfamily. All pumps extrude the substrate chemically unaltered and in an energy-dependent manner, using either an ion gradient (proton or Na+) or ATP. Although the drug is in many instances pumped from the cytoplasm (as depicted here), there is increasing evidence that RND pumps can also acquire substrates either directly from the periplasm or from the outer leaflet of the cytoplasmic membrane (not shown for clarity) [143,153]. Whereas the cytoplasmic membrane is a phospholipid bilayer, the outer membrane consists of a phospholipid inner leaflet and a lipid A-containing outer leaflet. Lipid A is the membrane-anchoring domain of LPS, which, together with porins, gives the outer membrane its characteristic permeability properties.

toxic compound extrusion (MATE) family [21]. Of these, the ABC and MFS families are very large and the other three are smaller families. Efflux transporters can be further classified into single- or multi-component pumps. Single component pumps transport their substrates across the cytoplasmic membrane. Multicomponent pumps, found in Gram-negative organisms, function in association with a periplasmic membrane fusion protein (MFP) component and an outer membrane protein (OMP) component, and efflux substrates across the entire cell envelope. In most instances multidrug efflux transporters are chromosomally encoded and therefore not readily transferable between bacteria. There are, however, examples in both Gram-positive and Gram-negative bacteria where these genes are found on mobile elements [22]. 2.1. Major facilitator superfamily (MFS) The MFS family is an ancient, large and diverse superfamily that includes more than a thousand sequenced members. Members of this family either catalyze uniport, solute/cation (H+ or Na+) symport,

solute/H+ antiport or solute/solute antiport and are involved in transport of sugars, metabolites, anions and drugs. These transporters usually function as single-component pumps, e.g., NorA of Staphylococcus aureus [23]. However, in some Gram-negative bacteria they function with membrane fusion proteins (MFPs) and outer membrane protein (OMP) components, e.g., EmrAB–TolC of E. coli [24]. The inner membrane proteins of the drug pumps belonging to this superfamily usually contain 12- or 14-transmembrane segments (TMS) [16]. The MFS proteins that catalyze drug efflux are from three subfamilies, drug/H+ antiporter (DHA) 1 (e.g., Bmr of Bacillus subtilis), DHA2 (e.g., QacA of S. aureus [25,26]) and DHA3 (e.g., MefA of Streptococcus pyogenes [27]). The DHA1 and DHA2 family of proteins are ubiquitous among prokaryotes and eukaryotes, and are known to efflux a very broad range of structurally distinct drugs. Members of the DHA1 family export sugars, polyamines, uncouplers, monoamines, acetylcholine, paraquat and methylglyoxal. In contrast, members belonging to the DHA2 family exhibit a more restricted substrate specificity, and transported substrates include bile salts and dyes.

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

Members of the DHA3 family are only found in prokaryotes, and are known to efflux antibiotics, including macrolides and tetracycline. Tetracycline efflux pumps constitute some of the best-characterized members of the MFS family. These pumps are found in both Gram-negative and Gram-positive bacteria. Most of them confer resistance to tetracycline, but not to minocycline or glycylcyclines. However, some Gram-negative Tet proteins confer resistance to both tetracycline and minocycline, but not to glycylcyclines [28]. 2.2. ATP-binding cassette (ABC) superfamily The ABC transporter superfamily contains both uptake and efflux transport systems. Members of this superfamily use energy derived from ATP hydrolysis to transport a variety of substances including sugars, amino acids, ions, drugs, polysaccharides and proteins [29,30]. Transporters of the ABC superfamily are multi-protein complexes consisting of integral membrane proteins (presumably forming a transport pore through the cytoplasmic membrane) and cytoplasmic proteins with ATPase activity. Bacterial ABC permeases generally contain 6 TMS each and associate in the membrane in pairs as either homo- or hetero-dimers. Two ATPase subunits associate with the permeases on the cytoplasmic face of the inner membrane to form a functional transporter. Drug efflux pumps belonging to the ABC-superfamily are rare in bacteria, with the LmrA pump of Lactococcus lactis probably being the best-studied example [31]. The current list of exporters belonging to the ABC superfamily includes 21 prokaryotic efflux systems [32]. 2.3. Small multidrug resistance (SMR) family Fitting their nomenclature, SMR family transporters consist of approximately 110 amino acid residues and contain 4 TMS and are energized by the protonmotive force. Due to their small size, SMR proteins were initially believed to function as trimers [19]. However, a recent study has shown that these proteins actually exist as tetramers [33]. Some of the wellcharacterized pumps of this family include the Smr pump of S. aureus [34] and the EmrE pump of E. coli [35], which efflux dyes, drugs and cations.

1489

2.4. Resistance-nodulation-cell division (RND) superfamily It was originally thought that proteins belonging to the RND superfamily were only found in eubacteria. However, they have now also been reported in eukaryotes and archaea [36]. RND drug transporters are typically encoded by chromosomal genes, but a plasmid-encoded RND drug transporter has recently been reported [37]. All members of the RND family characterized to date catalyze substrate efflux via a substrate/ H+ antiport mechanism. RND pumps play an important role in acquired and intrinsic resistance of Gram-negative bacteria to a variety of antimicrobials and all RND pumps studied to date are multidrug transporters. In Gram-negative bacteria, RND pumps function by forming complexes consisting of an RND membrane transport protein with 12 TMS, a MFP and an OMP. A characteristic feature of RND transporter topology is the presence of 2 large periplasmic loops between TMS 1 and 2 and TMS 7 and 8. The N-terminal halves of RND family proteins are homologous to the Cterminal halves and, as such, these proteins are believed to have arisen from an intragenic tandem duplication event that occurred before the divergence of the family members. There is, however, one example of an RND homologue from Mycobacterium jannaschii that has only 6 TMS and no internal duplication [36]. It is possible that this protein functions either as a homodimer or as a hetero-dimer, or by association with another protein. The best-studied members of these pumps are the AcrAB–TolC system of E. coli [38,39] and the MexAB–OprM system of Pseudomonas aeruginosa [40] that are known to efflux antibiotics, heavy metals, dyes, detergents, solvents, plus many other substrates. A more detailed description of Gram-negative RND pumps will follow in Section 2.7. 2.5. Multidrug and toxic compound extrusion (MATE) family Previously thought to be members of the MFS superfamily, the proteins belonging to the MATE family are now recognized as a separate family of transporters because, in spite of similar membrane topology, they show no sequence homology to MFS proteins. Examples of proteins belonging to this family include NorM of Vibrio parahaemolyticus and its

1490

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

homologue, YdhE of E. coli [41]. These proteins are approximately 450 amino acid residues in length and contain 12 TMS. Proteins belonging to this family use the Na+ gradient as the energy source to efflux cationic dyes and fluoroquinolones [41]. 2.6. Efflux pumps in Gram-positive bacteria The cell envelope of Gram-positive organisms shows a relatively simple structure when compared to that of Gram-negative organisms with the presence of a cytoplasmic membrane, which is surrounded by a thick layer of peptidoglycan. Therefore, efflux pumps of Gram-positive bacteria have only a single component located in the cytoplasmic membrane (Fig. 1). Despite the absence of an outer membrane diffusion barrier, drug-specific and multidrug efflux pumps have been described in Gram-positive bacteria, some of which make important contributions especially to macrolide and fluoroquinolone efflux. Some of the more important Gram-positive drug efflux pumps will therefore be briefly discussed in the following paragraphs. 2.6.1. S. aureus S. aureus is a major cause of hospital-acquired infections [42,43]. Efflux pumps characterized in this organism include QacA (MFS family) [19,25], Smr (SMR family) [34] and NorA (MFS family) [23]. QacA and Smr are examples of plasmid-encoded efflux pumps, while NorA is chromosomally encoded. QacA has been shown to efflux acriflavine, crystal violet, diamidines, ethidium bromide, and quaternary ammonium compounds [44]. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus [23], due to a weakly expressed norA gene [45]. This pump is responsible for resistance of S. aureus to hydrophilic fluoroquinolones only and does not extrude lipophilic substrates [46]. 2.6.2. Streptococcus pneumoniae S. pneumoniae is a major cause of respiratory tract infections. Some isolates are resistant to a wide range of antibiotics that include h-lactams, macrolides, quinolones and tetracycline [47]. Efflux mechanisms have been shown to play a role in quinolone resistance of this organism, though target mutations are believed to be the main contributors to this resistance [48,49].

The PmrA pump of S. pneumoniae is 24% identical to the NorA pump of S. aureus [50] and its expression causes a 2- to 4-fold increase in resistance to several fluoroquinolones. PmrA knockouts do not exhibit changes in antibiotic susceptibity patterns, suggesting that this pump is not expressed in the wild-type strains [50]. Macrolide resistance in S. pneumoniae is conferred by the MefE pump of the MFS family [51]. MefE [52] is 90% identical to MefA of S. pyogenes, and together these two pumps are referred to as Mef(A). They have been shown to efflux both 14and 15-membered macrolides and are responsible for approximately 70% of the macrolide resistance of S. pneumoniae observed in the United States [52]. The mef(A) gene of S. pneumoniae is part of a mobile element that is transferable by transformation [53]. 2.6.3. B. subtilis Two different MFS-type efflux pumps have been identified and characterized in B. subtilis: Bmr [54] and Blt [55]. Despite the fact that B. subtilis is not a clinically important organism, these two pumps provide excellent model systems for mechanistic studies of MFS-type multidrug efflux systems. Substrates for these two pumps include fluoroquinolones, ethidium bromide and energy inhibitors [56]. 2.7. Efflux pumps in Gram-negative bacteria Although Gram-negative bacteria contain efflux pumps representing the five superfamilies, RND pumps are most prominent in these bacteria. They not only play a major role in both intrinsic and acquired resistance of many Gram-negative bacteria to a variety of clinically significant antibiotics, but also export biocides, dyes, detergents and organic solvents (Table 1). These tripartite pumps span the entire Gram-negative cell envelope and are thus uniquely suited to synergize with reduced outer membrane permeability (uptake) to impart drug resistance. This may be the main reason why fewer non-RND family MDR efflux systems promote resistance to clinically relevant antibiotics. Notable exceptions may be the following: NorA (MFS) in Bacteroides fragilis [57]; MdfA (MFS) [58–60], MacAB–TolC (ABC) [61] and Dep (MFS) [62] and YhdE [41] in E. coli; NorM (MATE) in V. parahaemolyticus [41]; BcrA (MFS) in Burkholderia cepacia [63]; NorM

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

1491

Table 1 RND Family multidrug efflux systems of Gram-negative bacteriaa Organism

Efflux components

Regulator(s)

Substratesb

Reference

AG, CM, EB, FQ, NO, TC, TM CB, DC, NO, SDS CO ? CM, FQ, TM AG, ML AC, AG, ML AP, CM, CT, EB, EM, NA, FQ, TC, SDS AC, CM, FQ, MC, NO, SDS, TC FQ AC, BL, BS, CM, CV, EB, FA, FQ, ML, NO, OS, RF, SDS, TX AG, DC, FU, NO AC, BS, FQ, SDS, TX DC, NO CV, DC, NO, SDS AC, CV, EB, EM, NO, SDS FQ

[132] [273] [274] [275] [115] [119] [120] [141]

CV, EB, FA, TX FA, TX AC, EB, RF, SDS AC, EB, NO, SDS AC, AG, BL, CM, CV, EB, ML, NO, SDS, TC, TM, TR CM, CP, FQ, TC, TR CM, FQ AC, EB, HL?, NO, RD, Vanadium EM, TC, TR

[125,280] [281] [282] [283] [40,82,284]

AC, CM, EB, ER, FQ, TC AG, ML, TC OS OS OS OS AC, BS, BL, CH, CM, CV, DC, EM, FA, FQ, NA, NO, RF, SDS, TC, TX EB, CM, FQ, OS ? AC, EB, FQ, TC AG, BL, FQ EM, FQ, OS, TC

[285] [76,108,110] [286] [286–288] [289] [290] [11,291,292]

MFP

RND

OMP

Campylobacter jejuni

AdeA AmeA IfeA RagD CeoA AmrA BpeA CmeA

AdeB AmeB IfeB RagC CeoB AmrB BpeB CmeB

AdeC AmeC ? ? OpcM OprA OprB CmeC

AdeT, AdeSR AmeR IfeR RagAB ? AmrA BpeR(?) ?

Enterobacter aerogenes Enterobacter cloacae Escherichia coli

AcrA ? AcrA

AcrB AcrB AcrB

TolC ? TolC

AcrA AcrE MdtA YhiU AcrA AcrA

AcrD AcrF MdtBC YhiV AcrB AcrB

TolC TolC TolC TolC TolC ?

AcrR ? AcrR, MarA, SoxS, Rob, SdiA ? AcrS BaeSR EvaAS ? AcrR

MtrC FarA XepA AcrA MexA

MtrD FarB XepB AcrB MexB

MtrE MtrE XepC ? OprM

MtrR, MtrA ? ? AcrA MexR

MexC MexE MexH

MexD MexF MexI

OprJ OprN OpmD

NfxB MexT ?

MexJ

MexK

MexL

MexV MexX SrpA TgtA TgtD TgtG AcrA

MexW MexY SrpB TgtB TgtE TgtH AcrB

OprM/ OpmH OprM OprM SrpC TgtC TgtF TgtI TolC

? MexZ SrpSR TgtR ? ? AcrR

SdeA SdeC SdeX SmeA SmeD

SdeB SdeDE SdeY SmeB SmeE

? ? ? SmeC SmeF

? ? ? SmeSR SmeT

Acinetobacter baumannii Agrobacterium tumefaciens Bradyrhizobium japonicum Burkholderia cepacia Burkholderia pseudomallei

Haemophilus influenzae Klebsiella pneumoniae and K. oxytoca Neisseria gonorrhoeae Porphyromonas gingivalis Proteus mirabilis Pseudomonas aeruginosa

Pseudomonas putida

Salmonella enterica serovar Typhimurium Serratia marcescens

Stenotrophomonas maltophila

[138] [276] [38,277] [68,278] [169] [72,73] [176] [279] [139]

[74] [75] [78,113] [77]

[129,293] [129] [130] [122] [123,170]

a Only characterized efflux pumps are listed; those systems for which chromosomal annotations and presumed functions are available are not listed here, but some can be found in Ref. [11]. b AC, acriflavine; AG, aminoglycosides; AP, amplicillin; BL, h-lactams; BS, bile salts; CB, carbenicillin; CH, cholate; CM, chloramphenicol; CO, coumestrol; CP, cephalosporins; CT, cefotaximine; CV, crystal violet; DC, deoxycholate; EB, ethidium bromide; EM, erythromycin; FA, fatty acids; FQ, fluoroquinolones; FU, fusidic acid; HL, homoserine lactones; MC, mitomycin; ML, macrolides; NA, nalidixic acid; NO, novobiocin; OS, organic solvents; RD, rhodamine; RF, rifampicin; SDS, sodium dodecyl sulfate; TC, tetracycline; TM, trimethoprim; TR, triclosan; TX, Triton X-100; ?, unknown.

1492

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

(MATE) in Burkholderia vietnamensis [64]; and VcmA (MATE) in Vibrio cholerae [65] (for a listing of the substrates extruded by these systems see Table 3 in Ref. [12]). In the following we will therefore limit our remarks on RND systems, especially those involved in efflux of clinically significant antibiotics, and the reader is referred to the recent comprehensive reviews by Poole [10] and Li and Nikaido [12] describing the general role of the other types of drug efflux systems in conferring antimicrobial resistance on various bacteria. 2.7.1. RND pumps of E. coli Analysis of the E. coli genome has revealed the presence of seven RND transporters [66]. To date, five of these have been functionally characterized and confirmed to participate in drug efflux: AcrAB, AcrEF, AcrD, YhiUV and MdtABC. All E. coli RND pumps studied so far have been found to associate with the TolC OMP channel. The AcrAB–TolC system has been identified as the predominant drug efflux pump of this organism [67], and it is the bestcharacterized RND pump to date. Since knockout experiments with acrEF, yhiUV and mdtABCD did not change drug susceptibilities of the wild-type E. coli cells, this suggests that these pumps do not play a significant role in the antimicrobial resistance of this organism [67]. The AcrAB–TolC system demonstrates very broad substrate specificity. Substrates for this pump include acriflavine, h-lactams, bile salts, chloramphenicol, crystal violet, ethidium bromide, fatty acids, macrolides, organic solvents, fluoroquinolones and SDS. The AcrD pump was originally believed to function as a single component pump for the efflux of aminoglycosides [68], but more recent data showed that it actually requires AcrA and TolC to efflux bile salts, novobiocin and aminoglycosides [69]. The AcrEF pump is not expressed in wild-type cells, but is expressed in fluoroquinolone-resistant mutants that lack the AcrAB pump [70]. This system also confers resistance to solvents in E. coli [71]. Interestingly, the AcrF protein was shown to function with AcrA and TolC for solvent efflux [71], suggesting the components of the RND complex may be interchangeable. When overexpressed, the YhiUV pump is responsible for resistance to doxorubicin, erythromycin, deoxycholate and crystal violet, while the MdtABC

system confers resistance to bile salts and novobiocin [72,73]. Interestingly, the MdtABC system contains two different RND transporters, MdtB and MdtC, and both are required for drug extrusion. An MFS transporter-encoding gene, mdtD, has been found downstream of the mdtABC operon, but does not appear to play a role in antibiotic resistance [72]. 2.7.2. RND pumps of P. aeruginosa The first RND-type efflux pump reported in P. aeruginosa was MexAB–OprM [40]. Analysis of the P. aeruginosa genome has since revealed the presence of 12 RND pump-encoding operons, 7 of which have been characterized to date: MexAB–OprM, MexCD– OprJ [74], MexEF–OprN [75], MexXY–OprM [76], MexJK–OprM [77], MexGHI–OpmD [78] and MexVW–OprM [79]. Of these, MexAB–OprM, MexCD–OprJ, MexEF–OprM and MexXY–OprM are the major efflux systems contributing to intrinsic and acquired multidrug resistance. The other Mex systems are probably of more limited clinical significance because they have not yet been shown to be expressed in clinical isolates and export only a limited number of clinically significant antibiotics. Unlike E. coli, where only one OMP has been found to be working in conjunction with different pumps, at least 18 OprM homologs are known to be present and expressed to various degrees in P. aeruginosa [80]. 2.7.2.1. MexAB–OprM. The MexAB–OprM system of P. aeruginosa contributes to the antimicrobial resistance of wild-type strains and has the broadest substrate range of all characterized P. aeruginosa efflux pumps. Substrates of this pump include hlactams, h-lactamase inhibitors, quinolones, macrolides, tetracyclines, chloramphenicol, novobiocin, sulfonamides, trimethoprim and thiolactomycin [81–85], as well as non-antibiotic compounds including dyes, detergents, triclosan and organic solvents [85–87]. The export of h-lactams by the MexAB–OprM system is intriguing, as efflux-mediated resistance to this group of antibiotics in not very common. In some reports this pump has been shown to play a more important role than the AmpC h-lactamase of P. aeruginosa [88,89]. Of the h-lactams, only carbapenems appear to be poor substrates for MexAB–OprM. Clinical isolates of P. aeruginosa that overexpress the MexAB–OprM system are very common. One

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

study from British hospitals revealed that more than 80% of carbenicillin resistant isolates of this organism were overexpressing the MexAB–OprM system [90]. Similarly, one-third of ticarcillin resistant P. aeruginosa clinical isolates overexpressed MexAB–OprM [91], as did many multidrug resistant clinical veterinary isolates [92]. These studies suggest that exposure of P. aeruginosa to antibacterials can readily result in multidrug resistant strains, and that the MexAB– OprM system appears to be the major contributor to this phenotype. Overexpression of MexAB–OprM is typically the result of mutations in the adjacent repressor gene, mexR [93,94], but mexAB–oprM hyperexpression independent of mutations in mexR has also been reported [92,95,96]. 2.7.2.2. MexCD–OprJ. MexCD–OprJ is not expressed in wild-type P. aeruginosa [74], but is overexpressed in strains carrying nfxB mutations. This results in resistance to quinolones, tetracycline, chloramphenicol, acriflavine, ethidium bromide, triclosan and organic solvents [74,86,97]. P. aeruginosa nfxB mutants can be divided into two categories, A and B. A-type mutants are resistant to ofloxacin, erythromycin and some new cephalosporins, and type B mutants are resistant to tetracycline and chloramphenicol, in addition to the agents mentioned for type A mutants. It has been observed that type B nfxB mutants are four to eight times more susceptible to many penicillins, carbapenems and aminoglycosides than the wild-type strain of P. aeruginosa [98]. This may be due to down-regulation of the MexAB–OprM system [99] and AmpC h-lactamase [100] in MexCD–OprJ overexpressing mutants. In vivo selection of mutants overexpressing MexCD–OprJ could be achieved by exposure to newer fluoroquinolones, such as trovafloxacin [101]. MexCD–OprJ expression was shown to be inducible by the non-antibiotic compounds ethidium bromide, rhodamine 6G and acriflavine [102], as well as the clinically important disinfectants benzalkonium chloride and chorhexidine gluconate [103]. The latter finding suggests that clinically relevant biocides can result in the overexpression of MexCD–OprJ, thus contributing to multidrug resistance. 2.7.2.3. MexEF–OprN. The MexEF–OprN system is overexpressed in nfxC-type multidrug resistant strains, where it is responsible for resistance to fluoroquino-

1493

lones, tetracycline, chloramphenicol and trimethoprim [75]. NfxC mutants are also resistant to imipenem, although this is more likely due to a decrease in the expression of oprD [104], an outer membrane protein channel that acts as the passage for imipenem entry in P. aeruginosa, since expression of OprD has been shown to decline with the overexpression of MexEF–OprN [105]. The hypersusceptibility of nfxC strains to h-lactams and aminoglycosides [106] may result from decreased expression of MexAB–OprM and MexXY–OprM, as suggested for the nfxB mutants. In vitro and in vivo experiments involving exposure of P. aeruginosa to fluoroquinolones yielded mutants that overexpress the MexEF–OprN system [101,107], and clinical isolates were also shown to overexpress MexEF–OprN [92]. 2.7.2.4. MexXY. In contrast to the operons encoding the MexAB–OprM, MexCD–OprJ and MexEF–OprN systems, the mexXY operon does not contain an OMPencoding gene [76]. Instead, the MexXY system utilizes OprM as its outer membrane component [76,108]. Deletion of the mexXY genes results in increased susceptibility of P. aeruginosa strains to aminoglycosides, tetracycline and erythromycin [108]. Interestingly, when mexXY is expressed in E. coli, it promotes resistance to fluoroquinolones, although this efflux system does not contribute to intrinsic resistance to these agents in P. aeruginosa [76,108]. Overexpression of the MexXY–OprM pump has been observed in several impermeabilitytype aminoglycoside-resistant strains of P. aeruginosa, a result of inducible expression of mexXY by aminoglycosides [109], with deletion of mexY compromising this resistance [110]. A study of various aminoglycoside-resistant clinical isolates of P. aeruginosa demonstrated elevated levels of mexXY expression [92,111]. Contribution of this pump to aminoglycoside resistance of P. aeruginosa was also highlighted in a recent study in which aminoglycoside resistant isolates from cystic fibrosis (CF) patients were found to overexpress MexXY, accompanied with mutations in mexZ, the gene for the repressor of this system. Some non-CF isolates that were overexpressing MexXY, however, were found to exhibit mexZ-independent overexpression, suggesting the presence of an alternate mechanism in the up-regulation of this pump [112].

1494

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

2.7.2.5. MexJK. The MexJK pump exhibits one of the most restricted substrate profiles of all studied P. aeruginosa RND pumps, effluxing only triclosan, erythromycin and tetracycline [77]. This pump was identified as a result of exposure of P. aeruginosa to triclosan, an antimicrobial commonly used in various household products. This serves as an example where commonly used antimicrobial household products can result in multiple antibiotic-resistance phenotypes in this, and possibly other, clinically relevant organisms. A unique feature of this pump is that it has been shown to require OprM for extruding erythromycin and tetracycline [77], but requires OpmH for the efflux of triclosan (Chuanchuen et al., unpublished data). Clinical isolates expressing MexJK have not yet been described. 2.7.2.6. MexGHI–OpmD and MexVW. The MexGHI–OpmD system contains a MFP (MexH), a RND transporter (MexI) and an OMP channel (OpmD). This pump also contains a small integral membrane protein, MexG, whose function is unknown [78]. One study showed that the MexGHI–OpmD system is active in wild-type cells and confers mainly vanadium resistance [78]. However, a different study showed that this efflux system also confers resistance to norfloxacin, ethidium bromide, acriflavine and rhodamine 6G [113]. MexVW is the most recently characterized RND pump in P. aeruginosa and has been shown to work in conjunction with OprM [79]. MexVW confers resistance to fluoroquinolones, tetracycline, chloramphenicol, erythromycin, ethidium bromide and acriflavine [79]. To date, neither of these pumps has been shown to be of clinical relevance. 2.7.3. RND pumps from other Gram-negative bacteria Besides E. coli and P. aeruginosa, RND pumpmediated antibiotic efflux has also been reported for other clinically relevant bacteria. B. cepacia, originally identified as a plant pathogen, has recently emerged as an important human opportunistic pathogen, especially in cystic fibrosis patients [114]. This bacterium expresses the CeoAB–OpcM pump, which is highly homologous to MexAB–OprM of P. aeruginosa [115,116]. CeoAB–OpcM was found to efflux chloramphenicol, fluoroquinolones and trimethoprim. Burkholderia pseudomallei is the causative agent of melioidosis [117], which is difficult to treat because

of high intrinsic resistance of this organism to h-lactams, macrolides, polymyxin and aminoglycosides [118]. To date, two different RND pumps have been characterized in this bacterium, AmrAB–OprA [119] and BpeAB–OprB [120], and both confer resistance to aminoglycosides and, to a lesser extent, macrolides. Stenotrophomonas maltophila has emerged as an important nosocomial agent causing infections like endocarditis, bacteremia and septicemia [121]. The two RND pumps described to date in S. maltophila are SmeABC [122] and SmeDEF [123,124]. The SmeDEF system has been shown to efflux erythromycin, tetracyclines, macrolides, chloramphenicol and fluoroquinolones [123,124]. The SmeDEF system was found to be overexpressed in almost 50% of Spanish clinical isolates [124]. Interestingly, only SmeC, but not SmeAB of the SmeABC system is required for increased resistance to h-lactams, aminoglycosides and fluoroquinolones, suggesting that SmeC contributes to multidrug resistance as part of another, yet unidentified multidrug efflux pump [122]. Neisseria gonorrhoeae expresses the MtrCDE RND pump, which is involved in the efflux of antibiotics, dyes and detergents [125]. A study of clinical isolates of N. gonorrhoeae showed that the MtrCDE system was responsible for azithromycin and erythromycin resistance in at least 50% of the strains [126]. There is also evidence that the antibiotic resistance in N. gonorrhoeae rectal isolates is a function of overexpression of this system, likely a result of the selection of mtrCDE-expressing mutants by fatty acids and bile salts in the rectal environment [125]. Serratia marcescens has emerged as a notorious nosocomial agent, and incidents of multiple antibiotic resistance of this organism are fairly common [127,128]. Three different RND pumps, SdeAB, SdeCDE [129] and SdeXY [130], have been identified in this organism thus far. SdeAB was found to be overexpressed in a fluoroquinolone resistant clinical isolate of S. marcescens, and its expression was shown to be induced by exposure to fluoroquinolones in laboratory strains leading to a significant increase in resistance to fluoroquinolones, chloramphenicol, dyes and detergents [129]. SdeXY was shown to confer norfloxacin and tetracycline resistance [130], and appears to be a close homologue of the E. coli AcrAB–TolC pump. The substrate profile of SdeCDE, a homologue of the E. coli MdtABC system with two RND pumps, remains unknown. Acinetobacter bau-

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

mannii is a leading cause of nosocomial infections, particularly in intensive care units, and is implicated in various infections that include respiratory tract infections, bacteremia and meningitis [131]. This organism exhibits high resistance to various antibiotics, including h-lactams, aminoglycosides and quinolones. The first efflux pump to be identified from this organism was AdeABC, an aminoglycoside pump [132], which also confers resistance to fluoroquinolones, tetracyclines, chloramphenicol, erythromycin and trimethoprim. AdeDE, another recently identified pump, was shown to efflux amikacin, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, meropenem, rifampin and tetracycline [133]. Both of these pumps were identified in clinical isolates of A. baumannii, indicating the likely clinical significance of efflux-mediated resistance in this organism. Infections caused by Salmonella sp. are often fatal and antibiotic resistance of these organisms can compromise effective chemotherapy [134]. Fluoroquinolone resistance of Salmonella sp. can be explained by the presence of various efflux pumps. A homologue of the E. coli AcrAB pump identified in S. enterica serovar Typhimurium was found to be responsible for resistance to fluoroquinolones, tetracycline, chloramphenicol, carbenicillin and cefoxitin [135]. Subsequently, homologues of the AcrD and AcrEF pumps have also been identified in this organism [136,137]. The AcrAB–TolC pump found in E. aerogenes was found to be responsible for resistance of this organism to chloramphenicol, fluoroquinolones and tetracycline [138]. A study of clinical isolates of Klebsiella pneumoniae and K. oxytoca revealed overexpression of the AcrAB efflux system in these strains [139]. Campylobacter jejuni is the most common cause of bacterial human gastroenteritis worldwide [140]. To date, two different RND pumps CmeABC [141] and CmeDEF (Pumbwe and Piddock, unpublished data) have been demonstrated in this bacterium. The CmeABC pump confers resistance to fluoroquinolones, bile salts, ethidium bromide and heavy metals [141]. 2.8. Structure and function of RND efflux pumps The crystal structures of the three components of a tripartite RND pumps have now been solved: the TolC OMP structure in 2000 [142], the inner membrane AcrB antiporter in 2002 [143] and the periplasmic

1495

MFP in 2004 [144,145]. These structures advanced our knowledge of pump structure, substrate recognition, and revealed a mechanism by which RND pumps can bridge the entire cell envelope [146]. 2.8.1. Outer membrane channel proteins TolC is a trimer consisting of two joined barrel-like ˚ long 12 stranded h-barrel structures. At one end, a 40 A structure (each monomer contributing four h-strands) forms a pore with a wide opening. This h-barrel is embedded in the outer membrane and anchors a long ˚ ) tunnel structure formed by 12 antiparallel a (~100 A helices projecting far into the periplasm. This a tunnel contains an internal cavity which is open to the hbarrel, but closed at the end facing the cytoplasmic membrane. Unlike the rigid h-barrel structure, the closed end of the tunnel can be opened widely by a small uncoiling movement of the a helices [147], a feature that probably plays an important role in the function of the outer membrane protein component of the export complex. The distal end of the a tunnel is believed to interact with the bTolC-dockingQ domain of the RND transporter [143,146]. 2.8.2. Inner membrane RND transporters The inner membrane component of RND efflux pumps is responsible for the recognition of the molecule to be effluxed [148]. Recent studies conclusively showed that the two large periplasmic loops (about 300 amino acid residues each) of RND pumps contain the amino acid residues responsible for substrate recognition [149–152]. These studies were performed using chimeric constructs of different RND pump proteins from E. coli and P. aeruginosa. It was observed that the chimeric protein containing the periplasmic loops of one protein retained the substrate specificity of that particular protein. The AcrB crystal structure revealed that the RND transporter is composed of three protomers and contains three distinct domains, a transmembrane domain, a pore domain and a bTolC-dockingQ domain [143]. The pore and the bTolC-dockingQ domains protrude a ˚ into the periplasm, far enough to combined ~ 70 A enable physical contact with the distal end of the TolC a tunnel. Vestibules between the periplasmic domains of neighboring protomers are believed to be the sites of substrate entry, as described by the lateral substrate capture model of RND family pump function [153].

1496

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

One feature of RND pumps is their broad substrate specificity. Interesting insights regarding substrate recognition came from the crystal structures of AcrB with different ligands [154]. The resulting structures attribute the wide substrate specificity of AcrB to a large composite substrate-binding cavity of exceptional flexibility [153]. 2.8.3. The MFP proteins The structure of P. aeruginosa MexA revealed that it forms a ring-like structure interacting with both the outer membrane channel protein and the RND transporter [144–146]. Several fitting models were presented which mostly differ in the number of MexA monomers interacting with OprM and MexB [144,145]. Whatever the exact roles of the MFPs may be, these proteins are indispensable for pump assembly and function [39,155–157]. 2.8.4. Assembly of RND efflux pumps A recent study has demonstrated that the assembly of the AcrA, AcrB and TolC proteins into a functional AcrAB–TolC pump is constitutive [157], occurring in the presence or absence of substrate molecules. However, this is in contrast with findings from our laboratory that show assembly of a functional P. aeruginosa MexJK–OMP pump is dependent on substrate [Chuanchuen et al., unpublished observations]. In the presence of triclosan, MexJK functions with OpmH for assembly of a MexJK–OpmH triclosan efflux pump. Conversely, when erythromycin is present, MexJK functions with OprM and a MexJK– OprM erythromycin efflux pump is assembled. This suggests a great diversity with regards to the mechanisms of efflux pump architecture, and perhaps assembly, among different members of the RND protein family. This may be significant because half of the RND efflux pump operons of P. aeruginosa do not encode their own outer membrane channel proteins [158] and thus must rely on expression of other channel proteins, most likely of the OprM family, for functional efflux pump assembly. 2.9. Natural functions of RND pumps The natural functions of the different RND pumps are still a topic of debate. It was originally believed that these pumps evolved in Gram-negative bacteria

as a defense mechanism to counter the effects of environmental antimicrobials or other toxins. However, with phylogenetic studies pointing to the presence of RND homologs in Gram-positive bacteria, archaea and even humans, it has now been established that proteins belonging to RND pumps are part of an ancient family of proteins with representation in all major kingdoms [36]. The AcrAB pump of E. coli was found to have the highest affinity for bile salts, as demonstrated by the finding that AcrAB knockout strains are hypersusceptible to bile salts [159]. The natural habitat of E. coli is the enteric tract (which is rich in bile salts) and efflux as a protective mechanism therefore makes sense. Similarly, a protective function has been attributed to the MtrCDE system, which provides resistance to faecal lipids in rectal isolates of N. gonorrhoeae [160]. Additional natural functions suggested for efflux pumps include: removal of toxins, removal of metabolic byproducts (e.g., fermentation end products and toxins), and buffering the organisms against surges in pools of potentially toxic metabolites [161]. It has been suggested that the natural function of RND pumps might also include efflux of signal molecules required for cell-to-cell signaling (quorum sensing) [162]. P. aeruginosa secretes a quinolone, designated PQS ( Pseudomonas quinolone signal), which acts as a quorum-sensing signal [163] and overexpression of MexEF–OprN was shown to affect PQS signaling [164]. This is a very interesting finding as quinolones constitute a widely used group of antimicrobials and are substrates for RND pumps. Another study suggested that MexGHI–OmpD is involved in the homeostasis of N-acyl-homoserine lactone quorum-sensing molecules [78]. SdiA, a quorum-sensing regulator from E. coli, controls multidrug resistance by positively regulating the AcrAB pump [162]. One can therefore easily envision why RND pumps catalyze the efflux of certain antibiotics that structurally resemble these natural signaling molecules. MexAB– OprM was shown to play a role in the invasiveness of P. aeruginosa in mouse models [165], suggesting a possible role for it in the pathogenicity of this organism. Finally, a recent study suggested a correlation between the presence of the AcrAB–TolC pump in E. coli and calcium transport [166]. It is suspected that AcrAB–TolC could play a role in the transport of the calcium-channel components in the E. coli membrane.

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

2.10. Regulation of RND pump gene expression One can argue that an understanding of the environmental conditions involved in regulation of RND pump expression is an integral part of the drug discovery efforts aimed at overcoming efflux as a contributing factor to antibiotic resistance. These environmental conditions may yield clues as to the natural function of the respective efflux components and thus facilitate design of inhibitory compounds. Furthermore, the regulators themselves may be attractive drug targets, especially in those cases where specific ligands can be identified. The regulators involved in efflux gene expression are either local or global regulators. 2.10.1. Local regulators Many pump component-encoding operons contain a physically linked regulatory gene. For example, E. coli acrR is transcribed divergently from the acrAB genes and encodes a repressor of the TetR repressor family. AcrR is known to repress both its own and acrAB transcription, probably in a similar fashion as TetR represses tetA expression [167]. The role of AcrR in the fluoroquinolone resistance of E. coli clinical isolates was demonstrated in a study performed in China, which found a link between mutations in acrR (base changes, deletions, duplication) and high level of fluoroquinolone resistance as a result of acrAB overexpression [168]. It appears though that the major role of AcrR is to prevent the overexpression of acrAB, as its repression of acrAB is leaky and allows for its constitutive expression. Many other local repressors of multidrug efflux pumps belong to the TetR family, e.g., AcrS (AcrEF, E. coli) [169], MexL (MexJK, P. aeruginosa) [77], MexZ (MexXY, P. aeruginosa) [110] and SmeT (SmeDEF, S. maltophila) [170]. Most of the RND multidrug efflux systems of P. aeruginosa that have been studied to date are regulated by a linked regulatory gene, coding for either a repressor or an activator. Expression of the mexAB– oprM operon is regulated by the product of the upstream and divergently transcribed mexR gene. MexR is a transcriptional repressor of the MarR family [82] and the inactivation of mexR results in the overexpression of MexAB–OprM system [171]. Not surprisingly, clinical isolates that overexpress the MexAB– OprM system often carry mutations in the mexR

1497

gene [93,172]. MexCD–OprJ expression in P. aeruginosa is regulated by the NfxB repressor encoded by a gene located upstream of the mexCD–oprJ operon [74] and cystic fibrosis isolates expressing MexCD– OprJ as a consequence of long-term ciprofloxacin exposure contain mutations in nfxB [173]. MexT, a member of the LysR family of transcriptional regulators, is an activator of mexEF–oprN transcription [174] and thus mechanistically unique when compared to other P. aeruginosa RND operons whose expression is usually negatively regulated. The mexT gene is located upstream of and transcribed in the same direction as the mexEF–oprN genes. As mentioned previously in Section 2.7.2.3, overexpression of mexT induces the expression of mexEF–oprN, and decreases the expression of the OprD OMP channel [100]. There appears to be considerable diversity in strains that exhibit nfxC-type antibiotic resistance (i.e., overproduce MexEF–OprN) in terms of mexT-mediated expression of mexEF–oprN. Most PAO1 laboratory strains and its derivatives possess an 8-base pair insertional mutation in mexT that blocks the expression of MexEF–OprN [175]. nfxC-type mutants derived from such strains lack this insertion, resulting in the conversion of inactive MexT to its active form [175]. In some cases, additional mutations were identified in mexT of nfxC-type mutants that resulted in activation of MexT, while in other instances both wild-type and resistant mutants produced an active MexT and the nfx-type phenotype was attributed to some other, yet unknown mutation(s) [175]. An interesting mode of regulation of efflux pump expression was recently shown in S. enterica serovar Typhimurium [136]. It was observed that AcrB-deletion strains exposed to fluoroquinolones exhibited enhanced expression of the AcrEF pump. Further analysis revealed that this overexpression of acrEF was due to the insertion of insertion elements in the promoter region of acrEF that resulted in creation of new, stonger promoters for the operon. This is an example of the importance of transposable elements in the acquisition of multidrug resistance phenotypes in clinically relevant pathogens. Some efflux pumps are known to be regulated by two-component systems. These systems mediate the adaptive responses of bacterial cells to their environment. BaeSR regulates expression of the MdtABC pump of E. coli [72,73]; EvgAS the YhiUV pump

1498

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

studied example being the AcrAB–TolC system of E. coli. So far, several global transcriptional activators, including MarA, SoxS and Rob, were shown to be involved in the regulation of expression of this system (Fig. 2). The mar (multiple antibiotic resistance) locus consists of the marRAB operon and the divergently transcribed marC, both being expressed from a central operator/promoter region, marO [179]. MarR is a repressor and MarA is an activator. The functions of MarB and MarC remain unknown. MarA is a member of the AraC family of transcriptional activators, and activates its own transcription as well as a large number of other genes, including acrAB, tolC and micF, an antisense RNA that down-regulates the expression of ompF, a gene

of E. coli [176] plus a variety of other E. coli efflux pump-encoding genes: emrKY, yhiUV, acrAB, mdfA, as well as tolC [177]; SmeSR the SmeABC pump of S. maltophilia [122] and CzcRS the CzcCBA heavy metal efflux system of P. aeruginosa [178]. These examples illustrate the role that environmental conditions play in the expression of efflux pump-encoding genes. In most, but not all of these systems, the genes encoding two-component systems are located in very close proximity to the efflux pump gene operons and the response regulators function as activators. 2.10.2. Global regulators Expression of various efflux pumps is also controlled by different global regulators, with the best-

marRAB marC

marR

marA

-

?

soxRS

+

marB ?

rob

soxS

soxR

+

-

rob

Inducers (bile salts, fatty acids, dipyridyl)

+

Inactivation by oxidation Inactivation by salicylate binding micF ompF

tolC

Global acrR

acrA

acrB

Drug TolC

-

OM

Local AcrA

(fewer) OmpF

IM AcrB H+

Fig. 2. Major features of local and global regulation of Escherichia coli AcrAB–TolC expression (adapted from [12]). The acrAB operon is negatively regulated by a local repressor (acrR). acrAB, tolC and the antisense regulatory RNA micF are positively regulated by several activators (MarA, SoxS and Rob). The levels of MarA and SoxS are themselves regulated by the repressors MarR and SoxR, respectively. The activity of Rob is modulated by metabolites, such as bile salts and fatty acids. The micF transcript inhibits translation of the ompF porin mRNA, thus lowering expression of this outer membrane porin and reducing outer membrane permeability for many drugs. At the same time, all three activators increase expression of AcrA, AcrB and TolC, leading to increased drug efflux. Stippled arrows indicate negative regulation. For clarity, cross-regulation (e.g., upregulation of MarA by SoxS and Rob) and the minor regulators were omitted from this figure (for details consult the text and the review by Li and Nikaido [12]).

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

encoding an OMP channel that is responsible for entry of various antibiotics [180]. Lower OmpF levels combined with overexpression of AcrAB–TolC provide E. coli with a highly effective mechanism by which MarA can coordinate a response to the presence of antimicrobials. Overall, MarA activates expression of the mar regulon, including acrAB, tolC and marRAB, while MarR represses the mar regulon by repressing the synthesis of MarA. As in E. coli, overexpression of marA in E. aerogenes resulted in increased resistance to antibiotics, most likely due a decrease in the expression of porins and an increase in active efflux [181]. Elevated levels of SoxS and Rob, two MarA homologues, activate the transcription of acrAB, tolC and micF. SoxS is the effector of the soxRS global superoxide response (sox) regulon and Rob is a protein binding to the E. coli chromosomal origin of replication [182]. SoxS is only expressed after conversion of the transcriptional activator SoxR into an active form by superoxide-generating agents [183,184]. Unlike MarA and SoxS, Rob is synthesized constitutively and activates its target genes only after the binding of inducers, such as medium-chain fatty acids and bile salts [185]. Identification of MarA homologues in various bacterial species, e.g., E. coli O157:H7 [186] and other enterobacteriaceae [187], suggests that the intrinsic, and perhaps acquired, resistance of these organisms and many others with yet unidentified MarA-like regulatory cascades is probably dependent on synergy between outer membrane permeability and active efflux. RamA, another multiple antibiotic resistance global regulator, was first identified in K. pneumoniae [188], where it was shown to cause an increase in resistance to different antibiotics due to reduced porin and enhanced AcrAB expression [189]. RamA loci have also been identified in S. enterica serovar Paratyphi B [190] and E. aerogenes [191]. Global regulation of efflux pump expression is also evident from instances where interplay between different pumps is seen within the same organism. For example, in S. enterica serovar Typhimurium, increased expression of acrF and acrD was observed in response to the deletion of acrB [137]. This indicates a very tight coordination in the regulation of expression of efflux pumps with overlapping substrate profiles. The study showed that expression of AcrD

1499

and AcrF was derepressed upon deletion of AcrB, however, in the presence of a functional AcrB their expression is repressed, suggesting that the expression of these systems is regulated by environmental conditions. A similar scenario was observed with clinical isolates of P. aeruginosa that simultaneously expressed the MexAB–OprM and MexXY systems [192], thus broadening the resistance profile, which would be of great advantage in a clinical setting. There is mounting evidence that mex gene expression in P. aeruginosa is also controlled by a number of global regulators. Expression of MexGHI–OpmD is governed by the quorum sensing regulatory circuits [78]. Some clinical isolates express MexAB–OprM, although they do not contain mexR mutations [92,193]. Regulation of MexAB–OprM is quite complex and other regulators besides MexR are involved. Two types of MexAB–OprM overexpressing mutants have been characterized. The nalB-type mutants contain mutations in mexR, whereas nalC-type mutants do not. Recently, it was found that nalC mutants contain mutations in PA3721, a transcriptional regulator of the TetR family [96]. PA3721 is a repressor of the PA3719–PA3720 operon, and the two genes encode proteins of unknown function. Hyperexpression of PA3719 alone is sufficient for expression of the nalC phenotype, with PA3719 directly or indirectly impacting MexAB–OprM expression. Additionally, nalD-type mutants expressing MexAB– OprM without mutations in either mexR or PA3721 were isolated [192].

3. Overcoming efflux With the demonstrated importance of RND transporters in the development of clinically significant antimicrobial resistance in many Gram-negative bacteria, these efflux pumps play an important role in the drug discovery process and are considered bona fide drug targets for the development of combination therapies [194–196]. Inhibition of these pumps may be achieved at different levels: by inhibiting drug binding to the inner membrane pumps, by inhibiting the interactions of different components of a multi-component pump, by targeting the energy source of pumps, or by targeting the regulatory networks that control the expression of efflux pumps.

1500

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

Over the past decade, a series of efflux pump inhibitors (EPIs) have been identified. The first broad-spectrum RND pump inhibitor, MC-207,110 (phenylalanyl-arginyl-h-naphthylamide), potentiated the activity of levofloxacin against wild-type P. aeruginosa 8-fold and against a MexAB–OprM overexpressing strain 64-fold [197]. Several improved EPIs exhibited antibiotic potentiation activity for P. aeruginosa strains expressing MexAB–OprM, MexCD–OprJ and MexEF–OprN, and for E. coli expressing AcrAB–TolC [196,198]. Importantly, EPIs dramatically reduced the emergence of spontaneously levofloxacin-resistant bacteria [195] and were effective in animal models of P. aeruginosa infections [199]. Similar compounds have been shown to inhibit efflux pumps in E. aerogenes [200] and Campylobacter [201]. It is believed that these compounds act by inhibiting the specific binding sites of antibiotics within the pump molecule [196]. Benastatins obtained from fermentation of an actinomycete are another group of compounds that were found to be active against P. aeruginosa expressing MexAB–OprM [202]. EPIs have been identified for other bacteria and from other sources. 2,8-Dimethyl-4-(2V-pyrrolidinoethyl)-oxyquinolone, an alkoxyquinolone derivative, was shown to inhibit efflux pumps in E. aerogenes and K. pneumoniae. This EPI potentiated the efficacy of chloramphenicol, norfloxacin, tetracycline and cefepime by up to 8-fold [203]. Several tetracycline derivatives were described that inhibit the TetB efflux pump [204]. The most potent of these is a derivative of doxycycline, 13-cyclopentylthio-5-OH tetracycline (13-CPTC). Contrary to the EPIs described above, 13-CPTC has antibacterial properties of its own against LPS-deficient E. coli and S. aureus. Combination of 13-CPTC with doxycycline resulted in reduction of MIC values for doxycyline by 4- to 10-fold [204]. EPIs isolated from plant extracts [205,206] showed no intrinsic antibacterial activity, but were able to potentiate the activity of norfloxacin against S. aureus by inhibiting the NorA pump [205,207]. EPIs clearly show promise for developing combination therapies with existing antibacterials to restore their antibacterial activity against resistant bacteria. However, this approach presents its own challenges because of the potential undesirable effects on eukaryotic cells, for example toxicity and inhibition of

eukaryotic transporters that are structurally and functionally similar. Bypassing efflux pumps may be an available alternative to EPIs. This could be achieved by development of newer drugs that are poor substrates for these pumps. Indeed, some of the newer fluoroquinolones seem to be poor substrates for certain pumps found in Gram-positive bacteria [208,209]. However, it is not yet clear if the increased activity is due to higher affinity for the target or lower affinity for these pumps. As mentioned in Section 2.7.2.2, there is always the risk of inducing an alternate pump in response to exposure to an antibiotic that is a poor substrate for a particular pump. The glycylcycline tigecycline (GRA-936) is an example of a substrate that is a poorer substrate for Tet pumps [210]. However, glycylcyclines are substrates for RND pumps of many Gram-negative bacteria, including P. aeruginosa, K. pneumoniae, P. mirabilis and Morganella morganii [189,211–213], which render them ineffective against Gram-negative pathogens.

4. Outer membrane permeability Gram-negative bacteria possess an asymmetric outer membrane consisting of an inner leaflet containing phospholipids and an outer leaflet containing the lipid A moiety of LPS. This composition of the outer membrane (OM) renders it impermeable to many substrates and transport across the OM is achieved by porin proteins that form water-filled channels [14,214]. Drug molecules can penetrate the OM employing one of the following modes: by diffusion through porins, by diffusion through the bilayer or by self-promoted uptake. The mode of entry employed by a drug molecule largely depends on its chemical composition. For example, hydrophilic compounds either enter the periplasm through porins (e.g., hlactams) or self-promoted uptake (aminoglycosides). 4.1. LPS and antibiotic resistance Lipopolysaccharide (LPS) is largely responsible for the impermeability of the bilayer to hydrophobic molecules such as antibiotics and detergents. LPS is composed of lipid A, a core polysaccharide, and the O-antigen. Fatty acid substituents in the LPS are fully

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

saturated giving it a gel-like structure [215] with low fluidity. The highly charged (anionic) nature of the Oantigen and the cross-bridging of the core region via phosphate groups and divalent cations [216] also contribute to the low permeability of the LPS. For example, the core region of P. aeruginosa LPS is the most highly phosphorylated of all known Gram-negative cores, and as a result, the cell envelope of this organism is a strong diffusion barrier [217]. The role of LPS as a barrier to antibiotics is well documented. Strains of E. coli and S. enterica serovar Typhimurium defective in the synthesis of lipid A and the inner core of LPS have been found to be at least 4fold more susceptible to erythromycin, roxithromycin, clarithromycin and azithromycin than the wild-type strains [218]. Mutations in LPS that result in antibiotic hypersusceptibility have also been reported in P. aeruginosa [219] and V. cholerae [220]. The role of LPS in antibiotic resistance was also demonstrated in Brucella species which are susceptible to hydrophobic agents, but relatively resistant to cationic peptides [221]. This has been attributed to the nature of the LPS of this organism, which is a weaker barrier for hydrophobic agents, but binds polycations less efficiently [222]. LPS is also responsible for the difference in the susceptibilities of members of Yersinia sp. to hydrophobic agents and polycations [223,224]. Polymyxin-susceptible B. pseudomallei strains were shown to have defects in LPS core biosynthetic genes [225]. The most common LPS modification leading to resistance to polymyxin B, aminoglycosides and cationic antibacterial peptides is the modification of lipid A with a positively charged 4-amino-4-deoxy-arabinose moiety, as seen in S. enterica serovar Typhimurium [226–228]. The presence of aminoarabinose effectively reduces the net negative charge of the cell envelope, reducing the permeation of cationic antimicrobials. 4.2. Porins and antibiotic resistance Antibiotics such as h-lactams [229], chloramphenicol and fluoroquinolones [14] permeate the Gramnegative outer membrane via porins. As such, changes in porin copy number, size or selectivity will alter the rate of diffusion of these antibiotics [230]. One of the first examples of antibiotic resistance due to porin loss was a clinical isolate of S. marcescens that exhibited

1501

resistance to both aminoglycosides and h-lactams [231]. Additional examples have since been reported with various bacterial isolates, including E. coli [232], Enterobacter cloacae [233], S. enterica [234,235], Haemophilus influenzae [236], K. pneumoniae [237] and E. aerogenes [238]. In E. aerogenes, h-lactam resistant isolates often exhibit loss of a porin along with the expression of h-lactamase [239,240], but resistance can also result from mutations that lead to a narrowing of the porin channel [241,242]. In E. cloacae, meropenem resistance resulted from loss of porins [243]. h-Lactam resistance of K. pneumoniae often results from loss of porins as well, though usually in conjunction with h-lactamase production [244,245]. A mutation in a porin gene in N. gonorrhoeae was shown to be responsible for resistance of this organism to h-lactams and tetracycline [246]. P. aeruginosa possesses a much lower outer membrane permeability (approximately 8% that of E. coli), but a large exclusion limit (permitting passage of molecules of molecular weight up to 3000 Da compared with around 500 Da for E. coli) [247]. Although OprF is the major non-specific porin of P. aeruginosa and OprF-deficient clinical isolates with multidrugresistant phenotypes were previously identified [248], OprF contributed only minimally to antibiotic resistance in laboratory-generated mutants. Likewise, its role as a major determinant of antibiotic resistance in P. aeruginosa clinical isolates has not been established [249]. P. aeruginosa also contains various specific porins that have been shown to contribute to antibiotic resistance of this organism. OprD facilitates permeation of basic amino acids and the small h-lactam imipenem. In response to imipenem exposure, oprD expression is often down-regulated, resulting in resistance to the drug [250,251] and is now recognized as the major determinant of the P. aeruginosa imipenem resistance. The down-regulation has been attributed to base transitions, deletions, or insertional mutations leading to abberant oprD transcripts [252,253]. OprD is moderately expressed by P. aeruginosa, but is regulated by multiple systems. Its expression is repressed by salicylate, catabolite repression and MexT, an activator of MexEF–OprN efflux pump expression [104,174]. Its repression by MexT is an excellent example of synergy between permeability and efflux mechanisms. OprE, another specific channel of P. aeruginosa, has been found to be absent in quinolone

1502

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

and cephalosporin resistant mutants of this organism [254]. However, its role has not been examined very extensively to date. OprR is a relatively recently described OM protein from P. aeruginosa and its expression was found to be up-regulated in response to quaternary ammonium compound (QAC) exposure [255]. However, its contribution to clinically significant resistance has not yet been documented. 4.3. Overcoming restricted outer membrane permeability Permeabilization of the OM has been shown to increase the efficacy of antibiotics that otherwise cannot penetrate the outer membrane [256–258]. Various permeabilizing agents have been described in literature, including antimicrobial peptides, chelators and polycations [259–261]. Polycationic compounds are known to target the OM, particularly the LPS, making them a promising candidate for the dealing with the problem of permeability-related antibiotic resistance [262]. Many of these permealizers have been used to potentiate the activity of antimicrobials in E. coli [263–267], S. enterica [263,268], K. pneumoniae [264,265,268], E. cloacae [264,265,268], P. aeruginosa [265,267], Proteus vulgaris [265], S. marcescens [265] and A. baumanii [266]. However, use of OM permeabilizers is limited partly by the potential for development of resistance.

isms. The wide distribution of these efflux systems in bacteria, as experimentally documented and predicted from genome sequence analyses, illustrates that they are likely to be dealt with in a range of pathogens, including the mycobacteria [45,270]. Of particular concerns are the broad substrate specificities of these efflux pumps and the fact that they compromise the use of newer drugs, e.g., tigecycline, and even experimental agents, the majority of which are substrates of the E. coli AcrAB–TolC efflux pumps [271]. However, as paradoxical as it may sound, efflux pumps not only complicate drug discovery efforts, but can also serve as drug discovery assets. A recent study showed that an efflux-sensitized P. aeruginosa strain panel obtained by inactivation of all major efflux pumps allowed identification of a compound that is normally subjected to efflux and would therefore have been missed in conventional screens using wild-type cells. After identification of the compound, medicinal chemistry efforts afforded a derivative which evaded efflux, but retained antibacterial activity [272]. New therapeutics could not only include new drugs with improved uptake properties or compounds bypassing efflux pumps, but also broad-spectrum efflux pump inhibitors improving the potency of existing antimicrobials. Recent advances in genome sequence analyses, the availability of molecular structures and a more profound understanding of the function and regulation of efflux systems will facilitate exploitation of pumps as new drug targets.

5. Summary and conclusions Reduced OM permeability results in reduced antibiotic uptake, leading to low-level drug resistance. In the presence of drug efflux pumps, the resistance is amplified multiplicatively by synergism between reduced uptake and active efflux. This effect has been shown in P. aeruginosa [269], where either inactivation of the MexAB–OprM pumps or permeabilization of the OM alone resulted in a very drastic, but similar decrease in the antibiotic resistance of this organism. With the isolation of an ever increasing number of multidrug resistant isolates from clinical settings that lack porins and express various efflux pumps, it is now recognized that reduced OM permeability and active efflux are clinically relevant drug resistance mechan-

Acknowledgment Work in the HPS laboratory was funded by grant AI051588 from the National Institutes of Health.

References [1] H.C. Neu, The crisis in antibiotic resistance, Science 257 (1992) 1064 – 1073. [2] S.B. Levy, Antibiotic resistance: an ecological imbalance, Ciba Found. Symp. 207 (1997) 1 – 9. [3] B.H. Normark, S. Normark, Evolution and spread of antibiotic resistance, J. Intern. Med. 252 (2002) 91 – 106. [4] R.L. Juliano, V. Ling, A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants, Biochim. Biophys. Acta 455 (1976) 152 – 162.

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513 [5] P.R. Ball, I. Chopra, S.J. Eccles, Accumulation of tetracyclines by Escherichia coli K-12, Biochem. Biophys. Res. Commun. 77 (1977) 1500 – 1507. [6] S.B. Levy, L. McMurry, Plasmid-determined tetracycline resistance involves new transport systems for tetracycline, Nature 276 (1978) 90 – 92. [7] P.R. Ball, S.W. Shales, I. Chopra, Plasmid-mediated tetracycline resistance in Escherichia coli involves increased efflux of the antibiotic, Biochem. Biophys. Res. Commun. 93 (1980) 74 – 81. [8] L. McMurry, R.E. Petrucci Jr., S.B. Levy, Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 77 (1980) 3974 – 3977. [9] O. Lomovskaya, M.S. Warren, V. Lee, in: D. Hughes, D.I. Andersson (Eds.), Antibiotic Development and Resistance, Taylor and Francis, London, 2001, pp. 65 – 90. [10] K. Poole, Outer membranes and efflux: the path to multidrug resistance in Gram-negative bacteria, Curr. Pharm. Biotechnol. 3 (2002) 77 – 98. [11] K. Poole, Efflux-mediated multiresistance in Gram-negative bacteria, Clin. Microbiol. Infect. 10 (2004) 12 – 26. [12] X.Z. Li, H. Nikaido, Efflux-mediated drug resistance in bacteria, Drugs 64 (2004) 159 – 204. [13] I.T. Paulsen, K. Lewis, Microbial Drug Efflux, Horizon Press, Wynmondham, 2002. [14] H. Nikaido, Molecular basis of bacterial outer membrane permeability revisited, Microbiol. Mol. Biol. Rev. 67 (2003) 593 – 656. [15] I.T. Paulsen, M.K. Sliwinski, M.H. Saier Jr., Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities, J. Mol. Biol. 277 (1998) 573 – 592. [16] S.S. Pao, I.T. Paulsen, M.H. Saier Jr., Major facilitator superfamily, Microbiol. Mol. Biol. Rev. 62 (1998) 1 – 34. [17] M.D. Marger, M.H. Saier Jr., A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport, Trends Biochem. Sci. 18 (1993) 13 – 20. [18] H.W. van Veen, W.N. Konings, The ABC family of multidrug transporters in microorganisms, Biochim. Biophys. Acta 1365 (1998) 31 – 36. [19] I.T. Paulsen, R.A. Skurray, R. Tam, M.H. Saier, R.J. Turner, J.H. Weiner, E.B. Goldberg, L.L. Grinius, The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs, Mol. Microbiol. 19 (1996) 1167 – 1175. [20] M.H. Saier Jr., R. Tam, A. Reizer, J. Reizer, Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport, Mol. Microbiol. 11 (1994) 841 – 847. [21] M.H. Brown, I.T. Paulsen, R.A. Skurray, The multidrug efflux protein NorM is a prototype of a new family of transporters, Mol. Microbiol. 31 (1999) 393 – 395. [22] P. Butaye, A. Cloeckaert, S. Schwarz, Mobile genes coding for efflux-mediated antimicrobial resistance in Gram-positive and Gram-negative bacteria, Int. J. Antimicrob. Agents 22 (2003) 205 – 210.

1503

[23] H. Yoshida, M. Bogaki, S. Nakamura, K. Ubukata, M. Konno, Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones, J. Bacteriol. 172 (1990) 6942 – 6949. [24] O. Lomovskaya, K. Lewis, Emr, an Escherichia coli locus for multidrug resistance, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 8938 – 8942. [25] J.M. Tennent, B.R. Lyon, M. Midgley, I.G. Jones, A.S. Purewal, R.A. Skurray, Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus, J. Gen. Microbiol. 135 (1989) 1 – 10. [26] I.T. Paulsen, M.H. Brown, T.G. Littlejohn, B.A. Mitchell, R.A. Skurray, Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 3630 – 3635. [27] J. Clancy, J. Petitpas, F. Dib-Hajj, W. Yuan, M. Cronan, A.V. Kamath, J. Bergeron, J.A. Retsema, Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes, Mol. Microbiol. 22 (1996) 867 – 879. [28] I. Chopra, M. Roberts, Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance, Microbiol. Mol. Biol. Rev. 65 (2001) 232 – 260. [29] C.F. Higgins, ABC transporters: from microorganisms to man, Annu. Rev. Cell Biol. 8 (1992) 67 – 113. [30] M.J. Fath, R. Kolter, ABC transporters: bacterial exporters, Microbiol. Rev. 57 (1993) 995 – 1017. [31] H. Bolhuis, H.W. van Veen, J.R. Brands, M. Putman, B. Poolman, A.J. Driessen, W.N. Konings, Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP, J. Biol. Chem. 271 (1996) 24123 – 24128. [32] M.H. Saier Jr., I.T. Paulsen, Phylogeny of multidrug transporters, Semin. Cell Dev. Biol. 12 (2001) 205 – 213. [33] C. Ma, G. Chang, Structure of the multidrug resistance efflux transporter EmrE from Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 2852 – 2857. [34] L. Grinius, G. Dreguniene, E.B. Goldberg, C.-H. Liao, S.J. Projan, A staphylococcal multidrug resistance gene product is a member of a new protein family, Plasmid 27 (1992) 119 – 129. [35] S. Schuldiner, M. Lebendiker, H. Yerushalmi, EmrE, the smallest ion-coupled transporter, provides a unique paradigm for structure–function studies, J. Exp. Biol. 200 (1997) 335 – 341. [36] T.-T. Tseng, K.S. Gratwick, J. Kollman, D. Park, D.H. Nies, A. Goffeau, M.H. Saier Jr., The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins, J. Mol. Microbiol. Biotechnol. 1 (1999) 107 – 125. [37] L.H. Hansen, E. Johannesen, M. Burmolle, A.H. Sorensen, S.J. Sorensen, Plasmid-encoded multidrug efflux pump conferring resistance to olaquindox in Escherichia coli, Antimicrob. Agents Chemother. 48 (2004) 3332 – 3337.

1504

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

[38] D. Ma, D.N. Cook, M. Alberti, N.G. Pon, H. Nikaido, J.E. Hearst, Molecular cloning and characterization of acrA and acrE genes of Escherichia coli, J. Bacteriol. 175 (1993) 6299 – 6313. [39] D. Ma, D.N. Cook, M. Alberti, N.G. Pon, H. Nikaido, J.E. Hearst, Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli, Mol. Microbiol. 16 (1995) 45 – 55. [40] K. Poole, K. Krebes, C. McNally, S. Neshat, Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon, J. Bacteriol. 175 (1993) 7363 – 7372. [41] Y. Morita, K. Kodama, S. Shiota, T. Mine, A. Kataoka, T. Mizushima, T. Tsuchiya, NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli, Antimicrob. Agents Chemother. 42 (1998) 1778 – 1782. [42] C. Carbon, MRSA and MRSE: is there an answer? Clin. Microbiol. Infect. 6 (Suppl. 2) (2000) 17 – 22. [43] A. Marchese, G.C. Schito, E.A. Debbia, Evolution of antibiotic resistance in gram-positive pathogens, J. Chemother. 12 (2000) 459 – 462. [44] T.G. Littlejohn, I.T. Paulsen, M.T. Gillespie, J.M. Tennent, M. Midgley, I.G. Jones, A.S. Purewal, R.A. Skurray, Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus, FEMS Microbiol. Lett. 74 (1992) 259 – 265. [45] K. Poole, Efflux-mediated resistance to fluoroquinolones in gram-positive bacteria and the mycobacteria, Antimicrob. Agents Chemother. 44 (2000) 2595 – 2599. [46] E.Y. Ng, M. Trucksis, D.C. Hooper, Quinolone resistance mediated by norA: physiologic characterization and relationship to flqB, a quinolone resistance locus on the Staphylococcus aureus chromosome, Antimicrob. Agents Chemother. 38 (1994) 1345 – 1355. [47] G.V. Doern, S.D. Brown, Antimicrobial susceptibility among community-acquired respiratory tract pathogens in the USA: data from PROTEKT US 2000–01, J. Infect. 48 (2004) 56 – 65. [48] N.P. Brenwald, M.J. Gill, R. Wise, The effect of reserpine, an inhibitor of multi-drug efflux pumps, on the in-vitro susceptibilities of fluoroquinolone-resistant strains of Streptococcus pneumoniae to norfloxacin, J. Antimicrob. Chemother. 40 (1997) 458 – 460. [49] K.J. Madaras-Kelly, C. Daniels, M. Hegbloom, M. Thompson, Pharmacodynamic characterization of efflux and topoisomerase IV-mediated fluoroquinolone resistance in Streptococcus pneumoniae, J. Antimicrob. Chemother. 50 (2002) 211 – 218. [50] M.J. Gill, N.P. Brenwald, R. Wise, Identification of an efflux pump gene, pmrA, associated with fluoroquinolone resistance in Streptococcus pneumoniae, Antimicrob. Agents Chemother. 43 (1999) 187 – 189. [51] A. Tait-Kamradt, J. Clancy, M. Cronan, F. Dib-Hajj, L. Wondrack, W. Yuan, J. Sutcliffe, mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae, Antimicrob. Agents Chemother. 41 (1997) 2251 – 2255.

[52] P. Zhong, V.D. Shortridge, The role of efflux in macrolide resistance, Drug Resist. Updates 3 (2000) 325 – 329. [53] M. Santagati, F. Iannelli, M.R. Oggioni, S. Stefani, G. Pozzi, Abstract Presented at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. [54] A.A. Neyfakh, The multidrug efflux transporter of Bacillus subtilis is a structural and functional homolog of the Staphylococcus NorA protein, Antimicrob. Agents Chemother. 36 (1992) 484 – 485. [55] M. Ahmed, L. Lyass, P.N. Markham, S.S. Taylor, N. Vazquez-Laslop, A.A. Neyfakh, Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated, J. Bacteriol. 177 (1995) 3904 – 3910. [56] A.A. Neyfakh, The ostensible paradox of multidrug recognition, J. Mol. Microbiol. Biotechnol. 3 (2001) 151 – 154. [57] S. Miyamae, H. Nikaido, Y. Tanaka, F. Yoshimura, Active efflux of norfloxacin by Bacteroides fragilis, Antimicrob. Agents Chemother. 42 (1998) 2119 – 2121. [58] I.W. Nilsen, I. Bakke, A. Vader, O. Olsvik, M.R. El-Gewely, Isolation of cmr, a novel Escherichia coli chloramphenicol resistance gene encoding a putative efflux pump, J. Bacteriol. 178 (1996) 3188 – 3193. [59] C. Bohn, P. Bouloc, The Escherichia coli cmlA gene encodes the multidrug efflux pump Cmr/MdfA and is responsible for isopropyl-beta-d-thiogalactopyranoside exclusion and spectinomycin sensitivity, J. Bacteriol. 180 (1998) 6072 – 6075. [60] R. Edgar, E. Bibi, MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition, J. Bacteriol. 179 (1997) 2274 – 2280. [61] N. Kobayashi, K. Nishino, A. Yamaguchi, Novel macrolidespecific ABC-type efflux transporter in Escherichia coli, J. Bacteriol. 183 (2001) 5639 – 5644. [62] S. Phadtare, K. Yamanaka, I. Kato, M. Inouye, Antibacterial activity of 4,5-dihydroxy-2-cyclopentan-1-one (DHCP) and cloning of a gene conferring DHCP resistance in Escherichia coli, J. Mol. Microbiol. Biotechnol. 3 (2001) 461 – 465. [63] S.M. Wigfield, G.P. Rigg, M. Kavari, A.K. Webb, R.C. Matthews, J.P. Burnie, Identification of an immunodominant drug efflux pump in Burkholderia cepacia, J. Antimicrob. Chemother. 49 (2002) 619 – 624. [64] C.C. Miyamae, M.A. Valvano, Cloning and characterization of the Burkholderia vietnamensis norM gene encoding a multi-drug efflux system, FEMS Microbiol. Lett. 215 (2002) 279 – 283. [65] M.N. Huda, Y. Morita, T. Kuroda, T. Mizushima, T. Tsuchiya, Na+-driven multidrug efflux pump VcmA from Vibrio cholerae non-O1, a non-halophilic bacterium, FEMS Microbiol. Lett. 203 (2001) 235 – 239. [66] K. Nishino, A. Yamaguchi, Analysis of a complete library of putative drug transporter genes in Escherichia coli, J. Bacteriol. 183 (2001) 5803 – 5812. [67] M.C. Sulavik, C. Houseweart, C. Cramer, N. Jiwani, N. Murgolo, J. Greene, B. DiDomenico, K.J. Shaw, G.H. Miller, R. Hare, G. Shimer, Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

pump genes, Antimicrob. Agents Chemother. 45 (2001) 1126 – 1136. E.Y. Rosenberg, D. Ma, H. Nikaido, AcrD of Escherichia coli is an aminoglycoside efflux pump, J. Bacteriol. 182 (2000) 1754 – 1756. C. Elkins, H. Nikaido, Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops, J. Bacteriol. 184 (2002) 6490 – 6498. A.S. Jellen-Ritter, W.V. Kern, Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants selected with a fluoroquinolone, Antimicrob. Agents Chemother. 45 (2001) 1467 – 1472. K. Kobayashi, N. Tsukagoshi, R. Aono, Suppression of hypersensitivity of Escherichia coli acrB mutant to organic solvents by integrational activation of the acrEF operon with the IS1 or IS2 element, J. Bacteriol. 183 (2001) 2646 – 2653. N. Baranova, H. Nikaido, The BaeSR two-component regulatory system activates transcription of the yegMNOB (mdtABCD) transporter gene cluster in Escherichia coli and increases its resistance to novobiocin and deoxycholate, J. Bacteriol. 184 (2002) 4168 – 4176. S. Nagakubo, K. Nishino, T. Hirata, A. Yamaguchi, The putative response regulator BaeR stimulates multidrug resistance of Escherichia coli via a novel multidrug exporter system, MdtABC, J. Bacteriol. 184 (2002) 4161 – 4167. K. Poole, N. Gotoh, H. Tsujimoto, Q. Zhao, A. Wada, T. Yamasaki, S. Neshat, J. Yamagishi, X.Z. Li, T. Nishino, Overexpression of the mexC–mexD–oprJ effux operon in nfxB-type multidrug resistant strains, Mol. Microbiol. 21 (1996) 713 – 724. T. Kohler, M. Michea-Hamzehpour, U. Henze, N. Gotoh, L.K. Curty, J.C. Pechere, Characterization of MexE–MexF– OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa, Mol. Microbiol. 23 (1997) 345 – 354. T. Mine, Y. Morita, A. Kataoka, T. Mizushima, T. Tsuchiya, Expression in Escherichia coli of a new multidrug efflux pump, MexXY, from Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 43 (1999) 415 – 417. R. Chuanchuen, C.T. Narasaki, H.P. Schweizer, The MexJK efflux pump of Pseudomonas aeruginosa requires OprM for antibiotic efflux but not for efflux of triclosan, J. Bacteriol. 184 (2002) 5036 – 5044. S. Aendekerk, B. Ghysels, P. Cornelis, C. Baysse, Characterization of a new efflux pump, MexGHI–OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium, Microbiology 148 (2002) 2371 – 2381. Y. Li, T. Mima, Y. Komori, Y. Morita, T. Kuroda, T. Mizushima, T. Tsuchiya, A new member of the tripartite multidrug efflux pumps, MexVW–OprM, in Pseudomonas aeruginosa, J. Antimicrob. Chemother. 52 (2003) 572 – 575. J. Jo, F.S.L. Brinkman, R.E.W. Hancock, Aminoglycoside efflux in Pseudomonas aeruginosa: involvement of novel outer membrane proteins, Antimicrob. Agents Chemother. 47 (2003) 1101 – 1111.

1505

[81] X.Z. Li, D. Ma, D.M. Livermore, H. Nikaido, Role efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to b-lactam resistance, Antimicrob. Agents Chemother. 38 (1994) 1742 – 1752. [82] X.Z. Li, H. Nikaido, K. Poole, Role of mexA–mexB–oprM in antibiotic efflux in Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 39 (1995) 1948 – 1953. [83] X.-Z. Li, R. Srikumar, K. Poole, b-Lactamase inhibitors are substrates for the multidrug efflux pumps of Pseudmonas aeruginosa, Antimicrob. Agents Chemother. 42 (1998) 399 – 403. [84] T. Kohler, M. Kok, M. Michea-Hamzehpour, P. Plesiat, N. Gotoh, K. Nishino, L.K. Curty, J.C. Pechere, Multidrug efflux in intrinsic resistance to trimethoprim and sulfamethoxazole in Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 40 (1996) 2288 – 2290. [85] H.P. Schweizer, Intrinsic resistance to inhibitors of fatty acid biosynthesis in Pseudomonas aeruginosa is due to efflux: application of a novel technique for generation of unmarked chromosomal mutations for the study of efflux systems, Antimicrob. Agents Chemother. 42 (1998) 394 – 398. [86] X.Z. Li, L. Zhang, K. Poole, Role of the multidrug efflux systems of Pseudomonas aeruginosa in organic solvent tolerance, J. Bacteriol. 180 (1998) 2987 – 2991. [87] R. Srikumar, T. Kon, N. Gotoh, K. Poole, Expression of Pseudomonas aeruginosa multidrug efflux pumps MexA– MexB–OprM and MexC–MexD–OprJ in a multidrug-sensitive Escherichia coli strain, Antimicrob. Agents Chemother. 42 (1998) 65 – 71. [88] N. Masuda, N. Gotoh, C. Ishi, E. Sakagawa, S. Ohya, T. Nishino, Interplay between chromosomal b-lactamase and the MexAB–OprM efflux system in intrinsic resistance to hlactams in Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 43 (1999) 400 – 402. [89] T. Nakae, A. Nakajima, T. Ono, K. Saito, H. Yoneyama, Resistance to beta-lactam antibiotics in Pseudomonas aeruginosa due to interplay between the MexAB–OprM efflux pump and beta-lactamase, Antimicrob. Agents Chemother. 43 (1999) 1301 – 1303. [90] R.J. Williams, D.M. Livermore, M.A. Lindridge, A.A. Said, J.D. Williams, Mechanisms of beta-lactam resistance in british isolates of Pseudomonas aeruginosa, J. Med. Microbiol. 17 (1984) 283 – 293. [91] F. Bert, N. Lambert-Zechovsky, Comparative distribution of resistance patterns and serotypes in Pseudomonas aeruginosa isolates from intensive care units and other wards, J. Antimicrob. Chemother. 37 (1996) 809 – 813. [92] K.L. Beinlich, R. Chuanchuen, H.P. Schweizer, Contribution of multidrug efflux pumps to muliple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa, FEMS Microbiol. Lett. 198 (2001) 129 – 134. [93] K. Saito, H. Yoneyama, T. Nakae, nalB-type mutations cuasing the overexpression of the MexAB–OprM efflux pump are located in the mexR gene of the Pseudomonas aeruginosa chromosome, FEMS Microbiol. Lett. 179 (1999) 67 – 72. [94] P.G. Higgins, A.C. Fluit, D. Milatovic, J. Verhoef, F.J. Schmitz, Mutations in GyrA, ParC, MexR and NfxB in

1506

[95]

[96]

[97]

[98]

[99]

[100]

[101]

[102]

[103]

[104]

[105]

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513 clinical isolates of Pseudomonas aeruginosa, Int. J. Antimicrob. Agents 21 (2003) 409 – 413. I. Ziha Zafiri, C. Llanes, T. Kohler, J.-C. Pechere, P. Plesiat, In vivo emergence of multidrug resistant mutants of Pseudomonas aeruginosa overexpressing the active efflux system MexA–MexB–OprM, Antimicrob. Agents Chemother. 43 (1999) 287 – 291. L. Cao, R. Srikumar, K. Poole, MexAB–OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720–PA3719, Mol. Microbiol. 53 (2004) 1423 – 1436. R. Chuanchuen, K. Beinlich, T.T. Hoang, A. Becher, R.R. Karkhoff-Schweizer, H.P. Schweizer, Cross-resistance between triclosan and antibiotics in Pseudomonas aeruginosa is mediated by multidrug efflux pumps: exposure of a susceptible strain to triclosan selects nfxB mutants overexpressing MexCD–OprJ, Antimicrob. Agents Chemother. 45 (2001) 428 – 432. N. Masuda, N. Gotoh, S. Ohya, T. Nishino, Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 40 (1996) 909 – 913. X.Z. Li, N. Barre, K. Poole, Influence of the MexA–MexB– OprM multidrug efflux system on expression of the MexC– MexD–OprJ and MexE–MexF–OprN multidrug efflux systems in Pseudomonas aeruginosa, J. Antimicrob. Chemother. 46 (2000) 885 – 893. N. Masuda, E. Sakagawa, S. Ohya, N. Gotoh, K. Nishino, Hypersusceptibility of the Pseudomonas aeruginosa nfxB mutant to h-lactams due to reduced expression of the AmpC h-lactamase, Antimicrob. Agents Chemother. 45 (2001) 1284 – 1286. O.F. Join-Lambert, M. Michea-Hamzehpour, T. Kohler, F. Chau, F. Faurisson, S. Dautrey, C. Vissuzaine, C. Carbon, J. Pechere, Differential selection of multidrug efflux mutants by trovafloxacin and ciprofloxacin in an experimental model of Pseudomonas aeruginosa acute pneumonia in rats, Antimicrob. Agents Chemother. 45 (2001) 571 – 576. Y. Morita, Y. Komori, T. Mima, T. Kuroda, T. Mizushima, T. Tsuchiya, Construction of a series of mutants lacking all of the four major mex operons for multidrug efflux pumps or possessing each one of the operons from Pseudomonas aeruginosa PAO1: MexCD–OprJ is an inducible pump, FEMS Microbiol. Lett. 202 (2001) 139 – 143. Y. Morita, T. Murata, T. Mima, S. Shiota, T. Kuroda, T. Mizushima, N. Gotoh, T. Nishino, T. Tsuchiya, Induction of mexCD–oprN operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1, J. Antimicrob. Chemother. 51 (2003) 991 – 994. M.M. Ochs, M.P. McCusker, M. Bains, R.E. Hancock, Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids, Antimicrob. Agents Chemother. 43 (1999) 1085 – 1090. N. Masuda, E. Sakagawa, S. Ohya, Outer membrane proteins responsible for multiple drug resistance in Pseudomonas

[106]

[107]

[108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

[116]

[117] [118]

[119]

aeruginosa, Antimicrob. Agents Chemother. 39 (1995) 645 – 649. H. Fukuda, M. Hosaka, K. Hirai, S. Iyobe, New norfloxacin resistance gene in Pseudomonas aeruginosa PAO, Antimicrob. Agents Chemother. 34 (1990) 1757 – 1761. T. Kohler, M. Michea-Hamzehpour, P. Plesiat, A.L. Kahr, J.C. Pechere, Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 41 (1997) 2540 – 2543. J.R. Aires, T. Kohler, H. Nikaido, P. Plesiat, Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides, Antimicrob. Agents Chemother. 43 (1999) 2624 – 2628. N. Masuda, E. Sagagawa, S. Ohya, N. Gotoh, H. Tsujimoto, T. Nishino, Contribution of the MexX–MexY–OprM efflux system to intrinsic resistance in Pseudomonas aeruginosa, Antimicrob. Agents Chemother. 44 (2000) 2242 – 2246. S. Westbrock-Wadman, D.R. Sherman, M.J. Hickey, S.N. Coulter, Y.Q. Zhu, P. Warrener, L.Y. Nguyen, R.M. Shawar, K.R. Folger, C.K. Stover, Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability, Antimicrob. Agents Chemother. 43 (1999) 2975 – 2983. M.L. Sobel, G.A. McKay, K. Poole, Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates, Antimicrob. Agents Chemother. 47 (2003) 3202 – 3207. C. Vogne, J.R. Aires, C. Bailly, D. Hocquet, P. Plesiat, Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis, Antimicrob. Agents Chemother. 48 (2004) 1676 – 1680. H. Sekiya, T. Mima, Y. Morita, T. Kuroda, T. Mizushima, T. Tsuchiya, Functional cloning and characterization of a multidrug efflux pump, mexHI–opmD, from a Pseudomonas aeruginosa mutant, Antimicrob. Agents Chemother. 47 (2003) 2990 – 2992. J.R.W. Govan, V. Deretic, Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia, Microbiol. Rev. 60 (1996) 539 – 574. J.L. Burns, C.D. Wadsworth, J.J. Barry, C.P. Goodall, Nucleotide sequence analysis of a gene from Burkholderia (Pseudomonas) cepacia encoding an outer membrane lipoprotein involved in multiple antibiotic resistance, Antimicrob. Agents Chemother. 40 (1996) 307 – 313. L. Zhang, X.Z. Li, K. Poole, Fluoroquinolone susceptibilities of efflux-mediated multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Burkholderia cepacia, J. Antimicrob. Chemother. 48 (2001) 549 – 552. D.A.B. Dance, Meliodosis: the tip of the iceberg? Clin. Microbiol. Rev. 4 (1991) 52 – 60. A.J. Simpson, N.J. White, V. Wuthiekanun, Aminoglycoside and macrolide resistance in Bukholderia pseudomallei, Antimicrob. Agents Chemother. 43 (1999) 2332. R.A. Moore, D. DeShazer, S. Reckseidler, A. Weissman, D.E. Woods, Efflux-mediated aminoglycoside and macrolide

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

[120]

[121]

[122]

[123]

[124]

[125]

[126]

[127] [128] [129]

[130]

[131]

[132]

[133]

[134]

[135]

resistance in Burkholderia pseudomallei, Antimicrob. Agents Chemother. 43 (1999) 465 – 470. Y.Y. Chan, T.M.C. Tan, Y.M. Ong, K.L. Chua, BpeAB– OprB, a multidrug efflux pump in Burkholderia pseudomallei, Antimicrob. Agents Chemother. 48 (2004) 1128 – 1135. M. Denton, K.G. Kerr, Microbiological and clinical aspects of infection associated with Stenotrophomonas maltophilia, Clin. Microbiol. Rev. 11 (1998) 57 – 80. X.-Z. Li, L. Zhang, K. Poole, SmeC, an outer membrane multidrug efflux protein of Stenotrophomonas maltophilia, Antimicrob. Agents Chemother. 46 (2002) 333 – 343. A. Alonso, J.L. Martinez, Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia, Antimicrob. Agents Chemother. 44 (2000) 3079 – 3086. A. Alonso, J.L. Martinez, Expression of multidrug efflux pump SmeDEF by clinical isolates of Stenotrophomonas maltophilia, Antimicrob. Agents Chemother. 45 (2001) 1879 – 1881. K.E. Hagman, W. Pan, B.G. Spratt, J.T. Balthazar, R.C. Judd, W.M. Shafer, Resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux system, Microbiology 141 (1995) 611 – 622. L. Zarantonelli, G. Borthagaray, E.H. Lee, W. Veal, W.M. Shafer, Decreased susceptibility to azithromycin and erythromycin mediated by a novel mtr(R) promoter mutation in Neisseria gonorrhoeae, J. Antimicrob. Chemother. 47 (2001) 651 – 654. A. Hejazi, F.R. Falkiner, Serratia marcescens, J. Med. Microbiol. 46 (1997) 903 – 912. A. Kumar, E. Worobec, in: S.G. Pandalai (Ed.), Recent Res. Devel. Microbiol., vol. 7, 2003, pp. 307 – 324. A. Kumar, E.A. Worobec, Cloning, sequencing and characterization of the SdeAB efflux pump of Serratia marcescens, Antimicrob. Agents Chemother. 49 (2005) 1495 – 1501. J. Chen, T. Kuroda, M.N. Huda, T. Mizushima, T. Tsuchiya, An RND-type multidrug efflux pump SdeXY from Serratia marcescens, J. Antimicrob. Chemother. 52 (2003) 176 – 179. E. Bergogne-Berezin, K.J. Towner, Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features, Clin. Microbiol. Rev. 9 (1996) 148 – 165. S. Magnet, P. Courvalin, T. Lambert, Resistance-nodulationcell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454, Antimicrob. Agents Chemother. 45 (2001) 3375 – 3380. S.L. Chau, Y.W. Chu, E.T. Houang, Novel resistance-nodulation-cell division efflux system AdeDE in Acinetobacter genomic DNA group 3, Antimicrob. Agents Chemother. 48 (2004) 4054 – 4055. C.E. Cherubin, R.H. Eng, Quinolones for the treatment of infections due to Salmonella, Rev. Infect. Dis. 13 (1991) 343 – 344. E. Giraud, A. Cloeckaert, D. Kerboeuf, E. Chaslus-Dancla, Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar Typhimurium, Antimicrob. Agents Chemother. 44 (2000) 1223 – 1228.

1507

[136] A. Olliver, M. Valle, E. Chaslus-Dancla, A. Cloeckaert, Overexpression of the multidrug efflux operon acrEF by insertional activation with IS1 and IS10 elements in Salmonella enterica serovar Typhimurium DT204 acrB mutants selected with fluoroquinolones, Antimicrob. Agents Chemother. 49 (2005) 289 – 301. [137] D.J. Eaves, V. Ricci, L.J. Piddock, Expression of acrB, acrF, acrD, marA, and soxS in Salmonella enterica serovar Typhimurium: role in multiple antibiotic resistance, Antimicrob. Agents Chemother. 48 (2004) 1145 – 1150. [138] E. Pradel, J.M. Pages, The AcrAB–TolC efflux pump contributes to multidrug resistance in the nosocomial pathogen Enterobacter aerogenes, Antimicrob. Agents Chemother. 46 (2002) 2640 – 2643. [139] A. Mazzariol, J. Zuliani, G. Cornaglia, G.M. Rossolini, R. Fontana, AcrAB efflux system: expression and contribution to fluoroquinolone resistance in Klebsiella spp., Antimicrob. Agents Chemother. 46 (2002) 3984 – 3986. [140] B.M. Allos, Campylobacter jejuni Infections: update on emerging issues and trends, Clin. Infect. Dis. 32 (2001) 1201 – 1206. [141] J. Lin, L.O. Michel, Q. Zhang, CmeABC functions as a multidrug efflux system in Campylobacter jejuni, Antimicrob. Agents Chemother. 46 (2002) 2124 – 2131. [142] V. Koronakis, A. Sharff, E. Koronakis, B. Luisi, C. Hughes, Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export, Nature 405 (2000) 914 – 919. [143] S. Murakami, R. Nakashima, E. Yamashita, A. Yamaguchi, Crystal structure of bacterial multidrug efflux transporter AcrB, Nature 419 (2002) 587 – 593. [144] M.K. Higgins, E. Bokma, E. Koronakis, C. Hughes, V. Koronakis, Structure of the periplasmic component of a bacterial drug efflux pump, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 9994 – 9999. [145] H. Akama, T. Matsuura, S. Kashiwagi, H. Yoneyama, S.-I. Narita, T. Tsukihara, A. Nakagawa, T. Nakae, Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa, J. Biol. Chem. 279 (2004) 25939 – 25942. [146] J. Eswaran, E. Koronakis, M.K. Higgins, C. Hughes, V. Koronakis, Three’s company: component structures bring a closer view of tripartite drug efflux pumps, Curr. Opin. Struct. Biol. 14 (2004) 741 – 747. [147] C. Andersen, E. Koronakis, E. Bokma, J. Eswaran, D. Humphreys, C. Hughes, V. Koronakis, Transition to the open state of the TolC periplasmic tunnel entrance, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 11103 – 11108. [148] H.I. Zgurskaya, H. Nikaido, Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 7190 – 7195. [149] W. Mao, M.S. Warren, D.S. Black, T. Satou, T. Murata, T. Nishino, N. Gotoh, O. Lomovskaya, On the mechanism of substrate specificity by resistance nodulation division (RND)type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition, Mol. Microbiol. 46 (2002) 889 – 901.

1508

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

[150] C.A. Elkins, H. Nikaido, Substrate specificity of the RNDtype multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops, J. Bacteriol. 184 (2002) 6490 – 6498. [151] E.B. Tikhonova, Q. Wang, H.I. Zgurskaya, Chimeric analysis of the multicomponent multidrug efflux transporters from gram-negative bacteria, J. Bacteriol. 184 (2002) 6499 – 6507. [152] S. Eda, H. Maseda, T. Nakae, An elegant means of selfprotection in gram-negative bacteria by recognizing and extruding xenobiotics from the periplasmic space, J. Biol. Chem. 278 (2003) 2085 – 2088. [153] E.W. Yu, J.R. Aires, H. Nikaido, AcrB multidrug efflux pump of Escherichia coli: composite substrate-binding cavity of exceptional flexibility generates its extremely wide substrate specificity, J. Bacteriol. 185 (2003) 5657 – 5664. [154] E.W. Yu, G. McDermott, H.I. Zgurskaya, H. Nikaido, D.E. Koshland Jr., Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump, Science 300 (2003) 976 – 980. [155] H.I. Zgurskaya, H. Nikaido, AcrA is a highly asymmetric protein capable of spanning the periplasm, J. Mol. Biol. 285 (1999) 409 – 420. [156] T. Thanabalu, E. Koronakis, C. Hughes, V. Koronakis, Substrate-induced assembly of a contiguous channel for protein export from E. coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore, EMBO J. 17 (1998) 6487 – 6496. [157] T. Touze, J. Eswaran, E. Bokma, E. Koronakis, C. Hughes, V. Koronakis, Interactions underlying assembly of the Escherichia coli AcrAB–TolC multidrug efflux system, Mol. Microbiol. 53 (2004) 697 – 706. [158] C.K. Stover, X.-Q. Pham, A.L. Erwin, S.D. Mizoguchi, P. Warrener, M.J. Hickey, F.S.L. Brinkman, W.O. Hufnagle, D.J. Kowalik, M. Lagrou, R.L. Garber, L. Goltry, E. Tolentino, S. Westbrock-Wadman, Y. Yuan, L.L. Brody, S.N. Coulter, K.R. Folger, A. Kas, K. Larbig, R. Lim, D. Spencer, G.K.-S. Wong, Z. Wu, I.T. Paulsen, J. Reizer, M.H. Saier, R.E.W. Hancock, S. Lory, M.V. Olson, Complete genome sequence of Pseudomonas aeruginosa, an opportunistic pathogen, Nature 406 (2000) 959 – 964. [159] D.G. Thanassi, L.W. Cheng, H. Nikaido, Active efflux of bile salts by Escherichia coli, J. Bacteriol. 179 (1997) 2512 – 2518. [160] W.M. Shafer, J.T. Balthazar, K.E. Hagman, S.A. Morse, Missense mutations that alter the DNA-binding domain of the MtrR protein that occur frequently in rectal isolates of Neisseria gonorrhoeae that are resistant to fecal lipids, Microbiology 141 (1995) 907 – 911. [161] R.B. Helling, B.K. Janes, H. Kimball, T. Tran, M. Bundesmann, P. Check, D. Phelan, C. Miller, Toxic waste disposal in Escherichia coli, J. Bacteriol. 184 (2002) 3699 – 3703. [162] S. Rahmati, S. Yang, A.L. Davidson, E.L. Zechiedrich, Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA, Mol. Microbiol. 43 (2002) 677 – 685. [163] E.C. Pesci, J.B.J. Milbank, J.P. Pearson, S. McKnight, A.S. Kende, E.P. Greenberg, B.H. Iglewski, Quinolone signalling in the cell-to-cell cummunication system of Pseudomonas

[164]

[165]

[166]

[167]

[168]

[169]

[170]

[171]

[172]

[173]

[174]

[175]

[176]

[177]

aeruginosa, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 11229 – 11234. T. Kohler, C. van Delden, L. Kocjanic Curty, M.M. Hamzehpour, J.-C. Pechere, Overexpression of the MexEF–OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa, J. Bacteriol. 183 (2001) 5213 – 5222. Y. Hirakata, R. Srikumar, K. Poole, N. Gotoh, T. Suematsu, S. Kohno, S. Kamihira, R.E. Hancock, D.P. Speert, Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa, J. Exp. Med. 196 (2002) 109 – 118. H.E. Jones, I.B. Holland, A. Jacq, T. Wall, A.K. Campbell, Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca2+ channels in the membrane, Biochim. Biophys. Acta 1612 (2003) 90 – 97. P. Orth, D. Schnappinger, W. Hillen, W. Saenger, W. Hinrichs, Structural basis of gene regulation by the tetracycline inducible Tet repressor–operator system, Nat. Struct. Biol. 7 (2000) 215 – 219. H. Wang, J.L. Dzink-Fox, M. Chen, S.B. Levy, Genetic characterization of highly fluoroquinolone-resistant clinical Escherichia coli strains from China: role of acrR mutations, Antimicrob. Agents Chemother. 45 (2001) 1515 – 1521. D. Ma, D.N. Cook, J.E. Hearst, H. Nikaido, Efflux pumps and drug resistance in Gram-negative bacteria, Trends Microbiol. 2 (1994) 489 – 493. L. Zhang, X.-Z. Li, K. Poole, SmeDEF multidrug efflux pump contributes to intrinsic multidrug resistance in Stenotrophomonas maltophilia, Antimicrob. Agents Chemother. 45 (2001) 3497 – 3503. K. Evans, L. Adewoye, K. Poole, MexR repressor of the mexAB–oprM multidrug efflux operon of Pseudomonas aeruginosa: identification of MexR binding sites in the mexA– mexR intergenic region, J. Bacteriol. 183 (2001) 807 – 812. L. Adewoye, A. Sutherland, R. Srikumar, K. Poole, The MexR repressor of the mexAB–oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutants compromising activity, J. Bacteriol. 184 (2002) 4308 – 4312. S. Jalal, O. Ciofu, N. Hoiby, N. Gotoh, B. Wretlind, Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients, Antimicrob. Agents Chemother. 44 (2000) 710 – 712. T. Kohler, S.F. Epp, L.K. Curty, J.C. Pechere, Characterization of MexT, the regulator of the MexE–MexF–OprN multidrug efflux system of Pseudomonas aeruginosa, J. Bacteriol. 181 (1999) 6300 – 6305. H. Maseda, K. Saito, A. Nakajima, T. Nakae, Variation of the mexT gene, a regulator of the MexEF–OprN efflux pump expression in wild-type strains of Pseudomonas aeruginosa, FEMS Microbiol. Lett. 192 (2000) 107 – 112. K. Nishino, A. Yamaguchi, EvgA of the two-component signal transduction system modulates production of the YhiUV multidrug transporter in Escherichia coli, J. Bacteriol. 184 (2002) 2319 – 2323. Y. Eguchi, T. Oshima, H. Mori, R. Aono, K. Yamamoto, A. Ishihama, R. Utsumi, Transcriptional regulation of drug effux

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

[178]

[179]

[180]

[181]

[182]

[183]

[184]

[185]

[186]

[187]

[188]

[189]

[190]

genes by EvgAS, a two-component system in Escherichia coli, Microbiology 149 (2003) 2819 – 2828. K. Perron, O. Caille, C. Rossier, C. van Delden, J.-L. Dumas, T. Kohler, CzcR–CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa, J. Biol. Chem. 279 (2004) 8761 – 8768. M.N. Alekshun, S.B. Levy, Regulation of chromosomally mediated multiple antibiotic resistance: the mar operons, Antimicrob. Agents Chemother. 41 (1997) 2067 – 2075. N. Delihas, S. Forst, micF: an antisense RNA gene involved in response of Escherichia coli to global stress factors, J. Mol. Biol. 313 (2001) 1 – 12. R. Chollet, C. Bollet, J. Chevalier, M. Mallea, J.M. Pages, A. Davin-Regli, mar Operon involved in multidrug resistance of Enterobacter aerogenes, Antimicrob. Agents Chemother. 46 (2002) 1093 – 1097. D.G. White, J.D. Goldman, B. Demple, S.B. Levy, Role of the acrAB locus in organic solvent tolerance mediated by expression fo marA, soxS, or robA in Escherichia coli, J. Bacteriol. 179 (1997) 6122 – 6126. T. Nunoshiba, E. Hidalgo, C.F. Amabile Cuevas, B. Demple, Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein tiggers redox-inducible expression of the soxS regulatory gene, J. Bacteriol. 174 (1992) 6054 – 6060. R.G. Martin, J.L. Rosner, Fis, an accessorial factor for transcriptional activation of the mar (multiple antibiotic resistance) promoter of Escherichia coli in the presence of the activator MarA, SoxS, or Rob, J. Bacteriol. 179 (1997) 7410 – 7419. E.Y. Rosenberg, D. Bertenthal, M.L. Nilles, K.P. Bertrand, H. Nikaido, Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein, Mol. Microbiol. 48 (2003) 1609 – 1619. S. Yaron, D.G. White, K.R. Matthews, Characterization of an Escherichia coli O157:H7 marR mutant, Int. J. Food Microbiol. 85 (2003) 281 – 291. M.M. Tavio, J. Vila, M. Perilli, L.T. Casanas, L. Macia, G. Amicosante, M.T. Jimenez de Anta, Enhanced active efflux, repression of porin synthesis and development of Mar phenotype by diazepam in two enterobacteria strains, J. Med. Microbiol. 53 (2004) 1119 – 1122. A.M. George, R.M. Hall, H.W. Stokes, Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli, Microbiology 141 (1995) 1909 – 1920. A. Ruzin, M.A. Visalli, D. Keeney, P.A. Bradford, Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae, Antimicrob. Agents Chemother. 49 (2005) 1017 – 1022. M.A. Yassein, H.E. Ewis, C.-D. Lu, A.T. Abdelal, Molecular cloning and characterization of the Salmonella enterica serovar Paratyphi B rma gene, which confers multiple drug resistance in Escherichia coli, Antimicrob. Agents Chemother. 44 (2002) 10 – 13.

1509

[191] R. Chollet, J. Chevalier, C. Bollet, J.M. Pages, A. DavinRegli, RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes, Antimicrob. Agents Chemother. 48 (2004) 2518 – 2523. [192] C. Llanes, D. Hocquet, C. Vogne, D. Benali-Baitich, C. Neuwirth, P. Plesiat, Clinical strains of Pseudomonas aeruginosa overproducing MexAB–OprM and MexXY efflux pumps simultaneously, Antimicrob. Agents Chemother. 48 (2004) 1797 – 1802. [193] R. Srikumar, P.C.J.K. Poole, Influence of mutations in the mexR repressor gene on expression of the MexA–MexB– OprM multidrug efflux system of Pseudomonas aeruginosa, J. Bacteriol. 182 (2000) 1410 – 1414. [194] O. Lomovskaya, W.J. Watkins, Efflux pumps: their role in antibacterial drug discovery, Curr. Med. Chem. 8 (2001) 1699 – 1711. [195] O. Lomovskaya, W. Watkins, Inhibition of efflux pumps as a novel approach to combat drug resistance in bacteria, J. Mol. Microbiol. Biotechnol. 3 (2001) 225 – 236. [196] O. Lomovskaya, M.S. Warren, A. Lee, J. Galazzo, R. Fronko, M. Lee, J. Blais, D. Cho, S. Chamberland, T. Renau, R. Leger, S. Hecker, W. Watkins, K. Hoshino, H. Ishida, V.J. Lee, Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy, Antimicrob. Agents Chemother. 45 (2001) 105 – 116. [197] T.E. Renau, R. Leger, E.M. Flamme, J. Sangalang, M.W. She, R. Yen, C.L. Gannon, D. Griffith, S. Chamberland, O. Lomovskaya, S.J. Hecker, V.J. Lee, T. Ohta, K. Nakayama, Inhibitors of efflux pumps in Pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antibacterial levofloxacin, J. Med. Chem. 42 (1999) 4928 – 4931. [198] T.E. Renau, R. Leger, E.M. Flamme, M.W. She, C.L. Gannon, K.M. Mathias, O. Lomovskaya, S. Chamberland, V.J. Lee, T. Ohta, K. Nakayama, Y. Ishida, Addressing the stability of C-capped dipeptide efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa, Bioorg. Med. Chem. Lett. 11 (2001) 663 – 667. [199] D. Griffith, O. Lomovskaya, V. Lee, et al., Abstract presented at the 39th Interscience Conference on Antimicrobials Agents and Chemotherapy, San Francisco, CA, 1999. [200] M. Mallea, J. Chevalier, A. Eyraud, J.M. Pages, Inhibitors of antibiotic efflux pump in resistant Enterobacter aerogenes strains, Biochem. Biophys. Res. Commun. 293 (2002) 1370 – 1373. [201] L. Mamelli, J.P. Amoros, J.M. Pages, J.M. Bolla, A phenylalanine–arginine beta-naphthylamide sensitive multidrug efflux pump involved in intrinsic and acquired resistance of Campylobacter to macrolides, Int. J. Antimicrob. Agents 22 (2003) 237 – 241. [202] M.D. Lee, J.L. Galazzo, A.L. Staley, J.C. Lee, M.S. Warren, H. Fuernkranz, S. Chamberland, O. Lomovskaya, G.H. Miller, Microbial fermentation-derived inhibitors of efflux-pumpmediated drug resistance, Farmaco 56 (2001) 81 – 85. [203] J. Chevalier, J. Bredin, A. Mahamoud, M. Mallea, J. Barbe, J.M. Pages, Inhibitors of antibiotic efflux in resistant Enter-

1510

[204]

[205]

[206]

[207]

[208]

[209]

[210]

[211]

[212]

[213]

[214]

[215]

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513 obacter aerogenes and Klebsiella pneumoniae strains, Antimicrob. Agents Chemother. 48 (2004) 1043 – 1046. M.L. Nelson, S.B. Levy, Reversal of tetracycline resistance mediated by different bacterial tetracycline resistance determinants by an inhibitor of the Tet(B) antiport protein, Antimicrob. Agents Chemother. 43 (1999) 1719 – 1724. F.R. Stermitz, P. Lorenz, J.N. Tawara, L.A. Zenewicz, K. Lewis, Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5V-methoxyhydnocarpin, a multidrug pump inhibitor, Proc. Natl. Acad. Sci. U. S. A. (2000) 1433 – 1437. G. Tegos, F.R. Stermitz, O. Lomovskaya, K. Lewis, Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials, Antimicrob. Agents Chemother. 46 (2002) 3133 – 3141. N.R. Guz, F.R. Stermitz, J.B. Johnson, T.D. Beeson, S. Willen, J. Hsiang, K. Lewis, Flovaonolignan and flavone inhibitors of a Staphylococcus aureus multidrug resistance pump: structure activity relationship, J. Med. Chem. 44 (2001) 261 – 268. H. Fukuda, S. Hori, K. Hiramatsu, Antibacterial activity of gatifloxacin (AM-1155, CG5501, BMS-206584), a newly developed fluoroquinolone, against sequentially acquired quinolone-resistant mutants and the norA transformant of Staphylococcus aureus, Antimicrob. Agents Chemother. 42 (1998) 1917 – 1922. T.D. Gootz, R.P. Zaniewski, S.L. Haskell, F.S. Kaczmarek, A.E. Maurice, Activities of trovafloxacin compared with those of other fluoroquinolones against purified topoisomerases and gyrA and grlA mutants of Staphylococcus aureus, Antimicrob. Agents Chemother. 43 (1999) 1845 – 1855. Y. Someya, A. Yamaguchi, T. Sawai, A novel glycylcycline, 9(N,N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline, is neither transported nor recognized by the transposon Tn10encoded metal-tetracycline/H+ antiporter, Antimicrob. Agents Chemother. 39 (1995) 247 – 249. C.R. Dean, M.A. Visalli, S.J. Projan, P.E. Sum, P.A. Bradford, Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1, Antimicrob. Agents Chemother. 47 (2003) 972 – 978. M.A. Visalli, E. Murphy, S.J. Projan, P.A. Bradford, AcraAB multidrug efflux pump is associated with reduced levels of susceptibility to tigecycline (GAR-936) in Proteus mirabilis, Antimicrob. Agents Chemother. 47 (2003) 665 – 669. A. Ruzin, D. Keeney, P.A. Bradford, AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in Morganella morganii, Antimicrob. Agents Chemother. 49 (2005) 791 – 793. R.E.W. Hancock, F.S. Brinkman, Function of Pseudomonas porins in uptake and efflux, Annu. Rev. Microbiol. 56 (2002) 17 – 38. H. Labischinski, G. Barnickel, H. Bradaczek, D. Naumann, E.T. Rietschel, P. Giesbrecht, High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane, J. Bacteriol. 162 (1985) 9 – 20.

[216] A. Wiese, K. Brandenburg, A.J. Ulmer, U. Seydel, S. MullerLoennies, The dual role of lipopolysaccharide as effector and target molecule, Biol. Chem. 380 (1999) 767 – 784. [217] I. Sadovskaya, J.R. Brisson, J.S. Lam, J.C. Richards, E. Altman, Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chaindeficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5, Eur. J. Biochem. 255 (1998) 673 – 684. [218] M. Vaara, Outer membrane permeability barrier to azithromycin, clarithromycin, and roxithromycin in gram-negative enteric bacteria, Antimicrob. Agents Chemother. 37 (1993) 354 – 356. [219] P. Plesiat, J.R. Aires, C. Godard, T. Kohler, Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa, J. Bacteriol. 179 (1997) 7004 – 7010. [220] J. Nesper, C.M. Lauriano, K.E. Klose, D. Kapfhammer, A. Kraiss, J. Reidl, Characterization of Vibrio cholerae O1 El tor galU and galE mutants: influence on lipopolysaccharide structure, colonization, and biofilm formation, Infect. Immun. 69 (2001) 435 – 445. [221] I. Moriyon, I. Lopez-Goni, Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis, Int. Microbiol. 1 (1998) 19 – 26. [222] J. Velasco, J.A. Bengoechea, K. Brandenburg, B. Lindner, U. Seydel, D. Gonzalez, U. Zahringer, E. Moreno, I. Moriyon, Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance, Infect. Immun. 68 (2000) 3210 – 3218. [223] J.A. Bengoechea, B. Lindner, U. Seydel, R. Diaz, I. Moriyon, Yersinia pseudotuberculosis and Yersinia pestis are more resistant to bactericidal cationic peptides than Yersinia enterocolitica, Microbiology 144 (1998) 1509 – 1515. [224] J.A. Bengoechea, K. Brandenburg, U. Seydel, R. Diaz, I. Moriyon, Yersinia pseudotuberculosis and Yersinia pestis show increased outer membrane permeability to hydrophobic agents which correlates with lipopolysaccharide acyl-chain fluidity, Microbiology 144 (1998) 1517 – 1526. [225] M.N. Burtnick, D.E. Woods, Isolation of polymyxin Bsusceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance, Antimicrob. Agents Chemother. 43 (1999) 2648 – 2656. [226] W. Kaca, J. Radziejewska-Lebrecht, U.R. Bhat, Effect of polymyxins on the lipopolysaccharide-defective mutants of Proteus mirabilis, Microbios 61 (1990) 23 – 32. [227] I.M. Helander, I. Kilpelainen, M. Vaara, Increased substitution of phosphate groups in lipopolysaccharides and lipid A of the polymyxin-resistant pmrA mutants of Salmonella typhimurium: a 31P-NMR study, Mol. Microbiol. 11 (1994) 481 – 487. [228] K. Nummila, I. Kilpelainen, U. Zahringer, M. Vaara, I.M. Helander, Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain aminoarabinose in lipid A, Mol. Microbiol. 16 (1995) 271 – 278.

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513 [229] F. Yoshimura, H. Nikaido, Diffusion of beta-lactam antibiotics through the porin channels of Escherichia coli K-12, Antimicrob. Agents Chemother. 27 (1985) 84 – 92. [230] H. Nikaido, E.Y. Rosenberg, Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli, J. Gen. Physiol. 77 (1981) 121 – 135. [231] F.W. Goldstein, L. Gutmann, R. Williamson, E. Collatz, J.F. Acar, In vivo and in vitro emergence of simultaneous resistance to both beta-lactam and aminoglycoside antibiotics in a strain of Serratia marcescens, Ann. Microbiol. (Paris) 134A (1983) 329 – 337. [232] K.J. Harder, H. Nikaido, M. Matsuhashi, Mutants of Escherichia coli that are resistant to certain beta-lactam compounds lack the ompF porin, Antimicrob. Agents Chemother. 20 (1981) 549 – 552. [233] V. Werner, C.C. Sanders, W.E. Sanders Jr., R.V. Goering, Role of beta-lactamases and outer membrane proteins in multiple beta-lactam resistance of Enterobacter cloacae, Antimicrob. Agents Chemother. 27 (1985) 455 – 459. [234] A.A. Medeiros, T.F. O’Brien, E.Y. Rosenberg, H. Nikaido, Loss of OmpC porin in a strain of Salmonella typhimurium causes increased resistance to cephalosporins during therapy, J. Infect. Dis. 156 (1987) 751 – 757. [235] C.S. Toro, S.R. Lobos, I. Calderon, M. Rodriguez, G.C. Mora, Clinical isolate of a porinless Salmonella typhi resistant to high levels of chloramphenicol, Antimicrob. Agents Chemother. 34 (1990) 1715 – 1719. [236] J.L. Burns, A.L. Smith, A major outer-membrane protein functions as a porin in Haemophilus influenzae, J. Gen. Microbiol. 133 (Pt 5) (1987) 1273 – 1277. [237] B. Pangon, C. Bizet, A. Bure, F. Pichon, A. Philippon, B. Regnier, L. Gutmann, In vivo selection of a cephamycinresistant, porin-deficient mutant of Klebsiella pneumoniae producing a TEM-3 beta-lactamase, J. Infect. Dis. 159 (1989) 1005 – 1006. [238] M. Mallea, J. Chevalier, C. Bornet, A. Eyraud, A. DavinRegli, C. Bollet, J.M. Pages, Porin alteration and active efflux: two in vivo drug resistance strategies used by Enterobacter aerogenes, Microbiology 144 (1998) 3003 – 3009. [239] J.W. Chow, D.M. Shlaes, Imipenem resistance associated with the loss of a 40 kDa outer membrane protein in Enterobacter aerogenes, J. Antimicrob. Chemother. 28 (1991) 499 – 504. [240] C. Bornet, A. Davin-Regli, C. Bosi, J.M. Pages, C. Bollet, Imipenem resistance of Enterobacter aerogenes mediated by outer membrane permeability, J. Clin. Microbiol. 38 (2000) 1048 – 1052. [241] J. Chevalier, J.M. Pages, M. Mallea, In vivo modification of porin activity conferring antibiotic resistance to Enterobacter aerogenes, Biochem. Biophys. Res. Commun. 266 (1999) 248 – 251. [242] E. De, A. Basle, M. Jaquinod, N. Saint, M. Mallea, G. Molle, J.M. Pages, A new mechanism of antibiotic resistance in Enterobacteriaceae induced by a structural modification of the major porin, Mol. Microbiol. 41 (2001) 189 – 198.

1511

[243] G. Cornaglia, A. Mazzariol, R. Fontana, G. Satta, Diffusion of carbapenems through the outer membrane of enterobacteriaceae and correlation of their activities with their periplasmic concentrations, Microb. Drug Resist. 2 (1996) 273 – 276. [244] V.T. Cao, G. Arlet, B.M. Ericsson, A. Tammelin, P. Courvalin, T. Lambert, Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmidmediated CMY-4 and permeability alteration, J. Antimicrob. Chemother. 46 (2000) 895 – 900. [245] S. Hernandez-Alles, M. Conejo, A. Pascual, J.M. Tomas, V.J. Benedi, L. Martinez-Martinez, Relationship between outer membrane alterations and susceptibility to antimicrobial agents in isogenic strains of Klebsiella pneumoniae, J. Antimicrob. Chemother. 46 (2000) 273 – 277. [246] M.J. Gill, S. Simjee, K. Al-Hattawi, B.D. Robertson, C.S. Easmon, C.A. Ison, Gonococcal resistance to beta-lactams and tetracycline involves mutation in loop 3 of the porin encoded at the penB locus, Antimicrob. Agents Chemother. 42 (1998) 2799 – 2803. [247] H. Nikaido, R.E. Hancock, in: J.R. Sokatch (Ed.), The Bacteria: A Treatise on Structure and Function, Academic Press, London, 1986, pp. 145 – 193. [248] W.A. Woodruff, R.E. Hancock, Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene, J. Bacteriol. 170 (1988) 2592 – 2598. [249] L. Pumbwe, M.J. Everett, R.E. Hancock, L.J. Piddock, Role of gyrA mutation and loss of OprF in the multiple antibiotic resistance phenotype of Pseudomonas aeruginosa G49, FEMS Microbiol. Lett. 143 (1996) 25 – 28. [250] J.P. Quinn, E.J. Dudek, C.A. DiVincenzo, D.A. Lucks, S.A. Lerner, Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections, J. Infect. Dis. 154 (1986) 289 – 294. [251] L. Martinez-Martinez, A. Pascual, M.C. Conejo, L. Picabea, E.J. Perea, Resistance of Pseudomonas aeruginosa to imipenem induced by eluates from siliconized latex urinary catheters is related to outer membrane protein alterations, Antimicrob. Agents Chemother. 43 (1999) 397 – 399. [252] J.P. Pirnay, D. De Vos, D. Mossialos, A. Vanderkelen, P. Cornelis, M. Zizi, Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates, Environ. Microbiol. 4 (2002) 872 – 882. [253] D.J. Wolter, N.D. Hanson, P.D. Lister, Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance, FEMS Microbiol. Lett. 236 (2004) 137 – 143. [254] Y. Yamano, T. Nishikawa, Y. Komatsu, Outer membrane proteins responsible for the penetration of beta-lactams and quinolones in Pseudomonas aeruginosa, J. Antimicrob. Chemother. 26 (1990) 175 – 184. [255] A. Tabata, H. Nagamune, T. Maeda, K. Murakami, Y. Miyake, H. Kourai, Correlation between resistance of Pseudomonas aeruginosa to quaternary ammonium compounds and expression of outer membrane protein OprR, Antimicrob. Agents Chemother. 47 (2003) 2093 – 2099.

1512

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513

[256] R.E. Hancock, Alterations in outer membrane permeability, Annu. Rev. Microbiol. 38 (1984) 237 – 264. [257] S. Sukupolvi, M. Vaara, Salmonella typhimurium and Escherichia coli mutants with increased outer membrane permeability to hydrophobic compounds, Biochim. Biophys. Acta 988 (1989) 377 – 387. [258] M. Vaara, M. Nurminen, Outer membrane permeability barrier in Escherichia coli mutants that are defective in the late acyltransferases of lipid A biosynthesis, Antimicrob. Agents Chemother. 43 (1999) 1459 – 1462. [259] R.E. Hancock, The bacterial outer membrane as a drug barrier, Trends Microbiol. 5 (1997) 37 – 42. [260] R.E. Hancock, Antibacterial peptides and the outer membranes of gram-negative bacilli, J. Med. Microbiol. 46 (1997) 1 – 3. [261] H.M. Ayres, D.N. Payne, J.R. Furr, A.D. Russell, Effect of permeabilizing agents on antibacterial activity against a simple Pseudomonas aeruginosa biofilm, Lett. Appl. Microbiol. 27 (1998) 79 – 82. [262] R.M. Epand, H.J. Vogel, Diversity of antimicrobial peptides and their mechanisms of action, Biochim. Biophys. Acta 1462 (1999) 11 – 28. [263] M. Vaara, Agents that increase the permeability of the outer membrane, Microbiol. Rev. 56 (1992) 395 – 411. [264] M. Vaara, M. Porro, Group of peptides that act synergistically with hydrophobic antibiotics against gram-negative enteric bacteria, Antimicrob. Agents Chemother. 40 (1996) 1801 – 1805. [265] K. Kikuchi, E.M. Bernard, A. Sadownik, S.L. Regen, D. Armstrong, Antimicrobial activities of squalamine mimics, Antimicrob. Agents Chemother. 41 (1997) 1433 – 1438. [266] A. Giacometti, O. Cirioni, M.S. Del Prete, F. Barchiesi, A.M. Paggi, E. Petrelli, G. Scalise, Comparative activities of polycationic peptides and clinically used antimicrobial agents against multidrug-resistant nosocomial isolates of Acinetobacter baumannii, J. Antimicrob. Chemother. 46 (2000) 807 – 810. [267] A. Giacometti, O. Cirioni, M.S. Del Prete, A.M. Paggi, M.M. D’Errico, G. Scalise, Combination studies between polycationic peptides and clinically used antibiotics against Grampositive and Gram-negative bacteria, Peptides 21 (2000) 1155 – 1160. [268] C. Li, M.R. Lewis, A.B. Gilbert, M.D. Noel, D.H. Scoville, G.W. Allman, P.B. Savage, Antimicrobial activities of amine- and guanidine-functionalized cholic acid derivatives, Antimicrob. Agents Chemother. 43 (1999) 1347 – 1349. [269] X.Z. Li, L. Zhang, K. Poole, Interplay between the MexA– MexB–OprM multidrug efflux system and the outer membrane barrier in the multiple antibiotic resistance of Pseudomonas aeruginosa, J. Antimicrob. Chemother. 45 (2000) 433 – 436. [270] X.Z. Li, L. Zhang, H. Nikaido, Efflux pump-mediated intrinsic drug resistance in Mycobacterium smegmatis, Antimicrob. Agents Chemother. 48 (2004) 2415 – 2423. [271] X. Li, M. Zolli-Juran, J.D. Cechetto, D.M. Daigle, G.D. Wright, E.D. Brown, Multicopy suppressors for novel anti-

[272]

[273]

[274]

[275]

[276]

[277]

[278]

[279]

[280]

[281]

[282]

[283]

[284]

[285]

bacterial compounds reveal targets and drug efflux susceptibility, Chem. Biol. 11 (2004) 1423 – 1430. L. Duncan, T. Doyle, Q. Du, J. Ehrhardt, A.S. Lynch, K. Mduli, G.T. Robertson, Construction of a novel Pseudomonas aeruginosa strain panel deficient in fifty-two potential drug efflux systems, Abstract presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy Washington, DC, 2004. W.T. Peng, E.W. Nester, Characterization of a putative RNDtype efflux system in Agrobacterium tumefaciens, Gene 270 (2001) 245 – 252. J.D. Palumbo, C.I. Kado, D.A. Phillips, An isoflavonoidinducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots, J. Bacteriol. 180 (1998) 3107 – 3113. P. Krummenacher, F. Narberhaus, Two genes encoding a putative multidrug efflux pump of the RND/MFP family are cotranscribed with an rpoH gene in Bradyrhizobium japonicum, Gene 241 (2000) 247 – 254. H.J. Linde, F. Notka, C. Irtenkauf, J. Decker, J. Wild, H.H. Niller, P. Heisig, N. Lehn, Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae, J. Antimicrob. Chemother. 49 (2002) 625 – 630. J.A. Fralick, Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli, J. Bacteriol. 178 (1996) 5803 – 5805. C.A. Elkins, H. Nikaido, Substrate specificity of the RNDtype multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops, J. Bacteriol. 184 (2002) 6490 – 6498. L. Sanchez, W. Pan, M. Vinas, H. Nikaido, The acrAB homolog of Haemophilus influenzae codes for a functional multidrug efflux pump, J. Bacteriol. 179 (1997) 6855 – 6857. C.E. Lucas, J.T. Balthazar, K.E. Hagman, W.M. Shafer, The MtrR repressor binds the DNA sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae, J. Bacteriol. 179 (1997) 4123 – 4128. E.H. Lee, W.M. Shafer, The farAB-encoded efflux pump mediates resistance of gonococci to long-chained antibacterial fatty acids, Mol. Microbiol. 33 (1999) 839 – 845. T. Ikeda, F. Yoshimura, A resistance-nodulation-cell division family xenobiotic efflux pump in an obligate anaerobe, Porphyromonas gingivalis, Antimicrob. Agents Chemother. 46 (2002) 3257 – 3260. M.A. Visalli, E. Murphy, S.J. Projan, P.A. Bradford, AcrAB multidrug efflux pump is associated with reduced levels of susceptibility to tigecycline (GAR-936) in Proteus mirabilis, Antimicrob. Agents Chemother. 47 (2003) 665 – 669. K. Poole, K. Tetro, Q. Zhao, S. Neshat, D. Heinrichs, N. Bianco, Expression of the multidrug resistance operon mexA– mexB–oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression, Antimicrob. Agents Chemother. 40 (1996) 2021 – 2028. Y. Li, T. Mima, Y. Komori, Y. Morita, T. Kuroda, T. Mizushima, T. Tsuchiya, A new member of the tripartite multidrug efflux pumps, MexVW–OprM, in Pseudomonas aeruginosa, J. Antimicrob. Chemother. 52 (2003) 572 – 575.

A. Kumar, H.P. Schweizer / Advanced Drug Delivery Reviews 57 (2005) 1486–1513 [286] J. Kieboom, J.J. Dennis, J.A. de Bont, G.J. Zylstra, Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance, J. Biol. Chem. 273 (1998) 85 – 91. [287] J.L. Ramos, E. Duque, P. Godoy, A. Segura, Efflux pumps involved in toluene tolerance of Pseudomonas putida DOT1E, J. Bacteriol. 180 (1998) 3323 – 3329. [288] F. Fukumori, H. Hirayama, H. Takami, A. Inoue, K. Horikoshi, Isolation and transposon mutagenesis of a Pseudomonas putida KT2442 toluene-resistant variant: involvement of an efflux system in solvent resistance, Extremophiles 2 (1998) 395 – 400. [289] G. Mosqueda, J.L. Ramos, A set of genes encoding a second toluene efflux system from Pseudomonas putida DOT-1E is linked to the tod genes for toluene metabolism, J. Bacteriol. 182 (2000) 937 – 943. [290] A. Rojas, E. Duque, G. Mosqueda, G. Golden, A. Hurtado, J.L. Ramos, A. Segura, Three efflux pumps are

1513

required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E, J. Bacteriol. 183 (2001) 3967 – 3973. [291] H. Nikaido, M. Basina, V. Nguyen, E.Y. Rosenberg, Multidrug efflux pump AcrAB of Salmonella typhimurium excretes only those beta-lactam antibiotics containing lipophilic side chains, J. Bacteriol. 180 (1998) 4686 – 4692. [292] F.J. Lacroix, A. Cloeckaert, O. Grepinet, C. Pinault, M.Y. Popoff, H. Waxin, P. Pardon, Salmonella typhimurium acrBlike gene: identification and role in resistance to biliary salts and detergents and in murine infection, FEMS Microbiol. Lett. 135 (1996) 161 – 167. [293] A. Kumar, E.A. Worobec, Fluoroquinolone resistance of Serratia marcescens: involvement of a proton gradient-dependent efflux pump, J. Antimicrob. Chemother. 50 (2002) 593 – 596.