The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

Classes of Efflux PumpsBecause there are so many different efflux transporters, the only feasible way for their classification is to use phylogenetic grouping, based on protein sequences. Such a classification for all transporter proteins has been established by Milton Saier's group (17 – 19) and is available in the Transporter Classification Database (http://www.tcdb.org/). Transporter genes in hundreds of sequenced bacterial genomes are classified in Ian Paulsen's database (20) for each of these genomes (http://www.membranetransport.org/). Among many families of transporters, several contain prominent members of efflux transporters: especially important in bacteria are the RND, MFS (major facilitator superfamily), MATE (multidrug and toxic compound extrusion), SMR (small multidrug resistance), and ABC (ATP-binding cassette) superfamilies or families. ABC transporters utilize ATP hydrolysis as the energy source, but all others are dependent on proton motive force and are thus secondary transporters or proton/drug antiporters.

The transporters also differ in their subcellular organization. The RND pumps, which are all exporters of drugs and toxic cations, are located in the inner membrane (IM) (cytoplasmic membrane) but must interact with the periplasmic adaptor protein (also called membrane fusion protein) and the outer membrane (OM) channel, thus producing a tripartite complex spanning the IM, the periplasm, and the OM (represented by E. coli AcrAB-TolC and P. aeruginosa MexAB-OprM) (see the multicomponent pump depicted in Fig. 1). Some members of the ABC superfamily (e.g., MacB), the MATE family (e.g., MdtK), and even the MFS (e.g., EmrB) (all from E. coli) also are organized in this manner. The tripartite transporters excrete drugs directly into the external medium so that the reentry of drugs requires the slow traversal of the OM, an effective permeability barrier (21, 22). For this reason, these pumps are far more efficient in creating detectable resistance to antibiotics (especially AcrB, a constitutive RND transporter of E. coli [9]) (see Gammaproteobacteria: Enterobacteriaceae, below). In contrast, the pumps that are not organized in this manner and exist as single-component or “singlet” pumps in the IM (Fig. 1), including the vast majority of MFS and SMR pumps, are less effective in producing a detectable decrease in susceptibility, because the drug molecules are excreted only into the periplasm and can spontaneously diffuse back into the cytosol, since most antibiotics are relatively lipophilic molecules that can cross the phospholipid bilayer region of the IM. However, RND pumps, which are thought to capture antibiotics mostly from the periplasm (23, 24), can collaborate with the singlet pumps and thus increase their efficacy (25, 26).

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FIG 1

Location of drug efflux pumps and pathways of drug influx and efflux across the OM and IM in Gram-negative bacteria. The influx of drugs (shown as pills) through the OM occurs in one or more of the following three pathways: porin channels (e.g., OmpF of E. coli and OprF of P. aeruginosa), specific protein channels (e.g., CarO of A. baumannii and OprD of P. aeruginosa for carbapenems), and the LPS-containing asymmetric lipid bilayer region. After their entry into the periplasmic space, the drug molecules can further penetrate the IM via diffusion. However, these drugs can be extruded out of the cell by efflux transporters, which exist as either single-component pumps (“singlet”; e.g., Tet pumps) or multicomponent pumps (e.g., AcrAB-TolC and MexAB-OprM tripartite efflux systems that each typically contain a pump, an OM channel protein [OMP], and an accessory membrane fusion protein [MFP]). While the singlet pumps may take up the drug from the cytosol and the periplasm and function with porins or other types of protein channels to make the efflux process effective, the multicomponent exporters capture their substrates from the periplasm and the IM and directly pump them into the medium. The competition between the influx and efflux processes ultimately determines the steady state of drug molecules in bacterial cells. With the lipophilic drug molecules that cross the OM slowly or the hydrophilic drugs that penetrate the A. baumannii/P. aeruginosa low-permeability porins (i.e., “slow porins”), the efflux mechanism become very effective, thus being able to yield MDR. In contrast, with the less hydrophobic and smaller drug molecules that can rapidly penetrate, for example, E. coli porins, efflux is not effective to counteract drug influx, thus hardly decreasing the concentrations of the drug in the cell.

The most detailed information on the contribution of various pumps to drug susceptibility is available for E. coli K-12, and Table 1 lists data on known and predicted multidrug pumps identified in the Transporter Classification Database mentioned above. An obvious way to detect the contribution of individual pumps is to measure the MICs of drugs in defective mutants. This was done in 2001 by Sulavik and coworkers (27) and showed that the RND transporter AcrB (in cooperation with its periplasmic and OM partners AcrA and TolC) plays a truly predominant role in raising the MIC levels in a wild-type strain. This also creates a problem because deletion of other pumps rarely produces detectable changes in MICs in the presence of the active AcrB-AcrA-TolC system. A similar problem was reported in a study (28) examining the MIC values of nearly 4,000 deletion mutants of all nonessential E. coli genes (the “Keio collection” [29]). Thus, although that study showed that the functions of many metabolic genes have an unsuspected influence on drug sensitivity, in terms of transporter genes, it essentially identified the effect of only the acrAB-tolC complex and nothing else. One possible exception is the deletion of the ycdZ gene, which produced hypersusceptibility to tetracycline and may code for an exporter. However, this conclusion is not supported by a study from the Carol Gross group, who quantitated the growth phenotype of the same set of mutants in the presence of sub-MICs of various drugs (30). This approach is more sensitive than the determination of MIC values and, indeed, as presented in Table 1, showed that the deletion of practically all known and suspected pumps produces hypersusceptibility to at least one agent tested. These results, however, must be interpreted with care, since this approach is very sensitive and could produce false-positive results in spite of efforts to avoid them (Table 1).

A completely different approach is the plasmid-based overexpression of putative efflux genes. This analysis by Nishino and Yamaguchi (31) indeed detected efflux activity in those genes whose activity was difficult to detect by deletion-based approaches. However, these data do not tell us whether the pumps are functioning in the wild-type or even mutant cells, although the level of expression of most pumps can be increased by regulatory signals (see Regulation of Multidrug Efflux Pumps, below).

Reviews describing various types of efflux pumps include those written by Poole (32, 33), Piddock (34 – 36), Paulsen et al. (37), Saier and others (38), Van Bambeke and others (39), and Higgins (40). A review by Alekshun and Levy (41) is useful, as it also emphasizes the contribution of nonefflux mechanisms of resistance.

MFS TransportersMFS transporters can be classified into at least 74 families on the basis of sequence homology (130). E. coli K-12 contains 70 MFS transporters, 15 of which may be considered drug exporters, as they belong to families 2 and 3 (http://www.membranetransport.org/), which are composed of 12-TMS (transmembrane segment) and 14-TMS members, respectively, of drug/H⁺ antiporters (37, 130). Most of them, however, are free-standing transporters located in the IM and transport drugs from the cytosol to only the periplasm. Because most antimicrobial agents reach the cytosol usually by diffusion across the membrane bilayer, the pumped-out drug molecules have a good chance of reentering the cytosol through this free-diffusion process, and the transporters of this class are not expected to create high-level resistance. However, constitutive RND pumps, such as AcrAB-TolC and MexAB-OprM, may capture such pumped-out drug molecules in the periplasm and thus synergistically enhance the activity of singlet pumps in producing resistance (Fig. 1). This was first shown in P. aeruginosa (25) and was rediscovered in E. coli nearly a decade later (26). The latter study (26) also made an important point that the contribution of some singlet pumps may have escaped detection because of overlapping specificities; thus, the double deletion of an MFS pump, MdfA, and an SMR pump, EmrE, made E. coli as susceptible as or even more susceptible than the AcrB deletion mutant to cationic agents with intracellular targets, like acriflavine or ethidium. This finding suggests that RND pumps are usually rather inefficient in capturing drugs from the cytosol (although some contrary views have been presented [131]) and that the singlet pumps often play an important role in resistance to agents with intracellular targets. The fact that the plasmid-encoded TetA pump creates significant tetracycline resistance (8) suggests that this synergistic mechanism can sometimes be quite effective.

Although the MFS-type MDR pumps usually do not play a predominant role in resistance, as described below, the singlet drug pumps of the Tet group, usually plasmid encoded, are clinically important in creating tetracycline-specific resistance in many bacterial species. In the current nomenclature, TetA refers to the MFS exporter, and the phylogenetic group to which it belongs is specified by the group within parentheses, as in TetA(B). Currently, the 12-TMS TetA pumps, present in Gram-negative bacteria, contain 13 phylogenetic groups, whereas the 14-TMS Tet pumps, present in Gram-positive bacteria, contain at least 3 groups (132). The plasmid-encoded TetA pumps were the first bacterial drug efflux pumps identified (8, 133). Biochemical studies by the Yamaguchi group showed that their substrate was the magnesium salt of tetracycline (134), and cysteine-scanning mutagenesis followed by labeling studies identified the residues important for substrate binding and proton translocation (135). Interestingly, the Gram-positive pumps Tet(K) and Tet(L) were found to transport monovalent cations, such as Na⁺, and cation transport was hypothesized to be the original function of such pumps (136). Finally, glycylcyclines such as tigecycline were developed by selecting for derivatives that withstand the presence of Tet pumps and are indeed poor substrates for these tetracycline-specific transporters (137). However, tigecycline is a substrate for the RND pumps of many species, including E. coli, such as AcrAB or AcrEF (138).

A few MFS pumps, however, occur with their own periplasmic adaptor proteins and with OM channels, such as TolC, and presumably produce an efficient tripartite efflux system. In E. coli, these pumps include EmrB (occurring with the cognate adaptor EmrA) and EmrY (with EmrK), which indeed appear to be involved in the efflux of uncouplers and other substrates (Table 1) (15, 31, 139, 140). Importantly, the crystallographic structure of EmrD was determined (141). It is similar in general to those of the other MFS transporters but has a larger central cavity surrounded by hydrophobic and aromatic side chains. It was also noted that the loops connecting H4 and H5, and H10 and H11, protrude into the cytoplasm much more than in the inward transporters of the MFS, such as LacY and GlpT, and that these loops may play a role in substrate recognition and capture (141). A pH-dependent conformational change was also established for EmrD (142). EmrB that occurs with the periplasmic adaptor EmrA appears to assemble in vitro into a dimer of EmrAB dimers (143). If EmrA forms an intermediary channel similar to the AcrA and CusB channels (see above), perhaps the discrepancy between the trimeric TolC and dimeric EmrB may not matter. Alternatively, the dimeric arrangement could be an artifact of the in vitro assembly of the proteins. The EmrA protein was shown to form dimers and trimers in vitro, and interestingly, it bound an efflux substrate, carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a proton uncoupler), with a reasonable affinity (K_D of ∼1 μM) (144).

Among the singlet MFS transporters, MdfA, which confers MDR when overproduced (145), has been studied extensively in terms of biochemistry (146). However, its clinical relevance remains unknown, although it plays a major role with the SMR-type transporter EmrE in the efflux of cationic dyes (and presumably other cationic agents, such as quaternary ammonium compounds) (26).

ABC TransportersIn fungi and animal cells, most of the transporters involved in drug efflux belong to the ABC family (7, 147). In Gram-negative bacteria, there are only few examples of ABC family drug efflux pumps, although MsbA, the exporter of biosynthetic intermediates of lipopolysaccharide (LPS), was shown to pump out drugs, including erythromycin, when overexpressed in Lactococcus lactis (148).

The best-studied bacterial ABC drug exporter is MacB of E. coli, which functions together with the periplasmic adaptor MacA and the OM channel TolC (149). MacAB-TolC raises macrolide MIC values when overproduced (149). The isolated MacB shows only trace ATPase activity, which is not stimulated by substrates. However, ATP hydrolysis is very strongly stimulated by the simultaneous presence of MacA (150); these results were confirmed by another laboratory (151), which showed that MacB is a dimer, as expected for an ABC transporter. Finally, an analysis using surface plasmon resonance led to the conclusion that MacA binds to MacB with a nanomolar affinity, and the complex remains stable during the ATP hydrolysis cycle (152); the authors of this study assume that the MacA channel connects a TolC trimer and a MacB dimer, with no direct connection between the latter two. The expression of the MacAB system is stimulated by the heat shock sigma factor σ³² (153). Its physiological function might be related to the export of LPS or its biosynthetic intermediate (154).

SMR TransportersThe proton-motive force-driven SMR transporters belong to the drug/metabolite transporter (DMT) superfamily and are very small, each containing only four TMSs. Thus, unlike MFS transporters, which presumably function as monomers, SMR transporters, which typically exchange incoming H⁺ with the pumping out of either monocationic (ethidium and tetraphenylphosphonium, etc.) or dicationic (e.g., paraquat) compounds (155), must function as a dimer. They also appear to decrease susceptibility to aminoglycosides when the proteins are overproduced from plasmids (156). However, there was controversy on the issue of whether this was a parallel dimer in which each component monomer was embedded in the same direction within the bilayer or an antiparallel dimer. A crystallographic study clearly shows the antiparallel arrangement within an EmrE dimer (157), but chemical cross-linking favors a parallel arrangement, and it appears that the direction of insertion of the monomeric unit really does not matter for the efflux function (158).

EmrE is one of just a few transporters that produce a drug-hypersusceptible phenotype when the gene is deleted in wild-type E. coli still containing AcrAB (Table 1). One of the characteristic substrates of EmrE is a quaternary ammonium compound, including the endogenous osmoprotectant of E. coli, betaine (159), and thus, EmrE overproduction makes cells more susceptible to hyperosmolarity conditions as well as alkaline-pH media. Using these phenotypes, one study found that OmpW, an OM protein, apparently helps in the removal of such compounds pumped into the periplasm by EmrE (160). This is rather unexpected, as OmpW forms an 8-stranded β-barrel, which usually contains a channel too narrow for solute diffusion. Indeed, its structure shows that its central channel is truncated, although it may open up sideways into the interior of the OM (161). If (and how) quaternary ammonium compounds could diffuse through OmpW is thus an open question; however, we note that AcrAB-TolC is not needed for full resistance to paraquat (26).

There are a few reports suggesting the possible involvement of SMR transporters in resistance in clinical isolates of organisms other than E. coli. The deletion of abeS resulted in significant decreases in MICs of chloramphenicol, ciprofloxacin, and erythromycin in A. baumannii (162), and the deletion of a pair of genes, kpnEF, in K. pneumoniae makes cells hypersusceptible to a wide range of antimicrobials (163). An EmrE homolog contributes to MDR in P. aeruginosa (164). The contribution of SMR transporters to resistance seems to be an important future area of study for clinical microbiologists.

MATE TransportersThe MATE transporters have now become a part of a new superfamily, the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) flippase superfamily (19), because of their connection to transporters like the LPS flippase RfbX. These transporters are widespread in bacteria and are also found in higher animals and plants.

The first of these transporters identified was the Na⁺/cationic agent antiporter NorM from Vibrio parahaemolyticus (165), with 12 TMSs. Its homologs pump out cationic dyes, fluoroquinolones, and aminoglycosides into the periplasmic space. Many of these transporters use a gradient of Na⁺, H⁺, or both as the energy source. The crystal structure of NorM from Vibrio cholerae indicates an outward-facing conformation with two portals open to the outer leaflet of the IM (166). The structures of NorM from Neisseria gonorrhoeae (167) and its distant, proton-driven Pyrococcus furiosus homolog (168) bound to several substrates show a wide, central substrate-binding cavity. With the latter, protonation of Asp41 bends TMS1 so that the part of the cavity located in the N-terminal half of the protein becomes collapsed, suggesting that this will produce the extrusion of substrates. Furthermore, cyclic peptides representing prototype inhibitors were also cocrystallized, and the best inhibitor appears to bind tightly to the cavity, preventing the bending of TMS1 (168). Finally, the mechanism of the NorM pump was examined with MD simulation (169).

Although genes for the MATE pumps have been cloned from many pathogens, their contribution to resistance in the organisms of origin has not been studied in most cases. One exception is a transporter from Enterobacter cloacae, EmmdR, and its gene disruption contributed to susceptibility to fluoroquinolones and cationic dyes (170).

Pathways of Drug Influx across the OMIn Gram-negative bacteria, antimicrobial agents must first traverse the OM barrier in order to exert their action (Fig. 1). The OM usually functions as a very effective permeability barrier, because the porin channels are narrow, being only 7 by 11 Å in the E. coli OmpF porin at its constriction point (171), and the bilayer domain of the OM is asymmetric, with its outer leaflet composed only of LPS (172), producing an unusually impermeable bilayer. Furthermore, because several basic amino acid residues are on one side and acidic amino acids are on the opposite side at the constriction zone of the porin channel, the water molecules inside are thought to be strongly oriented in one direction, and this presumably hinders the diffusion of lipophilic drugs, as they must disorganize this assembly of water molecules for penetration (173). Indeed, measurement of influx rates of cephalosporins showed that lipophilicity strongly hinders diffusion through porin channels (174). These considerations suggest that only those drugs that are relatively small, and preferably not too lipophilic, pass through the porin channels relatively rapidly. This group includes β-lactams, fluoroquinolones, tetracycline, chloramphenicol, cycloserine, and aminoglycosides. Aminoglycosides can be fairly large, but as polycations they are likely to become “sucked into” the periplasm by the presence of the interior-negative Donnan potential (175). Aminoglycosides and β-lactams are also essentially prohibited from diffusion through the bilayer region because of the presence of multiple cationic groups and a strongly acidic group, respectively. β-Lactams allow us to measure their OM permeation process precisely by coupling their influx with their subsequent hydrolysis by the periplasmic β-lactamase (174), and the importance of the porin pathway can be ascertained by using porin-deficient mutants in this assay. For other classes of drugs, quantitative assays of the OM penetration process are difficult. However, one can artificially increase the permeability of the OM bilayer region, either by using an agent that disorganizes the LPS leaflet (polymyxin B nonapeptide) (176) or with mutants with partial defects in LPS synthesis (177). The MIC values of the agents mentioned above showed little change under these conditions, suggesting that they predominantly permeate through the nonbilayer pathway, i.e., through the porin channels, at least in E. coli.

The preference for the OM permeation pathway, however, is not absolute. We note in particular that among β-lactams, those that are more hydrophobic (e.g., oxacillin and cloxacillin) or larger (such as some third- or fourth-generation cephalosporins) tend to be hindered in their penetration through porin channels, and for them, permeation through the bilayer region, although slow, may become significant (176, 177).

It should be mentioned here that the situation is very different for organisms such as P. aeruginosa or Acinetobacter species, which do not produce classical E. coli-type trimeric porins that provide a fast influx of small drugs. The major nonspecific porin in these organisms is a homolog of E. coli OmpA, and its major function is structural, that is, to connect the OM to the underlying peptidoglycan (178). The porin function is produced by the alternative folding of only a small fraction of the protein (perhaps ∼2% of the population) to produce ∼16 transmembrane β-strands, and we proposed to call this class of porins “slow porins,” in order to distinguish them from the classical trimeric porins, in which every molecule produces an open channel (179, 180). Because the number of open channels is small, OM permeability is very low, and β-lactams cross the OM of P. aeruginosa at a rate ∼100 times lower than that for the E. coli OM (181, 182). Because of this slow permeation through slow porins, the endogenous RND system MexAB-OprM can compete well with the influx of hydrophilic β-lactams as well as other antibiotics (Fig. 1). Thus, the deletion of a component of this pump complex decreases the MICs of many antibiotics drastically (Table 2). The situation is similar for A. baumannii (183). Hence, with these organisms, even efflux at moderate rates is expected to produce significant increases in β-lactam MICs, and indeed, the genetic deletion of major efflux pumps decreases β-lactam MICs substantially (Table 2) (13, 184). If we increase the OM permeability of P. aeruginosa by adding polymyxin B nonapeptide, we see impressive decreases in antibiotic MICs, comparable to those obtained by the genetic deletion of MexAB-OprM (42).

Because LPS contains about six, usually all saturated, fatty acid chains in a single molecule, it is expected to produce a strong permeability barrier when organized into an LPS-only leaflet, as in the OM (22). Indeed, when the permeability of the OM bilayer to steroid probes (which are too large and too hydrophobic for passage through porin channels) was examined, it was found to be ∼2 orders of magnitude lower than that of the conventional phospholipid bilayer membranes (185). At the time when this study was carried out, the existence of multidrug efflux transporters was not known. More recently, however, such derivatives of steroid hormones were shown to be the substrates of AcrB (186). Thus, the permeability difference between the OM bilayer and the phospholipid bilayer might not quite reach 100-fold, yet it seems clear that the OM bilayer is an unusually impermeant barrier. Nevertheless, for large, lipophilic agents, the bilayer is the only possible pathway for OM permeation. These agents include macrolides, rifamycins, novobiocin, and fusidic acid (Table 3). Glycopeptides such as vancomycin and teicoplanin are large but not lipophilic. Nevertheless, because of their size, their only possible path to cross the OM is through the bilayer. Since these agents have so much difficulty in crossing the OM, they are usually considered agents effective against only Gram-positive bacteria. These agents can be active against Gram-negative bacteria if the LPS leaflet is breached (176, 177) or if the RND pump is inactivated (13, 187).

Drugs Traversing the OM Mainly through Porin ChannelsThe RND transporters, which play a predominant role in raising the MIC values of most antibiotics, pump out drugs mostly from the periplasmic space, as mentioned above (see Biochemistry and Genetics of Multidrug Efflux Pumps). They cannot create resistance if the drugs flow into the periplasm across the OM rapidly enough to counteract the rate of efflux. This is especially so because the RND pumps appear to have a relatively low velocity, for example, with AcrB having a velocity of ∼0.3 nmol/s/mg cells (dry weight) for penicillins (65) (or turnover rates of the order of 100/s [see reference 63]). (In E. coli, the chromosomally encoded class C β-lactamase is expressed at a low, constitutive level, and thus, β-lactam MIC values are essentially determined by the balance between influx and active efflux.) On the other hand, ampicillin was measured to cross the OM with a permeability coefficient (P) of 2.8 × 10⁻⁴ cm/s (64) or at a rate of P × A × Δc, where A is the area of cell surface (∼128 cm²/mg) and Δc is the concentration difference of the drug across the OM. If the external concentration of ampicillin is 10 μg/ml (3 × 10⁻⁵ M), the influx rate is expected to be ∼10 nmol/s/mg, which is much higher than the V _max of efflux. Thus, efflux has only a barely visible effect on the MIC of this drug in E. coli, as has been ascertained by the use of ΔacrAB mutants (Table 2) (188). Similarly, for relatively small antibiotics such as fluoroquinolones and tetracycline, which are expected to diffuse through the trimeric porin channels rapidly, AcrAB inactivation decreases their MIC values only minimally (Table 2).

Even among the β-lactams, however, more hydrophobic compounds, such as oxacillin or cloxacillin, diffuse through the E. coli porin channels presumably rather slowly. Consequently, active efflux by AcrB strongly affects the MIC values, and the cloxacillin MIC decreases 128-fold, from 256 to 2 μg/ml, upon the deletion of acrB (Tables 2 and 3) (188). Because the acrB deletion hardly affects the ampicillin MIC, we thought previously that cloxacillin must be an exceptionally good substrate of AcrB and that ampicillin was a very poor substrate. Nonetheless, quantitative determination of efflux kinetic parameters (65) showed that these two drugs have similar affinities for AcrB and that the V _max is higher for ampicillin by only ∼2-fold.

In contrast, in P. aeruginosa, where even small antibiotics must diffuse across the OM slowly via its slow porin, active efflux becomes very effective in increasing MICs, as seen from the fact that MICs of practically any antibiotic are drastically decreased upon the deletion of its major RND pump MexAB-OprM (Table 2). Here the situation with β-lactams becomes somewhat more complex, because with early compounds, hydrolysis by the powerful, inducible chromosomal β-lactamase plays a significant role. The relative lack of an effect of pump deletion on the fourth-generation cephems cefepime and cefpirome (Table 2) may suggest that they are poor substrates of the pump; it may also reflect the extreme stability of these compounds with the chromosomal class C enzyme (189). The imipenem MIC is hardly affected by the pump deletion, but this is because imipenem permeates across the OM much more rapidly than other compounds, by utilizing a specific channel, OprD (190). Thus, the efflux pump, even if it were capable of pumping out imipenem, would be overmatched by the rapid influx of the substrate (Fig. 1).

Drugs Traversing the OM through the Lipid Bilayer RegionLarge molecules that cannot diffuse through the porin channels must penetrate the OM by slowly diffusing through the asymmetric bilayer domains, which have similarly low permeability in E. coli and P. aeruginosa (185, 191). Because of their slow influx, active efflux can become extremely effective, particularly when these molecules are preferred substrates for the efflux pumps, as can be seen in the huge decreases of MICs upon genetic inactivation of the main RND pumps (novobiocin and erythromycin) (Tables 2 and 3).

To recapitulate, the multidrug pumps work in synergy with the OM barrier. The pumps can make Gram-negative bacteria resistant only when the influx of the drug across the OM is relatively slow, and thus, efflux should always be considered in relation to the OM penetration process (Fig. 1). This also underscores the problems presented by organisms that produce slow porins, such as P. aeruginosa and Acinetobacter, because the efflux processes there become extremely efficient in increasing the resistance level.

E. coli E. coli is a commensal resident of human and animal intestinal tracts but also includes various intestinal pathogenic types (enterotoxigenic, enterohemorrhagic, enteroinvasive, enteropathogenic, enteroaggregative, and diffusely adherent E. coli) as well as extraintestinal pathogenic E. coli (192). The major, constitutively expressed RND-type multidrug transporter is AcrB, although E. coli possesses a number of drug pumps of various families (Table 1) (15, 16). AcrB is described above as the prototype example for the structural and biochemical elucidation of RND pump transport mechanisms. The effect of acrAB genetic deletion on antimicrobial susceptibility is shown in Tables 1 to 3. Lipophilic (or large) compounds (e.g., erythromycin, novobiocin, fusidic acid, and cloxacillin) cannot diffuse easily through porins, and consequently, AcrB-catalyzed efflux becomes very effective in raising their MIC values to a range outside clinical utility (Table 3). Thus, efflux is responsible, in synergy with the OM barrier, for making E. coli intrinsically resistant to such compounds. In contrast, compounds that are smaller and usually hydrophilic (with the exception of nalidixic acid and chloramphenicol) (such as ampicillin, cephalothin, imipenem, and fluoroquinolones) can penetrate the OM barrier rapidly through porin channels. Therefore, in wild-type cells, AcrB cannot raise MIC values to a significant extent, although many of these drugs are likely to be good substrates of AcrB (for the case of ampicillin, see reference 65).

These small agents are of course useful in the treatment of E. coli infections. Regarding treatment options, a major advance in the 1960s was the introduction of semisynthetic penicillins and cephalosporins active against Gram-negative bacteria, such as ampicillin and amoxicillin, and the first-generation cephalosporins (e.g., cephalothin). Their efficacy decreased drastically with the spread of plasmids coding for class A β-lactamases (usually of the TEM or SHV type), which could hydrolyze these drugs rapidly. To counter this problem, in the 1980s, extended-spectrum cephems (third-generation cephalosporins) were introduced. They could withstand the assault of class A β-lactamases but were hydrolyzed at sufficient rates by overproduced, chromosomal, class C AmpC β-lactamases in Enterobacter and Proteus, etc. (but not in E. coli). In E. coli, these extended-spectrum cephems eventually became less useful because of the spread of plasmids producing TEM or SHV derivatives as extended-spectrum β-lactamases (ESBLs) that acquired a broadened substrate specificity. More recently, however, plasmids encoding class A CTX-M-type enzymes, which apparently originated from a chromosomal gene in an obscure genus called Kluyvera, have become so prevalent as to replace the older ESBLs (193). One common type, CTX-M-15, hydrolyzes a third-generation agent, cefotaxime, much faster than it does a first-generation compound, cephalothin. Other agents also entered the market around the time of the introduction of the extended-spectrum cephems or somewhat later. These agents include fluoroquinolones, semisynthetic aminoglycosides, and carbapenems.

It should be noted that there has been a steady increase in the resistance of E. coli isolates to the agents mentioned above. In a survey covering 30 years of isolates in Sweden (194), the prevalence of isolates showing “non-wild-type” MICs of ciprofloxacin increased from 0% to 40% in 2009. Drugs that have become essentially useless in recent years include ampicillin (70% showing non-wild-type MIC values), tetracyclines, and trimethoprim (up to 60%). It is even more alarming that these statistics are from Sweden, a country with one of the lowest frequencies of drug-resistant bacteria. The prevalence of E. coli isolates resistant to extended-spectrum cephalosporins was 4.4% in Sweden in 2012 but was much higher in some other European countries, e.g., 31% in Slovenia (195). Efflux mechanisms likely contribute to such a rapid emergence of resistance in the presence of antimicrobial selection pressure, as discussed below.

Salmonella spp.A clinical Salmonella enterica serovar Typhimurium isolate that lost OmpC and became resistant to the earlier-generation cephems was reported in 1987 (221). A patient who received a kidney transplant developed septicemia involving this organism, which developed cephalexin resistance only 3 days after this drug was added to the regimen. The pretherapy strain produced OmpF and OmpC in the laboratory, but the posttherapy isolate produced only OmpF. Presumably, the synthesis of the OmpF porin was strongly repressed by the relatively high salt content within the human body and was thus irrelevant during therapy, and hence, the posttherapy strain allowed only a minimal influx of drugs, which made it more resistant to cephalexin, which was hydrolyzed readily by the plasmid-encoded TEM β-lactamase in both isolates.

In Salmonella, OmpF and OmpC are the major porins, and AcrB is also the major, constitutive multidrug pump, although there are a total of 5 chromosomally encoded RND systems identified (i.e., AcrAB-TolC, AcrA-AcrD-TolC, AcrEF-TolC, MdtABC-TolC, and MsdABC or MsdAB-TolC) (58, 222 – 224). Importantly, RamA, a positive regulator of the acrAB transcription that does not exist in E. coli, seems to play a major role in AcrAB overproduction (see Regulation of Multidrug Efflux Pumps, below). Thus, for fluoroquinolones, we would expect situations similar to those that have been found for E. coli. In 1993, Piddock and associates found that posttherapy, ciprofloxacin-resistant isolates accumulated much less drug inside the cells (225), a phenomenon that is now interpreted to be a result of AcrB-driven efflux. Although mutations in target DNA topoisomerases are important, very high-level resistance to fluoroquinolones seems to require the additional contribution of increased efflux (188, 226), and this was the case in the DT204 clone (227). Among nontyphoid Salmonella isolates from Spain, ∼40% were nalidixic acid resistant, although their ciprofloxacin MICs were below the clinical resistance breakpoint; in all of these strains, MICs of nalidixic acid (and ciprofloxacin) decreased strongly in the presence of the AcrB inhibitor PAβN at 20 μg/ml (228) (see Efflux Pump Inhibitors, below). In laboratory-selected ciprofloxacin-resistant S. enterica serovar Enteritidis mutants, AcrB overproduction was present, and in one mutant, a deficiency of the OmpF porin was also present (229). About 5% of S. enterica serovar Typhi strains isolated in South Africa were nalidixic acid resistant, and their nalidixic acid and ciprofloxacin MICs decreased strongly in the presence of PAβN, although this inhibitor was used at a high concentration of 40 μg/ml (230). With β-lactams, one might also expect a situation similar to that found in E. coli, i.e., decreased porin expression and efflux contributing to resistance together with hydrolysis by β-lactamases encoded by R plasmids. In fact, in a plasmid-free strain, the contribution of efflux is more pronounced because of the total absence of enzymatic hydrolysis, caused by the absence of the chromosomal ampC gene in this species (231). Thus, in S. enterica serovar Typhimurium, inactivation of acrAB decreases MIC values of penicillins and cephalosporins quite strongly (58).

Efflux is a significant factor for most β-lactams, with the exception of cefazolin, which is too hydrophilic to be a substrate for AcrB (58, 63). These data agree with the actual measurements of AcrB-catalyzed efflux of β-lactams (63 – 65). However, changes in porins and efflux have been rarely reported for β-lactam-resistant Salmonella isolates. These results may be related to the ecology of these organisms. Salmonella, as a pathogen of farm animals, tends to exist in a strongly clonal manner (232). Therefore, once a clone acquires a resistance plasmid, it will remain and can become the major mechanism of resistance. During the recent period in which extended-spectrum cephems were among the most important agents for therapy, strains containing plasmid-encoded AmpC β-lactamases (e.g., CMY enzymes) and ESBL β-lactamase (e.g., CTX-M) were prevalent among resistant strains (233). They have an advantage over porin-depressed or efflux-enhanced strains, which may have to pay heavily for these unfavorable metabolic changes. In addition, with E. coli (bloodstream) infections, resistant mutants can be selected during the course of therapy, thus favoring porin or efflux mutants. Infections with nontyphoid Salmonella may be caused frequently by strains already containing R plasmids (232, 234). In this regard, there are increasing numbers of reports of multidrug-resistant or extensively drug-resistant isolates of Salmonella that carry the plasmid-encoded OqxAB RND pump with other resistance determinants such as aac(6′)-Ib-cr and/or CTX-M genes (228, 235, 236).

Regarding porin mutants, there are a few reports implicating porin downregulation in β-lactam resistance. Armand-Lefèvre et al. (237) showed that OmpF was completely deficient in an imipenem-resistant, CMY-4 β-lactamase-producing isolate of S. enterica serovar Wien and that the resistance can be reproduced in an OmpF-deleted E. coli strain. Su et al. (234) reported that treatment of a patient infected by S. enterica serovar Typhimurium with ertapenem led to the development of carbapenem resistance concomitant with the mutational inactivation of OmpC, which was the only strongly expressed porin in the parent strain; this confirms the above-mentioned prediction that porin loss would be one mechanism of resistance development during the course of therapy. In a report with results more difficult to interpret, the downregulation of OmpD was correlated with an increased MIC of ceftriaxone (238). Unfortunately, nothing is known about the permeability of the channels of OmpD, although its E. coli homolog, NmpC, showed less single-channel conductance in NaCl than with OmpF and OmpC (239).

Citrobacter spp.The genus Citrobacter is closely related to Salmonella, and one would expect rather similar situations. Thus, some fluoroquinolone-resistant isolates of Citrobacter freundii were found to accumulate less drug (240). In vitro selection of fluoroquinolone-resistant mutants showed the importance of increased efflux in practically all cases (241). Intrinsic resistance to linezolid is mediated by AcrB in this species as well as others (242). A C. freundii isolate containing the plasmid-encoded RND pump was reported recently (243) (see Drug Efflux Genes on Plasmids, below).

Enterobacter aerogenes and Enterobacter cloacae In contrast to E. coli, the chromosomally encoded ampC β-lactamase in Enterobacter aerogenes and Enterobacter cloacae is strongly inducible, and thus, penicillins and the first-generation cephalosporins were ineffective for them. The third-generation cephalosporins were initially thought to be resistant to AmpC-catalyzed hydrolysis. However, strongly overproduced AmpC turned out to have a low V _max but a very high affinity for these drugs (244) and to be capable of producing significant resistance. The fourth-generation cephems and carbapenems are quite effective against non-plasmid-containing strains. Enterobacter spp. produce the constitutive AcrB pump as in E. coli. (Another RND pump gene, eefB, was cloned from E. aerogenes, but its inactivation had no effect on the MICs of antibiotics [245].) They also possess the E. coli-like porins OmpF and OmpC, and hence, one would expect situations in these organisms similar to those seen in E. coli regarding their mechanisms of resistance to β-lactams and fluoroquinolones.

When a large number of β-lactam-resistant strains of Enterobacteriaceae from French hospitals was examined for the production of porins by Pagès and associates in 1996 (246), 6% of resistant strains of E. cloacae and 44% of resistant strains of E. aerogenes lacked detectable porins. The very high fraction of porin deficiency in E. aerogenes is striking. As these bacteria are commensal organisms, they presumably had less chance to acquire R plasmids than did obligatory pathogens like Salmonella, at least up to 1996 (although the plasmids producing ESBLs have become increasingly common in recent years). Furthermore, the resistant strains were probably selected mostly during therapy, favoring mutational mechanisms. Still, the difference in the frequency of porin-deficient strains between E. aerogenes (20 out of 44 resistant strains) and E. cloacae (1 out of 17 resistant isolates) begs for an explanation. The porins of at least one strain of E. cloacae had a five-times-lower permeability than did those of E. coli (244), so perhaps, in this organism, the influx of drugs across the OM is already limited, even without any mutation. Cephaloridine permeation of E. cloacae was found to be 25-fold lower than that of E. coli by another laboratory (247). (The OM permeability of E. cloacae to β-lactams, measured by a novel method [248], was reported to be 20 to 1,000 times lower than that of E. coli. However, the E. coli values were not obtained in the same way, and it is uncertain if a quantitative comparison is warranted.) In contrast, E. aerogenes was found to have a permeability very similar to that of E. coli (249), and thus, the creation of significant levels of resistance may need the downregulation of porins in addition to drug hydrolysis and/or efflux. (OmpF and OmpC from E. aerogenes were studied in detail, and the repression of OmpF biosynthesis by high osmotic pressure resulted in MIC increases of 8- to 16-fold [250] for slowly penetrating compounds such as cefotaxime, ceftazidime, and ceftriaxone [208].) An imipenem-resistant isolate of E. cloacae was deficient in both OmpF and OmpC (247). A carbapenem-resistant mutant of E. cloacae, selected during imipenem therapy, lacked both OmpF and OmpD porins (251); furthermore, ertapenem and meropenem MIC values were decreased strongly in the presence of 40 μg/ml PAβN, although overexpression of AcrB was not evident. (Imipenem does not appear to be a substrate of the P. aeruginosa MexAB-OprM system [13], but meropenem, a derivative with a lipophilic side chain, is a substrate [252]. Ertapenem, another carbapenem with a lipophilic side chain, is also expected to be a substrate for MexAB-OprM and homologous AcrAB.) In vitro selection for E. cloacae mutants with increased MICs of ceftazidime, cefepime, or cefpirome often resulted in strains defective in OmpF (253, 254). A study of ertapenem-resistant isolates from Taiwan (255) found that 43% were altered in the expression of porins (and 96% expressed a multidrug pump). In another study of carbapenem-resistant isolates (256), both OmpF and OmpC porins were totally lacking in two E. aerogenes isolates, whereas variable decreases in porin levels were found in E. cloacae isolates. Similarly, an imipenem-resistant E. aerogenes isolate lacked both OmpF and OmpC (257). A more recent study demonstrated that porin expression often becomes altered when E. aerogenes infection is treated with imipenem (258).

The Pagès group continued their work on E. aerogenes clinical isolates, which resulted in several studies. In one cephalosporin-resistant isolate, there was a missense mutation leading to an altered porin with decreased permeability (259). They also identified an increased efflux of fluoroquinolones and chloramphenicol in some multidrug-resistant strains (260), which is likely to be due to AcrB (261). Finally, frequencies of chloramphenicol-resistant isolates susceptible to the AcrB inhibitor PAβN appeared to have increased between 1995 and 2003, suggesting a larger role for the broad-specificity efflux mechanism in recent years (262).

As would be expected from results with E. coli and Salmonella, a large fraction of fluoroquinolone-resistant isolates of E. cloacae appeared to overproduce a multidrug pump, presumably AcrB (263). Deletion of acrA predictably decreased the MICs of oxacillin, erythromycin, clindamycin, linezolid, ciprofloxacin, chloramphenicol, tetracycline, and tigecycline (264). All multidrug-resistant E. aerogenes strains tested (also resistant to β-lactams) were found to overproduce AcrAB (265), but the effect of an EPI on β-lactam MICs was not examined. Among the large, lipophilic agents that have little activity against these species, macrolides are AcrAB substrates, but a ketolide, telithromycin, appeared to also be pumped out by an additional pump that is susceptible to PAβN inhibition (266). Enterobacter spp. develop resistance to tigecycline through AcrAB overproduction (during ciprofloxacin treatment in one study [267]), and this was caused by the overproduction of the RamA regulator (268), although one study concluded that the overexpression of RamA, an not necessarily that of AcrAB, correlated with the resistance, suggesting the involvement of other efflux pumps (269).

Klebsiella pneumoniae Klebsiella pneumoniae produces the E. coli-like trimeric porins OmpK36 (an OmpC homolog) (270) and OmpK35 (an OmpF homolog) (271). Their permeability has not been measured in a way that could be compared with that of E. coli porins. However, our recent measurement of ampicillin and benzylpenicillin permeation in strain ATCC 11296 showed that the OM of this species is vastly more permeable to benzylpenicillin than E. coli OmpF, yet the permeability to ampicillin is actually similar (S. Kojima, E. Sugawara, and H. Nikaido, unpublished data), an observation that explains why porin deficiency is needed for resistance in K. pneumoniae. It also produces the ubiquitous AcrAB-TolC pump, whose deletion predictably produces hypersusceptibility to erythromycin, chloramphenicol, nalidixic acid, fluoroquinolones, cefoxitin, and cefotaxime and surprisingly produces hypersusceptibility to gentamicin (272). Thus, some aspects of the resistance mechanisms are expected to be similar to what was found for the species discussed above. Indeed, the loss of porins was reported frequently to be the contributing mechanism of resistance, beginning with a 1985 report by Gutmann and associates (273) that one-step selection with nalidixic acid, trimethoprim, or chloramphenicol yielded mutants showing cross-resistance to all three agents and with detectable changes in OM protein patterns. Later, the selection of a porin-deficient mutant in a patient treated with cefoxitin was reported by the same group (274). During the 1990s, in vitro selection of porin mutants by cefoperazone-sulbactam (275) or cefoxitin (276) was confirmed, and clinical porin-deficient isolates resistant to cefoxitin and extended-spectrum cephems (277, 278) were reported. Porin loss is often caused by the insertion of an insertion sequence (IS) element (279). In the present century, reports on porin loss are so numerous that it is impossible to give an exhaustive list of the literature; we give only a few representative examples (280, 281). We note, however, that clinical isolates resistant to carbapenems often lack porins (257, 282, 283). The deletion of OmpC alone has little effect on antibiotic MICs, but the loss of OmpF has a large effect, as predicted (284). When OmpC or OmpF was expressed from plasmids in a strain expressing an ESBL but lacking both porins, MIC values for most agents decreased significantly, but the extent of the decrease was greater with the expression of OmpF (OmpK35) (271). The difference was larger with more bulky agents: for cefpirome (515 Da), the MIC of 512 μg/ml in the porin-deficient strain was reduced to 4 μg/ml by the expression of OmpC, whereas OmpF expression reduced it to 0.5 μg/ml. The MIC of a large agent, ceftazidime (547 Da), which was >512 μg/ml in the porin-deficient parent strain, was reduced only to 256 μg/ml by the expression of OmpC, whereas OmpF expression decreased it to 2 μg/ml. On the other hand, for a smaller agent, meropenem (383 Da), the expression of either porin decreased the MIC of 4 μg/ml in the parent strain to the same value, 0.03 μg/ml. These results suggest that OmpF produces a more permissive, possibly larger, diffusion channel than does OmpC. (This concept may also explain why there have been some confusing data on the role of OmpF versus OmpC for carbapenem susceptibility [see reference 285].) A similar explanation was invoked to explain the higher level of resistance to a more bulky ceftazidime than a smaller cefotaxime in a strain lacking only OmpF (286); this seems to be a valid hypothesis since the strain produced an ESBL enzyme that hydrolyzed cefotaxime much better, on the basis of V _max/K_m values.

In recent years, K. pneumoniae has become a major nosocomial pathogen causing outbreaks of multidrug-resistant clones. Such clones typically express ESBLs (more recently of the CTX-M type) and/or AmpC-type enzymes. These enzymes obviously make predominant contributions to general β-lactam resistance, thus making carbapenems some of the few remaining effective drugs. Recently, however, porin deficiency combined with enzymes with marginal carbapenemase activity has attracted attention. One of the often encountered types combines a frameshift null mutation of OmpF with a mutational change in OmpC, which inserts a few residues into loop 3 that create the narrow constriction of the channel (for example, see references 283, 285, 287, and 288). The role of porin deficiency is often underestimated. For example, Zhang et al. (285) conclude that porin alterations play only a minor role because the removal of the KPC-2 plasmid reduces the carbapenem MICs drastically. Still, unlike the presence of β-lactamase, the porin deficiency by itself cannot raise the carbapenem MICs substantially. (The proper experiment should have been the addition of the same plasmid to the wild-type and porin-deficient strains.) Considering that active efflux contributes to resistance to agents other than imipenem in organisms with a low-permeability OM, such as P. aeruginosa (252), efflux is likely to also make a contribution in porin-deficient K. pneumoniae, but this remains a topic for future study. Finally, LamB appeared to allow some permeation of carbapenems in a strain deficient in both OmpF and OmpC (289).

The genome of K. pneumoniae shows the presence of a large number of drug efflux pumps similar to those observed in E. coli (290). The overexpression of efflux pumps, most probably that of AcrAB, is important in resistance to certain agents, such as chloramphenicol and tetracycline but especially fluoroquinolones (see references 291 and 292). In an isolate lacking porins and showing high-level fluoroquinolone resistance, an efflux pump was apparently expressed strongly, as judged from the low level of norfloxacin accumulation in the absence of energy poison (293). In an important study of ESBL-producing strains from outbreaks of nosocomial infections (294), ciprofloxacin resistance (caused in part by AcrB overproduction) was strongly correlated with cefoxitin resistance, suggesting that for β-lactam resistance, not only the β-lactamases and porin depletion but also efflux may make a significant contribution. Similarly, ESBL-producing strains often lack porins and show a stronger efflux of fluoroquinolones (295). The significance of efflux in β-lactam resistance was pursued later by the Pagès group (296), who examined cefoxitin-resistant but ceftazidime-susceptible clinical isolates. Although β-lactam resistance is most frequently caused by the plasmid-encoded ESBLs, the ceftazidime susceptibility of these strains did not fit with this idea. Indeed, all the strains examined were devoid of plasmid-encoded β-lactamases and produced only the chromosomal class A enzyme. However, in the presence of PAβN (50 μg/ml [unfortunately an excessive concentration]), the MICs of cefoxitin (and cloxacillin), erythromycin, chloramphenicol, nalidixic acid, and ofloxacin decreased strongly, implicating active efflux as a major factor in resistance. There was little decrease in the ceftazidime MIC; as we mention above, ceftazidime is too hydrophilic to be a good substrate of AcrB. Similarly, studies by Källman et al. (297, 298) are important because they show that AcrAB-mediated efflux contributes to cefuroxime resistance in both K. pneumoniae and E. coli, together with the porin deficiency and β-lactamase-catalyzed hydrolysis. Additionally, in vitro selection with cefoxitin and fluoroquinolones, starting from the drug-susceptible revertants of the clinical strains described above, resulted in AcrB overproduction caused mainly by mutations in ramR and in one case in soxR (299). A recent study linked ramR mutation-driven overexpression of AcrAB to tigecycline resistance in KPC-producing strains (300), although one study suggested that RamA-AcrB overexpression occurred only in about one-half of the tigecycline-resistant isolates (301).

Finally, efflux pumps other than AcrB also contribute to resistance. In addition to MDR mediated by the chromosomally encoded OqxAB pump (302 – 304), overexpression of the newly identified RND-type KpgABC pump was involved in tigecycline nonsusceptibility due to an IS element insertion in the promoter region of kpgABC (305). The KexD RND pump expressed by cloning provides MDR with the requirement of AcrA accessory and KocC OM proteins (306). KpnEF, an SMR pump, contributes to MDR and may be important in the infection process, because its expression is regulated by the Cpx regulatory system, which also affects capsule production (163). Inactivation of the MFS-type KpnGH pump results in 4- to 10-fold MIC reductions of the third- and fourth-generation cephalosporins, spectinomycin, streptomycin, and tetracycline (307).

Proteus, Providencia, and Morganella spp.In an extensive study in 1987 (308), it was shown that two major OM proteins (presumably porins) are produced in Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens and that the major porin in every case had channel properties similar to those of E. coli OmpF. Furthermore, in vitro selection with cefoxitin led to the isolation of mutants deficient in the major porin in each species. Since the chromosomal AmpC β-lactamases of these species can hydrolyze cefoxitin only extremely slowly and cefoxitin passes through the porin channel only slowly, this selection is similar to what has been achieved with carbenicillin and E. coli K-12, resulting in the loss of the porin with a larger channel. Interestingly, in 4 out of 5 strains, the MIC of tetracycline also increased upon selection with cefoxitin, suggesting that strains derepressed for a multidrug pump, presumably AcrB, were also selected. In a fluoroquinolone-resistant M. morganii isolate selected in the laboratory, there was a downregulation of what appeared to be OmpF and increased norfloxacin uptake in the presence of CCCP, indicating active efflux (309).

The substrate range of AcrB from P. mirabilis includes, as expected, a number of antibiotics, dyes, and detergents (310, 311). A similar range was found in a tigecycline-resistant clinical isolate of M. morganii overexpressing AcrAB, whose inactivation has led to hypersusceptibility to multiple agents, including a 130-fold reduction of the tigecycline MIC value (312). In a 1992 study (313), a cefoxitin-resistant laboratory isolate of P. vulgaris not only lacked OmpF but also accumulated smaller amounts of most fluoroquinolones; although multidrug pumps were not known at that time, it seems clear that resistance was the result of synergy between the decreased OM permeability and AcrB-catalyzed efflux. This study found that porin loss did not diminish the intracellular accumulation of two agents, sparfloxacin and tosufloxacin. Since it is difficult to imagine that these compounds cross the OM by a unique, unconventional route, this result, if confirmed, may indicate that the major pump discriminates between these substrates. Isolates of P. rettgeri from larvae of the oil fly have shown a correlation between natural resistance to a variety of antimicrobials and organic solvent tolerance, a phenomenon indicating strong efflux involvement (314).

Serratia marcescens Porins of Serratia marcescens are similar to OmpF/OmpC of E. coli in terms of cephalosporin permeation (315). S. marcescens isolates selected in increasingly higher concentrations of cephalosporins, often those compounds that are not easily hydrolyzed by the endogenous β-lactamase, were found to lack one or more porins (316, 317). Similar porin-deficient S. marcescens isolates were also selected by using chloramphenicol, nalidixic acid, or trimethoprim and showed cross-resistance to β-lactams (273); from the vantage point of today, it seems quite likely that at least some mutants were overexpressing efflux pumps. Selection with moxalactam yielded a mutant strain lacking a “42-kDa” porin, or OmpF, and its OM showed a permeability to cephaloridine >100-fold lower than that of the parent strain, in spite of the presence of the “40-kDa” or OmpC porin (318). In contrast, OmpC deletion had little influence on antibiotic susceptibility. Salicylate induction of fluoroquinolone resistance was ascribed to the decreased expression level of OmpF (319); however, increased production of AcrB is likely to have played a more important role.

As expected, fluoroquinolone-resistant strains produced higher levels of an RND pump in Serratia (15). Cosmid cloning of the genes responsible for this phenotype identified sdeA and sdeB, which confer resistance to fluoroquinolones, chloramphenicol, sodium dodecyl sulfate (SDS), and ethidium (320). SdeAB's function is dependent on the TolC-like OM protein HasF, and sdeB inactivation increases the susceptibility to fluoroquinolones and other drugs listed above (321). Exposure to a biocide (cetylpyridinium chloride) led to a mutational upregulation of SdeAB and thus antibiotic resistance (322). SdeAB expression is controlled by a BadM-type repressor, SdeS (323), and a putative MarA-like regulator, SdeR (321). Another RND system (SdeCDE) requires two paired pump genes, sdeDE, but their deletion did not seem to affect the MICs of common antibiotics (320).The RND pump SdeXY produces resistance to fluoroquinolones as well as many substrates of AcrB (16). Its overexpression (with HasF participation) is responsible for tigecycline and fluoroquinolone resistance (324). SdeY, however, has a sequence 84% identical to that of E. coli AcrB and should more properly be called AcrB. Thus, at present, the relative importance of AcrB and SdeB in fluoroquinolone efflux remains unclear, and the expression levels of sdeB in clinical isolates had no clear correlation with their fluoroquinolone resistance levels (325). An MFS pump (SmfY), an SMR pump (SsmE), and an ABC pump (SmdAB) increased the MICs of several drugs when expressed from a plasmid in E. coli (16).

Shigella flexneri For Shigella flexneri, the involvement of efflux, including AcrAB overproduction, leading to high-level resistance to fluoroquinolones, was reported (326, 327). Inactivation of the MFS pump MdfA-encoding gene led to an 8-fold decease in the norfloxacin MIC (328).

Yersinia enterocolitica and Yersinia pestis The genus Yersinia, although relatively distantly related to E. coli, produces the classical trimeric porins OmpF and OmpC (329). It also contains genes for RND pumps. In one study, all 41 nalidixic acid-resistant isolates of Y. enterocolitica showed significant decreases in nalidixic acid MICs in the presence of 20 μg/ml PAβN, suggesting a strong contribution of efflux (330). A Mar homolog was identified in Y. pestis, and its overexpression increased, albeit to a small degree, the MICs of several antibiotics, including tetracycline and rifampin (331), presumably by the increased expression of the AcrB-type pump. Moreover, a homolog of a well-known activator of acrAB transcription, Rob, increased ofloxacin MICs >10-fold (332). Indeed, the acrAB deletion resulted in large decreases in MICs of many antimicrobials, including aminoglycosides (333). Lastly, an MFS pump, together with a KefC-like exporter, pumped out cationic antimicrobial peptides in Y. enterocolitica, and their deletion made cells more susceptible to novobiocin and tetracycline (334).

Vibrio spp. Vibrio spp. are outside the Enterobacteriaceae but not very far in terms of phylogeny. V. cholerae has attracted attention as the causative agent of cholera. Its OM appears to be significantly different from that of Enterobacteriaceae. First, its outer leaflet appears to contain phospholipids in addition to LPS (335), which should allow an unusually rapid permeation of large, lipophilic antibiotics. Indeed, V. cholerae is far more susceptible to such agents, e.g., erythromycin, novobiocin, and rifampin, than are members of the Enterobacteriaceae (336). Second, its major porins, OmpU and OmpV, albeit belonging to the OmpF/OmpC family, are quite different from their enterobacterial representatives in sequences (22) and in permeability. An early study on OmpU (337) found that its single-channel conductivity was ∼3 times higher than that of E. coli OmpF, consistent with the results of a liposome swelling assay revealing a much larger channel than that of OmpF (338). Finally, some of the bile salts apparently bind to the channel interior and block permeation (339), suggesting that these large, planar, lipophilic molecules diffuse through the channel, at least up to the constriction zone. These high-permeability characteristics of the V. cholerae OM are surprising for a gastrointestinal pathogen but may be related to the ecology of this organism as a water dweller.

Because of their genetic relatedness to E. coli, cloning of Vibrio genes in E. coli was not difficult, and a number of efflux systems have been identified in this manner. The V. cholerae genome contains 6 RND transporters (VexAB, VexCD/BreAB, VexEF, VexGH, VexIJK, and VexLM), and most of them require TolC for drug efflux (340 – 345). VexAB seems to play a predominant role in the efflux of antibiotics (benzylpenicillin, erythromycin, and polymyxin B) and detergents (cholate, SDS, and Triton X-100) (341), although among the 6 RND systems cloned from a non-O1 strain, VexEF conferred the strongest resistance to an E. coli host (343). VexCD (confusingly “renamed” BreAB) is involved in bile efflux (340) and is induced by bile (342). A drug-susceptible phenotype could also be created by adding EPI PAβN or NMP to the parent strain (346). VexGH shows an overlapping substrate specificity and also contributes to the production of cholera toxin (344). In an interesting study (345), it was shown that not only was the expression of VexAB and VexGH enhanced by the CpxAR system, but the expression of this system also was stimulated by the deletion of efflux pumps, presumably due to the accumulation of intracellular metabolites. In V. parahaemolyticus, 12 RND pumps were identified, and when expressed in hypersusceptible E. coli, two-thirds of them produced an MDR phenotype (347 – 349), although the deletion of one of them, VmeAB, produced little increase in drug susceptibility (347). The isolation of laboratory mutants resistant to deoxycholate showed that VmeTUV is important for the efflux of bile salts (349).

Among the MFS transporters, VceB was the earliest discovered drug transporter in V. cholerae (350); it occurs together with a periplasmic accessory protein, VceA, and a TolC-like OM protein, VceC (16), and contributes to the intrinsic levels of resistance to deoxycholate, CCCP, pentachlorophenol, and nalidixic acid (350). Another MFS transporter, called EmrD-3, was found by cloning in a drug-hypersusceptible E. coli host and produced resistance to various lipophilic agents, such as linezolid (351). Five MFS transporters (each under the control of a Lys-type MfsR regulator) whose deletion causes tetracycline and bile salt hypersusceptibility are known (352). V. parahaemolyticus was the organism in which the first MATE family transporter, NorM, was discovered by cloning in E. coli (353), and in V. cholerae, 5 members of this family were described (16), including the NorM homolog VcmA (15), which increases MICs of fluoroquinolones, ethidium bromide, acriflavine, and doxorubicin when overproduced in E. coli. Two MATE transporters from a multidrug-resistant V. fluvialis isolate provided 2-fold increases in MIC values of ciprofloxacin and norfloxacin when expressed in E. coli (354).

Although many potential efflux transporters are known, there is not much knowledge on their role in clinically relevant situations, possibly because for V. cholerae, antimicrobial chemotherapy plays only a subsidiary role to fluid repletion therapy. The incidence of drug-resistant V. cholerae appears to be increasing, but the mechanisms presumably involve plasmids and integrons in many cases. In a study of fluoroquinolone-resistant V. cholerae clinical isolates, decreased accumulation of norfloxacin was found together with target gene mutations, suggesting the involvement of efflux (15).

Aeromonas spp. Aeromonas belongs to another order, Aeromonadales. Although the pore-forming activity of an OmpA-like, monomeric protein has been reported (355), this protein presumably contributes only a minor activity to OM permeability, because OmpF/OmpC-like trimeric porins appear to exist in the genome sequences of Aeromonas salmonicida and Aeromonas hydrophila. The genome of A. hydrophila contains 10 RND pump genes as well as genes for MFS, MATE, SMR, and ABC efflux transporters (16). Apparently, an RND system, AheABC, plays a major role in the maintenance of the basal level of intrinsic resistance to most antimicrobials, which is rather similar to that of E. coli, except that A. hydrophila is more susceptible to macrolides like erythromycin and pristinamycin (16). An SMR pump, SugE, produces resistance to tributyltin, a compound that was used in the past as a biocide to prevent biofouling of ships; when introduced into E. coli, this gene increased resistance to chloramphenicol, tetracycline, as well as ethidium bromide (356). Fluoroquinolone-resistant isolates were often seen to have enhanced efflux activity (16). In contrast, another study found little evidence of the contribution of efflux to fluoroquinolone resistance (357), perhaps not surprisingly, because most strains had relatively low (<0.4 μg/ml) MICs of ciprofloxacin.

Legionella spp. Legionella pneumophila contains the major oligomeric OM protein of 28 kDa (358). Although its single-channel conductance was low (0.1 nS) compared with that of E. coli OmpF (0.7 nS), this may have been caused by the low salt concentration (0.1 M rather than 1 M), and the channel size is difficult to ascertain. L. pneumophila strains show high levels of in vitro susceptibility to a variety of antibiotics, including macrolides, rifamycins, fluoroquinolones, aminoglycosides, and β-lactams (359), probably suggesting a limited role of either OM permeability or an efflux mechanism in intrinsic resistance. However, the fact that in vitro exposure of Legionella to erythromycin or ciprofloxacin selects mostly low-level resistance may be an indication of efflux participation (360), while target mutation-derived moxifloxacin resistance causes high-level MIC increases (8- to 512-fold) (361). The genomes of L. pneumophila strains (3.4 to 3.5 Mb) are smaller than that of E. coli K-12 (4.64 Mb) but still possess a large number of genes encoding efflux pumps, membrane fusion proteins, and OM channel proteins (362, 363). Inactivation of the TolC efflux channel (36% identity to E. coli TolC) increases the susceptibility to erythromycin (16-fold reduction in the MIC) and to benzalkonium chloride, deoxycholate, ethidium bromide, norfloxacin, novobiocin, and rhodamine 6G (2- to 8-fold decreases in MIC values) (362, 363). Moreover, TolC is also involved in the secretion of a lipid-containing surfactant that promotes Legionella motility and displays activity against Legionella (363). Given that intracellular killing of L. pneumophila by antibiotics is required for legionellosis therapy, drug efflux could be an important factor affecting the efficacy of in vivo antibiotic regimens. Particularly, the multiplication of L. pneumophila within macrophages has already limited the choice of antibiotics to those that can penetrate phagocytic cells, such as macrolides, rifamycins, and fluoroquinolones, which are generally good substrates of typical drug pumps.

Pseudomonas aeruginosa In its natural habitat as well as in the hospital, the notorious opportunistic pathogen Pseudomonas aeruginosa is often exposed to fluctuating and hostile external conditions. To maintain cell homeostasis and thrive in challenging environments, it has evolved a strong and selective OM permeability barrier whose effectiveness is reinforced by broad-spectrum exporters. No fewer than 12 different RND-type drug efflux systems have been recognized and characterized in strain PAO1. Although most of these multispecific pumps accommodate antibiotic molecules and confer some degree of resistance when overexpressed from recombinant plasmids, only a minority of them appear to be therapeutically relevant. Indeed, significant constitutive or drug-induced expression levels are required for efflux pumps to contribute to intrinsic and/or acquired resistance to antimicrobial agents. Involvement of other families of transporters in resistance to antibiotics appears to be restricted to a few examples (MATE-type transporter PmpM and SMR-type transporter EmrE) (16, 164).

Acinetobacter spp. Acinetobacter spp. and particularly those belonging to the A. baumannii-A. calcoaceticus complex have emerged globally as common nosocomial and community pathogens with high levels of MDR or pandrug resistance (494, 495). The modest genome size of A. baumannii of ca. 4 Mb has shown the acquired genetic diversity (including at least two dozen genomic resistance islands [A. baumannii resistance islands {AbaR}] of 22 to 121 kb) that provides the molecular basis of almost all types of resistance mechanisms and renders the organism significantly resistant to a large number of antibiotics, biocides, and heavy metals (496 – 502). Multidrug-resistant isolates or their epidemic clones are frequently isolated from patients after treatment with ciprofloxacin, co-trimoxazole, colistin, imipenem, and/or tigecycline (503 – 506), highlighting the rapid in vivo evolution of MDR in Acinetobacter.

Stenotrophomonas maltophilia Stenotrophomonas maltophilia, found in various environments, including hospital patients and animal sources, is a key emerging opportunistic pathogen in humans that is highly versatile and adaptable. Its genome contains genes encoding numerous major mechanisms of resistance, including drug efflux pumps (565, 566). The MDR phenotypes are attributed to the interplay between low OM permeability (567) and efflux mechanisms (15). The reduction of LPS synthesis is associated with modestly increased susceptibility to several antibiotics (568). The efflux mechanism was initially suggested by the selection of multidrug-resistant isolates by any of several structurally unrelated agents (565, 569). Eight RND-type Sme systems and several other types of drug exporters have been identified (570).

The first RND pump, SmeABC, identified in this species via the construction of a cosmid-based genomic library (571), is attributable to acquired MDR (572, 573). The subsequently characterized SmeDEF pump plays a major role in both intrinsic and acquired MDR in clinical isolates from various sources (392, 572, 574 – 576), and its overexpression can be readily selected in vitro by conventional antibiotics and also by biocides such as triclosan (565, 577, 578). Inactivation of SmeDEF usually leads to a 2- to 8-fold reduction of the MIC values of fluoroquinolones (including ciprofloxacin, clinafloxacin, and moxifloxacin), tetracyclines (including minocycline and tigecycline), macrolides (erythromycin and azithromycin), chloramphenicol, novobiocin, dyes, and SDS against wild-type and multidrug-resistant isolates (574). In mutants carrying a genetic inactivation of the class B L1 metallo-β-lactamases and class A L2 β-lactamases, the role of RND pumps in resistance to aztreonam, piperacillin, cefepime, and cefpirome (4-fold MIC increases) was demonstrated (565, 571, 574). SmeC overexpression was also associated with L2 β-lactamase production (571). Intriguingly, SmeDEF disruption has little or only a minimal impact on susceptibility to penicillins, cephalosporins, carbapenems, and monobactams (i.e., no or merely a 2-fold MIC decrease in mutants that are also deficient in L1 and L2 β-lactamases) and does not alter rifampin and trimethoprim susceptibility (574).

There are 4 additional RND Sme pumps that all lack a genetically linked OM component (i.e., SmeGH, SmeIJK [paired SmeJK pump], SmeMN, and SmeYZ) and 2 RND pumps containing an OM protein (SmeOP-TolC and SmeVWX) (570, 579). Hypersusceptibility data with the selective inactivation of the SmeC or SmeF OM protein suggest that these OM proteins may function in multiple drug exporters (571, 574), similar to the situations observed for OprM of P. aeruginosa or TolC of E. coli. Although a TolC homolog was also identified in S. maltophilia (580), it is phylogenetically distinct from the SmeC, SmeF, and SmeX OM channels, and hence, its role in any Sme system (except SmeOP [579]) remains unknown. The tolC gene and an upstream pcm gene (encoding protein-l-isoaspartate O-methyltransferase) likely form the pcm-tolC operon, but only TolC inactivation renders the organism susceptible to aminoglycosides and macrolides (580). In fact, the Smlt3926 gene, encoding a TetR repressor, SmeRo, of SmeOP is located immediately upstream of the pcm-tolC operon, and thus, there is a 5-gene cluster comprised of tolC-pcm-smeRo-smeO-smeP. SmeOP-TolC was recently demonstrated to provide resistance to several antibiotics, CCCP, dyes, and detergents (579). The simultaneous hyperexpression of SmeJK (forming one exporter) and SmeZ pumps increases the substrate profiles of a clinical isolate (581). Inactivation of SmeJK significantly increases the susceptibility to aminoglycosides (amikacin, gentamicin, kanamycin, and tobramycin) and the macrolide leucomycin (8- to 16-fold reduction in MIC values), yet the disruption of either SmeJ or SmeK produces merely a 2-fold decrease in the MICs of the tested aminoglycoside agents (582), likely suggesting that SmeJ or SmeK alone may still be functional.

S. maltophilia also possesses ABC and MFS transporters (570). The ABC-type tripartite FuaABC system mediates fusaric acid-inducible resistance to fusaric acid, and this resistance is dependent on the FhuR regulator, which functions as a repressor in the absence of fusaric acid but as an activator in its presence (583). ABC-type MacABC causes intrinsic resistance to aminoglycosides, macrolides, and polymyxins, as its inactivation resulted in 2- to 8-fold reduction of the MIC values of these agents (570, 584). Another SmrA ABC pump conferred resistance to fluoroquinolones, tetracycline, doxorubicin, and dyes when expressed in E. coli (585), and an antibody developed against this pump enhanced antibiotic susceptibility in S. maltophilia (586). An MFS pump, EmrCAB, is involved in the extrusion of hydrophobic toxic agents but is not well expressed intrinsically (587).

Brucella spp. Brucella spp. are facultative intracellular coccobacilli with six recognized species (Brucella abortus, B. canis, B. melitensis, B. neotomae, B. ovis, and B. suis) that display distinct host-pathogen associations. These species are of high clinical significance given their role as causative agents of zoonotic brucellosis, which is reemerging, can be transmitted to humans, and is commonly associated with laboratory-acquired infections (particularly with B. melitensis) (588). Brucella spp. show similar genomes with two circular chromosomes and contain a number of putative drug efflux transporters (589, 590). These species contain trimeric porins homologous to E. coli porins with comparable permeability (591, 592).

Two RND systems, BepDE and BepFG, are involved in resistance to antibiotics and other toxic agents (including doxycycline, a major choice for the treatment of brucellosis) in collaboration with the OM channel BepC (593, 594). Repressed by the BepR regulator, the expression of BepDE was induced by deoxycholate. Inactivation of BepFG produced an increased susceptibility to toxic agents such as dyes but not to conventional antibiotics (594). The inclusion of PAβN resulted in variable reductions in erythromycin and moxifloxacin MIC values (595, 596), consistent with the RND pump's contribution to resistance. A putative BicA macrolide pump is present in B. abortus and B. suis (590). Two MATE pumps, NorMI and NorMII, were identified in B. melitensis, with NorMI being confirmed to yield MDR when expressed in E. coli (16). However, they were not involved in clinical isolates resistant to fluoroquinolones and rifampin (597).

Bartonella spp. Bartonella spp. exist in mammalian host reservoirs and are linked to several human infections, such as cat scratch disease. While Bartonella species are susceptible to most conventional antibiotic classes, the major resistance mechanism characterized to date is related to target modifications for resistance to fluoroquinolones, macrolides, and rifamycins (598). A VceA MDR pump was identified in Bartonella australis (GenBank accession number YP_007461659). OM porins and efflux components, including a TolC homolog as well as RND systems, were identified (599 – 601); nevertheless, none of the efflux pumps have been characterized for their role in resistance.

Rickettsia spp. Rickettsia spp. are obligately intracellular bacteria, including those causing spotted fevers. Rickettsia spp. are often resistant to β-lactams, aminoglycoside, and sulfonamide-trimethoprim but show variable susceptibilities to macrolides (602). Genome comparison of Rickettsia species shows the presence of a number of drug efflux pump genes, such as 6 genes encoding RND pumps and 20 genes encoding ABC transporters in Rickettsia conorii as well as an SMR pump in Rickettsia bellii (602, 603).

Achromobacter spp.The nonfermentative Achromobacter bacilli include an emerging opportunistic pathogen in cystic fibrosis patients, Achromobacter xylosoxidans, whose genome contains a range of resistance genes encoding drug-modifying enzymes and efflux pumps (605). The latter include 4 RND systems, additional tripartite efflux systems, and an ABC-type macrolide-specific MacAB transporter (605). Two RND pumps, AxyABM and AxyXY-OprZ, were identified to mediate MDR (606). Inactivation of AxyB produced up to a 20-fold reduction of the MIC values of several third-generation cephalosporins, with no changes in susceptibility to the aminoglycosides amikacin and tobramycin (606). However, the disruption of AxyY rendered a wild-type strain highly susceptible to aminoglycosides (16- to 128-fold MIC decreases for amikacin, gentamicin, netilmicin, and tobramycin), doripenem, erythromycin, and tetracycline (all with 4-fold MIC reductions). In an aminoglycoside-resistant mutant, AxyY inactivation restored its aminoglycoside susceptibility (20- to 192-fold MIC changes), thus revealing that AxyXY pumps out antibiotics often used for treatment of pulmonary infection of cystic fibrosis patients (607).

Burkholderia spp.The genus Burkholderia contains >40 species that are present in a variety of ecological niches, including soil, water, plants, and animals. MDR is an emerging feature of many isolates such as those of Burkholderia cepacia. A comparison of the OM proteomes of Burkholderia mallei and Burkholderia pseudomallei suggests many similarities, which include a number of porins and efflux pumps (608). The major trimeric porin (Omp38) is a distant relative of E. coli OmpF, but its permeability is likely lower, by 1 or 2 orders of magnitude, than that of OmpF (609). However, another study showed that the single-channel conductance of Omp38 was similar to that of OmpF (610). The genomes of Burkholderia contain multiple replicons such as those in the B. cenocepacia genome that has three chromosomes in each strain with RND pumps encoded in all chromosomes (611). A recent study assessed all 8 RND families with 471 putative RND sequences in 26 completely sequenced Burkholderia genomes (including the virulent, epidemic cystic fibrosis strain J2315) (612). The drug-related HAE-1 RND pumps in the Burkholderia genus vary very much in number, ranging from only 4 in 3 strains of B. mallei to 15 in Burkholderia sp. strain CCGE1002 and 11 to 16 in B. cenocepacia (611 – 613).

The first RND pump characterized in Burkholderia, CeoAB-OpcM of B. cenocepacia (i.e., RND-10 of the 14 RND systems) (611), mediates resistance to multiple antibiotics, including tigecycline, and is inducible by salicylate, iron starvation, and chloramphenicol (16). While ceoB expression appears weak, the expression of 4 RND pump genes (rnd-3, -9, -11, and -13) is readily detectable. Expression of the rnd-2 gene is also inducible by chloramphenicol (611). RNDs 6 and 7 are twin pumps encoded by the same operon, similar to MdtBC of E. coli (127, 611). Inactivation of the rnd-4 gene led to a 4- to 8-fold increase in susceptibility to aztreonam, ciprofloxacin, levofloxacin, gentamicin, tobramycin, and chloramphenicol (614, 615). With 16 RND pumps present in B. cenocepacia strain J2315, their differential roles in drug resistance of planktonic and biofilm cells were investigated (613). Another study showed the high prevalence of RND pump-overproducing clinical isolates (particularly RND-3 overproducers) (616). However, the data regarding the effect of PAβN on antibiotic susceptibility appeared contradictory. While PAβN at 40 μg/ml did not alter the antibiotic susceptibility of several strains, as also noted with erythromycin in B. pseudomallei (614, 617), a newer study showed that PAβN at 64 μg/ml increased the susceptibility to tigecycline (mostly 32- to 64-fold) in the B. cepacia complex (618). The MICs of PAβN against several strains are 30 to 640 μg/ml (614), suggesting various susceptibilities of the individual strains to PAβN. The RND system encoded by the operon containing the BCAM0925 to BCAM0927 genes is upregulated by chlorpromazine and mediates resistance to azithromycin and chlorhexidine (619).

In B. pseudomallei, containing a dozen RND systems, the widespread expression of 7 RND pumps in clinical strains was evident (612, 620). Three pumps, AmrAB-OprA, BpeAB-OprB, and BpeEF-OprC, were characterized (617, 621 – 623). While both AmrAB-OprA and BpeAB-OprB mediate intrinsic resistance to aminoglycosides and macrolides (617, 621), expression of these pumps is commonly seen in clinical isolates (620). BpeEF-OprC is responsible for the widespread trimethoprim resistance in both clinical and environmental isolates (624). Interestingly, aminoglycoside- and macrolide-hypersusceptible strains (with gentamicin and azithromycin MICs of ca. 0.5 to 2 μg/ml) were isolated from both clinical and environmental samples and contained a point mutation in amrB that inactivated AmrAB-OprA. The reversion of this mutation via in vitro exposure of the susceptible strains to gentamicin and kanamycin (40 and 30 μg/ml, respectively) was able to restore aminoglycoside and macrolide resistance (625), highlighting the clinical concerns on current gentamicin use (e.g., 4 μg/ml) in medium to isolate B. pseudomallei (626). In B. thailandensis, multidrug-resistant mutants selected by doxycycline overproduced AmrAB-OprA and BpeEF-OprC. When either of these pumps was inactivated, a third pump, BpeAB-OprB, was hyperexpressed, indicating the interplay among the three RND pumps (627), similar to those observed in P. aeruginosa (628) and Salmonella (629). Interestingly, an antagonistic effect between PAβN (at 50 and 200 μg/ml) and aminoglycosides/β-lactams was observed, and the speculation was that PAβN might have induced the overproduction of the AmrAB-OprA and BpeAB-OprB pumps (627). It remains to be determined whether an induction process exists for explaining the above-mentioned observations that PAβN at 40 μg/ml did not alter antibiotic susceptibility in B. pseudomallei (614, 617) and that PAβN at 20 μg/ml did not affect the efflux activity of SmeDEF toward ciprofloxacin and tetracycline in S. maltophilia (630).

Neisseria spp. Neisseria spp. are commensal bacteria in many animals, with two significant human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, which have shown resistance emergence (for a recent review, see reference 631). These species possess trimeric porins that display high permeability with anion selectivity (632), and they may render the species more susceptible to anionic penicillins. Low-level MDR is mediated by mutations in genes encoding porins or efflux pumps (15, 633). In fact, simultaneous porin mutation and RND pump overproduction are needed for gonococcal penicillin or ceftriaxone resistance, again supporting the significance of the synergistic interplay between the OM and efflux pumps (634 – 636). Mutations in the “multiple transferable resistance” gene (mtrR) had long been associated with MDR in N. gonorrhoeae, with an assumed alteration in OM permeability (15), but the gene was then found to control the expression of MtrCDE, the most characterized RND pump in Neisseria that contributes to resistance to antibiotics (including β-lactams, macrolides, and rifampin), detergents, bile salts, gonadal steroidal hormones, as well as host cationic peptides (15, 633, 635 – 639). Either the deletion or overexpression of MtrCDE produces a 4-fold rifampin MIC change (639). MtrCDE deficiency renders gonococci more susceptible to progesterone (640).

Overexpression of MtrCDE due to mtrR or other regulatory mutations (see Regulation of Multidrug Efflux Pumps, below) has widely been observed in multidrug-resistant clinical isolates (16, 641). This includes interpatient transmission of high-level ceftriaxone-resistant/multidrug-resistant N. gonorrhoeae with MtrCDE overproduction (636). A new study also showed the global gene transcriptional changes as a consequence of MtrCDE overproduction, and these changes included an upregulation of the gene encoding cytochrome c peroxidase that is associated with tolerance to peroxides (642). The newly available crystal structures of MtrD and MtrE (643, 644) also suggest that the transport mechanisms of AcrAB-TolC discussed above (see Biochemistry and Genetics of Multidrug Efflux Pumps) are applicable to the MtrCDE pump. Another RND pump, FarAB-MtrE, of both N. gonorrhoeae and N. meningitidis mediates resistance to antibacterial fatty acids and cationic peptides that are likely present on mucosal surfaces of the host (16, 645). Since many substrates of MtrCDE and FarAB-MtrE are present in the host, these efflux pumps are also involved in bacterial pathogenesis such that in vivo fitness or survival is enhanced by these pumps (640, 641). The fatty acids and bile salts in the gut may likely facilitate Mtr-mediated resistance, as observed for isolates from rectal infections often possessing the MDR phenotype (646). Also, MATE and ABC pumps were also identified with additional tetracycline-specific exporters (16). The clinical importance of three MDR pumps (i.e., MtrCDE, MacAB, and NorM) was recently demonstrated by their impact on extensive or multiple drug resistance of clinical isolates in comparison with clinical susceptibility breakpoints (647). For example, in an extensively resistant isolate, the inactivation of MtrCDE changed the susceptibility status for azithromycin, penicillin, and tetracycline from R to S or I. MacAB inactivation alone also rendered the resistant isolate S, providing an important clinical example of the role of ABC transporters in bacterial drug resistance. The NorM deficiency was also able to make an R-to-I status change for tetracycline (8-fold MIC decrease) and to yield a >32-fold MIC reduction for solithromycin, a fluoroketolide for which the susceptibility breakpoints are as yet unavailable (647). Lastly, the gene encoding an MFS transporter, the Mef macrolide pump, on conjugative plasmids is present mainly in Gram-positive bacteria (15) but was also found in N. gonorrhoeae and Acinetobacter junii. These plasmids were readily transferred in vitro to several Gram-positive and Gram-negative bacteria, including Neisseria (648).

Campylobacter spp.The Campylobacter group contains the two very important human enteropathogens Campylobacter jejuni and Campylobacter coli, which are among the top pathogens responsible for foodborne illnesses worldwide. There is an increasing emergence of resistance to clinically relevant antibiotics such as fluoroquinolones and macrolides, where drug efflux makes a major contribution (16).

Campylobacters possess a major porin, major outer membrane protein (MOMP), which appears to exist in a monomeric form as well as in a weakly associated trimeric form (650, 651) and likely yields OM permeability channels smaller than those of E. coli porins (652, 653). The marked susceptibility of wild-type campylobacter strains to large, hydrophobic antibiotics such as macrolides may suggest the presence of an unusually permeable bilayer domain in the OM.

The genome of C. jejuni indicates the presence of a large number of drug exporters (16). The CmeABC system is the most characterized RND pump in campylobacters and is responsible for both intrinsic and acquired MDR, including resistance to bile salts (654, 655). It is distributed in diverse isolates of animals and humans that are resistant to macrolides, fluoroquinolones, and tetracyclines (16, 656 – 661). Inactivation of CmeABC reduces significantly the in vitro emergence of fluoroquinolone-resistant mutants, while its overproduction facilitates such selection (659). Although the contribution of another RND pump, CmeDEF, to intrinsic resistance is likely masked by CmeABC, the two pumps interplay synergistically in providing resistance (662). CmeABC also interplays with 23S rRNA target alterations or ribosomal protein modifications in increasing macrolide resistance levels (658). The contribution of CmeABC to MDR was further observed with the synergistic interplay between anti-CmeA and anti-CmeB peptide nucleic acids to sensitize C. jejuni to multiple antimicrobials, thus also suggesting a potential novel approach to combat efflux-mediated MDR by using the pump-component-specific antisense peptide nucleic acids (663). The combinational use of anti-CmeA and anti-CmeB peptide nucleic acids, each at 1 to 4 μM, decreased the MIC values of ciprofloxacin and erythromycin against a wild-type strain 4- to >32-fold, while their separate use alone yielded merely a 2-fold MIC reduction. Although these peptide nucleic acids were effective in reducing the MIC of ciprofloxacin against a gyrA mutant 16-fold, they were able to produce only a 4-fold reduction of the erythromycin MIC for a high-level erythromycin-resistant mutant with a 23S rRNA mutation. An optimization study of these antisense peptide nucleic acids showed the importance of the ribosome-binding sites as the target (664). However, the entry of antibacterial peptides, including peptide nucleic acids, may require importers such as the SbmA/BacA IM proteins that were identified in several Gram-negative bacteria but remain unknown in campylobacters (665).

Tolerance of campylobacters to trisodium phosphate, a highly alkaline agent used to reduce pathogen prevalence on meat, is mediated by the NhaA1/NhaA2 cation/proton transporter and can be hindered by PAβN. This phenomenon was proposed to be attributable to the contribution of RND pumps (666). However, the effect of PAβN on OM permeability could also provide an alternative explanation, since the PAβN concentration used, 64 μg/ml, was quite high.

Campylobacters are widely distributed in food-producing animals, particularly in poultry. Thus, resistance in campylobacters of poultry origin has drawn much attention. Macrolides and fluoroquinolones are the drugs of choice to treat human campylobacteriosis. The exposure of chickens to either a fluoroquinolone (enrofloxacin) in drinking water or a macrolide (tylosin) in animal feed readily resulted in the isolation of CmeABC-overproducing mutants with an MDR phenotype (656, 667). In vitro stepwise exposures of C. jejuni to escalating levels of erythromycin or tylosin have generated a variety of macrolide-resistant mutants (668). The mutations in ribosome proteins L4 and L22 occurred early during this selection (low resistance level, with an erythromycin MIC of 8 to 16 μg/ml), followed by CmeABC overproduction (intermediate level, with an erythromycin MIC of 32 to 256 μg/ml), and finally accompanied by mutations in the 23S rRNA genes (high level, with an erythromycin MIC of ≥256 μg/ml), highlighting that efflux mechanisms likely facilitate the emergence of high-level macrolide resistance, similar to that of high-level fluoroquinolone resistance (659). CmeABC overexpression was partially a result of single-residue changes in the C terminus of the repressor CmeR (668). It should be noted that the multiple-stepwise-selection approach could yield pleiotropic mutations, thus underestimating the importance of the CmeABC-overproducing mutants that also possessed ∼200 up- or downregulated genes (including the upregulation of genes encoding an SMR pump [Cj1173] and two MFS pumps [CmeG and Cj0035c]), and some of these changes may affect physiology and metabolism (668). The latter offers an explanation regarding the growth burdens and fitness cost of macrolide-resistant campylobacters reported by the groups of Zhang and Yuan (669 – 671).

Additional pumps that are independent of CmeABC and CmeEFG also likely mediate MDR (16) but remain to be further studied. Similar to the above-mentioned gene expression changes in erythromycin-resistant mutants (668), the transcriptional response of C. jejuni to an inhibitory concentration of erythromycin (4 μg/ml; 16× MIC) involved more than a hundred up- or downregulated genes, including two upregulated putative drug efflux operons (Cj0309c-Cj0310c and Cj1173-Cj1174), each encoding a paired SMR transporter. Inactivation of these operons impaired cell growth under conditions of high oxygen levels and colonization in chickens but did not alter the drug susceptibility toward 14 agents tested (including erythromycin) (672). The only MFS drug pump characterized in this species to date is CmeG, and its inactivation renders C. jejuni more susceptible to erythromycin, ciprofloxacin, and H₂O₂ (673). Finally, a novel ABC transporter, ArsP, was recently identified to mediate resistance to nitarsone and roxarsone, the organic arsenic agents used in poultry production (674).

Helicobacter spp. Helicobacter species, including Helicobacter pylori, are associated with several important human and animal illnesses. As a dominant species of the human gastric microbiota, H. pylori causes a persistent inflammatory response and is linked to the development of ulcers and gastric cancers (675). While H. pylori infections need to be treated with antibiotics (676), acquired resistance to fluoroquinolones, macrolides, and metronidazole, agents used for the treatment of H. pylori infections, has emerged and is associated with antibiotic consumption (677, 678).

H. pylori displays intrinsic resistance to multiple antimicrobials, including glycopeptides, nalidixic acid, polymyxins, sulfonamides, and trimethoprim (676), suggesting that limited access to drug targets may constitute part of the mechanisms of resistance. Despite its relatively small genome size (1.7 Mb), H. pylori has genes encoding a large number of OM proteins (679), with several porins, including one that forms a large nonspecific channel (680). Nevertheless, the exquisite susceptibility of H. pylori to macrolides (MIC₉₀ of <0.1 μg/ml [676]) suggests, as with campylobacter, that many drugs may permeate the bilayer regions of the OM. Of several RND systems (681), HefABC contributes to MDR (682, 683), and HefDEF (CznBAC) is a metal exporter for cadmium, nickel, and zinc resistance and gastric colonization (684). Four TolC homologs (HefA, HefD, HefG, and HP1489) were identified, and only HefA inactivation rendered the mutants more susceptible to deoxycholate and novobiocin. The simultaneous disruption of HefA and HefD increased metronidazole susceptibility (685), and laboratory-selected resistant mutants overexpressed HefA (686). The expression of HefA was increased following exposure to metronidazole in clinical isolates (687). In an interesting study, H. pylori cultured in the presence of cholesterol became resistant to bile salts and their analogs, and this phenotype required the HefC pump (688). Intriguingly, hefC missense mutations were among the several gene mutations in in vitro-selected isolates with high-level resistance to amoxicillin, and the introduction of a mutated allele to the wild type increased resistance (689). Through examining the whole-genome sequences of clarithromycin-resistant isolates, a new study revealed the gene clusters of TolC homologs involved in clarithromycin susceptibility profiles in individual isolates (690). Given the extremely acidic environments where H. pylori colonizes, the effect of this environment on secondary transporters in in vivo antimicrobial resistance remains to be determined. In this regard, there are a number of putative ABC transporters in H. pylori (679). Inactivation of the ABC-type transporter MsbA rendered the strain more susceptible to erythromycin and glutaraldehyde. MsbA also interplays synergistically with the glutaraldehyde-resistant protein Ost/Imp to enhance hydrophobic drug resistance and LPS biogenesis (691). Various types of putative drug transporters (e.g., 2 RND pumps, 1 MFS pump, 2 MATE pumps, 4 SMR pumps, and 1 ABC pump) were also identified in Helicobacter cinaedi, a pathogen increasingly known for infections in immunocompromised patients (692).

Salmonella Efflux PumpsSimilar to the regulation of its E. coli homolog, the Salmonella AcrAB-TolC system is controlled through several regulatory pathways, such as AcrR, MarA, and SoxS (16, 770 – 773). Paraquat can induce AcrAB production, and this induction is dependent on SoxS (774). A recent study also showed that the expression level of the acrB, acrD, and/or acrF gene was increased when one or multiple acr genes were deleted (629), and this observation was similar to the situation found among the Mex pump genes of P. aeruginosa (628). However, the compensatory acr expression level changes appeared to have only a minimal impact on the drug susceptibility phenotype (except some aminoglycosides) (629).

Another gene locus of ramRA that is widespread in Enterobacteriaceae except E. coli also significantly influences the expression of not only AcrAB but also AcrEF and MdtABC in Salmonella (16, 229, 773, 775, 776). ramRA are transcribed divergently, with RamR repressing ramA expression (775 – 779). Induced by a variety of environmental signals, including bile salts, indole, and phenothiazines, RamA serves as a small activator protein to contribute to increased AcrAB production (776, 780 – 782). Bile binds to RamR (778) and in this way is thought to increase the expression of AcrAB. Although an earlier study (780) claimed that cholate and indole bound directly to RamA, the binding of an effector ligand to such a small regulator (only 129 amino acids) is highly unusual. Indole induces AcrAB production by also increasing RamA expression (780). RamA expression can be also increased by the inactivation of AcrAB and is regulated by the ATP-dependent Lon protease (782, 783). (The ramA sequence was present, but escaped notice, within the E. cloacae genomic fragment that was found to downregulate E. coli OmpF in 1990 [784] and was correctly identified in K. pneumoniae later [785].) The TetR-type RamR repressor negatively controls the expression of ramA and ramR itself by binding to the intergenic ramA-ramR promoter region (778), and mutations in ramR lead to increased production of RamA (777). RamR forms complexes with multiple agents (e.g., berberine, crystal violet, dequalinium, ethidium bromide, and rhodamine 6G), and this interaction occurs in a common residue of Phe115 and reduces the DNA-binding affinity of RamR (779). A recent review also highlighted the role of RamRA in the regulation of AcrAB (36). Additionally, the expression of the ABC-type MacAB pump is negatively controlled by PhoPQ (222).

K. pneumoniae Efflux PumpsSimilar to that in E. coli and Salmonella, AcrAB expression in K. pneumoniae, as shown in various clinical isolates or laboratory mutants, involves both negative control by AcrR and positive control by RamA and SoxS (292, 299, 501, 786). RamA and SoxS expressions are affected by RamR and SoxR regulators, respectively (299, 787). RamA can also autoregulate its own expression through binding to two ramA promoter sites (788). ramR is located upstream of the romA and ramA genes and is transcribed divergently (787), and RamR binds to two sites of the romA-ram locus (788). Mutations in ramR can yield hyperproduction of RamA and AcrAB-TolC (300, 787, 788).

Another AraC regulator, RarA, which is encoded by a gene upstream of the chromosomal oqxAB pump genes (chromosomal oqxAB genes are found only in K. pneumoniae to date), also positively controls the expression of AcrAB and OqxAB. RarA is present not only in K. pneumoniae but also in Enterobacter and Serratia (789, 790), and it functions in a regulon that affects the expression of 66 genes (791). The expression of OqxAB is additionally downregulated by a GntR-type regulator, OqxR, encoded by a gene located downstream of oqxAB (790). A point mutation in oqxR was linked to the hyperexpression of rarA and oqxB (792). Intriguingly, the genetic arrangement of rarA-oqxAB-oqxR is observed both in chromosomes and on a plasmid (790). Nearly all tested clinical tigecycline-nonsusceptible isolates (25 out of 26) from China contained mutations in ramR and/or acrR, with about one-third of the isolates carrying the simultaneous overexpression of ramA-acrB and rarA-oqxB (301). The expression of eefABC appears to be induced by an acidic or hyperosmolar environment but not by bile salts (793). The CpxAR system and the LysR-type OxyR regulator are also involved in the positive control of the expression of the acrB, acrD, and/or eefB efflux gene (794, 795).

A. baumannii Efflux PumpsThe AdeABC, AdeFGH, and AdeIJK RND pumps are regulated by the AdeRS system (858), AdeL repressor (547), and AdeN repressor (543), respectively. While adeRS and adeL are located upstream of the relevant operons, adeN is found ca. 800 kbp upstream of adeIJK (543). Mutations in AdeS and AdeR (including AdeS truncated by an ISAba1 insertion) are related to AdeABC overproduction (538, 858 – 860). Functional mutations were found in the conserved domains of AdeRS in AdeABC-hyperexpressing isolates with two mutational hot spots, one in AdeS near His149 and another in the DNA-binding domain of AdeR (861). Another study also identified diverse mutations in AdeRS in extensively drug-resistant isolates with the shared residue substitutions of Gly186Val in AdeS and Ala136Val in AdeR (860).

Inactivation of adeN led to 5-fold adeJ overexpression with resistance to aztreonam, ertapenem, meropenem, minocycline, and tigecycline, and mutants carrying adeN mutations in the region required for dimerization of TetR-type proteins displayed elevated levels of AdeIJK production (543), suggesting that remotely encoded AdeN negatively controls AdeIJK expression. The BaeSR regulatory system was found to positively influence the expression of the AdeA-AdeA2-AdeB system (548). Transcriptional upregulation of the MFS-type transporters MdfA and Tet(A) was observed among >200 up- or downregulated genes in a multidrug-resistant mutant after its exposure to a subinhibitory concentration of tigecycline (862).

S. maltophilia Efflux PumpsOf 8 RND Sme pumps, two two-component system regulators are linked to SmeABC and SmeYZ, while three TetR family repressors are found for SmeDEF, SmeGH, and SmeOP. SmeABC is positively controlled by SmeSR, as is evident with the reduced MDR due to the smeR deletion (571). Located upstream of the smeDEF operon, smeT encodes the most studied pump repressor SmeT in S. maltophilia (863, 864). The promoter regions of smeDEF and smeT are overlapping, and SmeT provides negative control to SmeDEF and its own expression. A Leu166Gln substitution in SmeT results in higher expression levels of both smeDEF and smeT (863). SmeT functions as a dimer, like other TetR proteins (727), but has two extensions at the N and C termini, with a small binding pocket observed in the TetR family repressors (864). SmeDEF overproduction was also found in an isolate with an IS1246-like element in the smeDEF promoter where SmeT likely binds, suggesting that an IS element may also influence smeDEF expression (576). Triclosan not only selects SmeDEF-overproducing multidrug-resistant isolates (577) but also induces SmeDEF production via its binding to SmeT and subsequent release from the smeT promoter (865). Similarly, natural flavonoids bind to SmeT and thus influence SmeDEF expression (866). While fusaric acid-inducible FuaABC is positively controlled by its local regulator, FuaR (583), another ABC-type MacABC pump was not experimentally proven to be affected by the MacRS two-component system encoded by genes divergently transcribed from macABC (584). The MFS EmrCAB pump is downregulated by the EmrR repressor (587).

Neisseria Efflux PumpsExpression of the MtrCDE pump is regulated both positively and negatively by several cis- and trans-acting elements (631). The MtrR repressor binds to the mtrR-mtrC intergenic region and downregulates mtrCDE expression (15, 867). MtrR also inhibits the production of FarR, a regulator of the FarAB pump (16). (A genome-wide microarray analysis revealed ∼70 genes whose expression can be repressed or activated by MtrR. One of the repressed genes is rpoH, encoding a stress response sigma factor, and as such, inducible MtrR production also increases gonococcal susceptibility to H₂O₂ [868].) Two AraC family regulators, the MtrA activator and MpeR repressor, also influence MtrCDE expression either directly or indirectly (637, 869). Several membrane-damaging agents can induce MtrCDE expression through their interaction with MtrA (637). This induction is also dependent on an envelope protein, MtrF, whose expression is negatively controlled by MtrR and MpeR (16, 869). A single-base-pair change located 120 bp upstream of the mtrC start codon generates a second promoter for mtrCDE expression and is sufficient not only to increase mtrCDE expression but also to render such expression independent of the control by MtrR or MtrA (870).

In N. meningitidis, mtrCDE expression appears to be independent of either MtrR or MtrA and is modulated by the Correia repeat enclosed element (CREE) that is inserted in the regulatory region for mtrCDE (871). This modulation involves the posttranscriptional regulation of the mtrCDE transcript by cleavage in the inverted repeat of the CREE (871). The CREEs are repetitive sequences identified in Neisseria spp. (commonly 153 to 157 bp or 104 to 108 bp) with an inverted repeat and a characteristic core (such as the integration host factor) that are involved in gene regulation (872). Intriguingly, CREE deletions do not show an effect on susceptibility to ciprofloxacin, erythromycin, and rifampin (873). However, whether these deletions could affect mtrCDE expression remains unknown.

C. jejuni Efflux PumpsLocated upstream of the cm