Efflux-mediated antimicrobial resistance

Keith Poole*

Department of Microbiology & Immunology, Queen's University, Kingston, ON, Canada K7L 3N6


* Tel: +1-613-533-6677; Fax: +1-613-533-6796; E-mail: poolek{at}post.queensu.ca


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

Keywords: efflux , resistance , antimicrobials , antibiotics , biocides , multidrug


    Introduction
 Top
 Abstract
 Introduction
 Efflux-mediated resistance to...
 Efflux-mediated resistance to...
 Evolution and natural function...
 Overcoming efflux-mediated...
 Concluding remarks
 References
 
While antimicrobials have proven invaluable in the management of bacterial infectious disease, resistance to these agents actually predates the introduction of the first true antibiotic (penicillin) into clinical usage,1 and resistance continues to compromise the use of old and new antimicrobials alike.28 The clinical impact of resistance is immense, characterized by increased cost, length of hospital stay and mortality,919 often as a result of inappropriate initial antimicrobial therapy.1924 Resistance to antibiotics occurs typically as a result of drug inactivation/modification, target alteration and reduced accumulation owing to decreased permeability and/or increased efflux.2527 It may be an innate feature of an organism or, when it is not, occurs as the result of mutation or the acquisition of exogenous resistance genes.28,29 Specific growth states (e.g. biofilm formation3034 and anaerobiosis35,36) can also negatively impact antimicrobial susceptibility. While biocidal agents generally remain effective at ‘at use’ concentrations, numerous mechanisms of reduced susceptibility have, nonetheless, been reported in bacteria.25 This review provides an overview of efflux determinants of antimicrobial (antibiotic and biocide) resistance, both agent-specific and multidrug, emphasizing recent advances and discussing all efflux mechanisms as determinants of resistance to specific, clinically-relevant antimicrobials. It is hoped that this will provide some insights vis-à-vis the probable clinical significance of drug-specific versus multidrug efflux systems as regards resistance to a given antimicrobial. While the emphasis is on the clinical relevance of efflux mechanisms of resistance, the probable role of Gram-negative multidrug efflux systems in other cellular processes is also addressed. The interested reader is referred to recent reviews of antimicrobial37 and multidrug3741 efflux for additional information.


    Efflux-mediated resistance to antibiotics
 Top
 Abstract
 Introduction
 Efflux-mediated resistance to...
 Efflux-mediated resistance to...
 Evolution and natural function...
 Overcoming efflux-mediated...
 Concluding remarks
 References
 
The last of the resistance mechanisms to be identified, efflux was first described as a mechanism of resistance to tetracycline in Escherichia coli,42,43 with the plasmid-encoded single component Tet protein export of tetracycline (complexed with Mg2+ it turns out) across the cytoplasmic membrane sufficient for resistance. In the intervening years, numerous plasmid- and chromosome-encoded efflux mechanisms, both agent- or class-specific and multidrug have been described in a variety of organisms where they are increasingly appreciated as important determinants of antimicrobial resistance. Bacterial efflux systems capable of accommodating antimicrobials generally fall into five classes, the major facilitator (MF) superfamily, the ATP-binding cassette (ABC) family, the resistance-nodulation-division (RND) family, the small multidrug resistance (SMR) family [a member of the much larger drug/metabolite transporter (DMT) superfamily] and the multidrug and toxic compound extrusion (MATE) family (see Reference 44 for an in-depth review of drug efflux families) (Figures 1 and 2). Though not unique to Gram-negative bacteria, RND family transporters are most commonly found in such organisms,45 and typically operate as part of a tripartite system that includes a periplasmic membrane fusion protein (MFP) and an outer membrane protein [now called outer membrane factor (OMF)], an organization also seen on occasion with ABC [e.g. the macrolide-specific MacAB-TolC efflux system (Table 1)] and MF [e.g. the VceAB multidrug efflux system of Vibrio cholerae46] family exporters (Figure 2). Members of all but the ABC family (whose members hydrolyse ATP to drive drug efflux) function as secondary transporters, catalysing drug–ion (H+ or Na+) antiport (Figures 1 and 2). Drug efflux systems can be drug-/class-specific as for the original Tet pump and the more recently described Mef exporters of macrolides and various chloramphenicol exporters (reviewed briefly in Reference 47) or capable of accommodating a range of chemically-distinct antimicrobials as for the chromosomally-encoded NorA-like MF transporters prevalent in Gram-positive bacteria or RND transporters of Gram-negative bacteria (Tables 1 and 2).



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Figure 1. Schematic diagram of representative drug exporting systems in Gram-positive bacteria, highlighting the different families of pumps involved in resistance. FQ, fluoroquinolone; CM, chloramphenicol; TC, tetracycline; ML, macrolides, MD, multidrug. While NorA is, strictly speaking, a multidrug transporter, it exports only FQs (and biocides) as clinically relevant agents and so it is highlighted here as an MF family efflux determinant of FQ resistance.

 


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Figure 2. Schematic diagram of representative drug exporting systems in Gram-negative bacteria, highlighting the different families of pumps involved in resistance. (a) Pumps localized solely to the cytoplasmic membrane; (b) Pumps that span the cell envelope. FQ, fluoroquinolone; CM, chloramphenicol; TC, tetracycline; ML, macrolides; BL, ß-lactams. While many of the indicated efflux system are actually multidrug exporters, they are highlighted here for their contribution to resistance to specific agents because their expression in mutants is reportedly selected by those agents. They have been shown to contribute to meaningful resistance to the indicated agent in clinical isolates or the indicated agent is the only clinically-relevant antimicrobial exported by the efflux system.

 

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Table 1. Efflux-mediated resistance to non-fluoroquinolone antimicrobials

 

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Table 2. Efflux-mediated resistance to fluoroquinolonesa

 
Chloramphenicol

Owing to a number of adverse affects associated with its use, chloramphenicol is now used sparingly in human medicine and is restricted in veterinary medicine to pets and non-food-producing animals.48 The fluorinated analogue, florfenicol, is, however, used in cattle, pigs and salmon, though not human medicine.48 Non-enzymic resistance to chloramphenicol has been known for >25 years, being first described in Pseudomonas aeruginosa carrying transposon Tn1696, an element later shown to encode the CmlA chloramphenicol exporter of the MF superfamily49 (Table 1). Related MF-family chloramphenicol exporters have been reported in a number of Gram-negative bacteria, usually encoded by genes present on mobile elements (i.e. plasmids, transposons, integrons) that often carry additional resistance genes.5058 This may explain the persistence of this (and other) chloramphenicol resistance determinants in, for example, food animals59 despite the longstanding prohibition of chloramphenicol use in these animals. Although the nomenclature of chloramphenicol efflux genes has not been standardized, which complicates ready appreciation of relationships between these determinants, several distinct groups of chloramphenicol exporters have been identified in bacteria, with a number of unrelated exporters described in Gram-positive bacteria (i.e. Streptomyces, Corynebacterium and Rhodococcus spp.). The CmlA family remains the largest family of chloramphenicol-specific exporters although an equally large group of transporters, also unique to Gram-negative bacteria, accommodates both chloramphenicol and the fluorinated chloramphenicol analogue, florfenicol (usually designated Flo) (see Reference 48 for a review of chloramphenicol and florfenicol exporters). The Flo transporters can be plasmid- or chromosome-encoded, though the flo determinants are typically associated with mobile genetic elements that, as is the case for cmlA determinants, carry additional resistance genes.6063 flo is an important determinant of florfenicol resistance in animal isolates of E. coli6468 but is also found in human pathogens (e.g. Salmonella enterica6062,69 and V. cholerae63). Recently, a unique plasmid- and transposon-encoded chloramphenicol–florfenicol exporter, FexA, was described in Staphylococcus lentus, the first report of a chloramphenicol–florfenicol exporter in Gram-positive bacteria.70,71 Interestingly, too, fexA expression is inducible, with FexA-mediated resistance to chloramphenicol and florfenicol increasing 2- to 4-fold in the presence of these agents.70 This is reminiscent of chloramphenicol induction of the chloramphenicol efflux genes of Tn169672 and Rhodococcus fascians,73 which seems to occur via a posttranscriptional translational attenuation mechanism.

In addition to chloramphenicol/florfenicol-specific exporters, a number of chromosome-encoded, broadly-specific, multidrug transporters of the RND family that are widely distributed amongst Gram-negative bacteria74 have been shown to accommodate chloramphenicol.48,7477 Moreover, mutant derivatives of P. aeruginosa that hyperexpress the RND family MexEF-OprN multidrug efflux system, which exports and provides resistance to fluoroquinolones and trimethoprim, in addition to chloramphenicol, are readily selected in vitro using chloramphenicol.7880 In vitro-selected chloramphenicol-resistant Burkholderia cepacia (cenocepacia) also shows a multidrug resistance phenotype81 that might be explained by production of this organism's RND family multidrug transporter, CeoAB-OpcM,82 inasmuch as CeoAB-OpcM is known to export chloramphenicol and has been implicated in the chloramphenicol resistance of a clinical (cystic fibrosis) isolate of this organism.83 Recently, a plasmid-encoded RND family multidrug transporter responsible for resistance of a swine isolate of E. coli to the growth enhancer olaquindox was described that also provided for substantial resistance to chloramphenicol.84 The MF family multidrug transporters MdfA of E. coli and VceAB of V. cholerae74 and the ABC type multidrug transporter LmrA of Lactococcus lactis85 have been reported to accommodate chloramphenicol as well. Although infrequently tested, RND family exporters also appear to export florfenicol.75,86

Tetracyclines

Tetracyclines were discovered in the 1940 s and have been used clinically to treat a variety of infections since the 1950 s and are still widely used today.87,88 Since the original reports of TetA-mediated efflux of and resistance to tetracycline in E. coli, numerous other Tet proteins have been described, in Gram-negative and Gram-positive organisms, with at least 20 different types presently known (reviewed in References 47 and 89) (Table 1). These MF family exporters are almost invariably encoded by genes present on mobile genetic elements (plasmids, transposons, IS elements and integrons47,87), often together with additional resistance genes (e.g. in multidrug-resistant S. enterica,50,5456,58,60,62 E. coli90 and V. cholerae63), although those found in tetracycline-producing microorganisms (as probable mechanisms of self-defence, see below) are chromosome-encoded.9193 Recently, a chromosomal tet efflux determinant, tet38, was reported in Staphylococcus aureus, its overexpression in a mutant strain providing for markedly enhanced tetracycline resistance.94 The vast majority of the different Tet proteins have been reported in Gram-negative bacteria (includes the Tet proteins A–E, G, H, I, J, Z and Tet(30) which are found exclusively in Gram-negative bacteria47,89) with two, TetK and TetL, found predominantly in Gram-positive bacteria.89 Novel Tet efflux proteins, Tet(V)95 and Tet(39)96 have also been reported in Mycobacterium smegmatis and Mycobacterium fortuitum95 and Acinetobacter spp.,96 respectively. Still, efflux is a very uncommon mechanism of tetracycline resistance in Gram-positive pathogens (e.g. Streptococcus97104 and Enterococcus105,106 spp.) though it is reported in pathogenic staphylococci,107109 especially veterinary isolates where it is very common.110,111 A recent study of tetracycline resistance in Enterococcus faecalis isolated from raw food did, however, note a high frequency of isolates with tetL.112 In Gram-negative pathogens, efflux is the predominant mechanism of tetracycline resistance in several organisms (e.g. Salmonella spp.,58,107,113116 Shigella spp.,117,118 E. coli,117,119,120 Acinetobacter spp.121 and Chlamydia spp.122) and has been noted in Helicobacter pylori,123 but not, for example, in Campylobacter124 or Neisseria125,126 spp. Efflux determinants are also present in tetracycline-resistant fish pathogens of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter and Salmonella spp.127

The Tet efflux proteins typically export and provide resistance to tetracycline, oxytetracycline and chlortetracycline, with TetB and TetL the only known Tet family exporters of minocycline.47,87 Unfortunately, however, TetB has the widest host range among Gram-negative pathogens.87 Expression of the various tet genes is inducible by tetracyclines, although the induction mechanisms differ for Gram-negative versus Gram-positive tetracycline efflux genes; Gram-negative tet genes are controlled by the tetracycline responsive TetR repressor whereas Gram-positive tet genes are controlled by a ‘translational attenuation’ mechanism.47

Many of the RND family multidrug resistance efflux systems of Gram-negative bacteria also accommodate tetracyclines7477,128 as do the MF family MdfA,74 and the Tap (also known as RV1258c)129,130 and P55131 multidrug exporters of E. coli and the mycobacteria, respectively (Table 1). The L. lactis ABC family multidrug transporter LmrA85 and the E. coli SMR family multidrug transporter EmrE74 also export tetracyclines as does the recently described MF family exporter NorB of S. aureus, though only weakly.94 Still, there are few reports of multidrug exporters as primary determinants of tetracycline resistance (i.e. selected by tetracyclines in vitro or in vivo) although tetracycline selection of RND family MexAB-OprM-overproducing multidrug-resistant isolates of P. aeruginosa has been demonstrated in vitro.78,132,133 Similarly, multidrug-resistant Stenotrophomonas maltophilia, including mutants overproducing the RND family SmeDEF efflux system134 can also be selected with tetracycline in vitro.135,136 Tetracycline has also been shown to positively influence expression of the mexXY genes encoding an RND family multidrug efflux system that contributes to intrinsic resistance to this agent as well as aminoglycosides and macrolides in P. aeruginosa.137

Macrolide–lincosamide–streptogramin antibiotics

Antibiotics of the macrolide–lincosamide–streptogramin (MLS) group138 are employed widely in the treatment of Gram-positive infections (mainly staphylococci and streptococci) and infections caused by anaerobic microorganisms.88,139143 While most Gram-negative bacteria and the mycobacteria are generally resistant to these agents, there are notable exceptions (macrolide susceptibility of mycobacteria144 and macrolide/lincosamide susceptibility of Bordetella, Campylobacter, Chlamydia, Helicobacter, Legionella, Neisseria, Haemophilus and Moraxella spp.140). A variety of individual efflux mechanisms of resistance to one or more of these agents has been reported (reviewed in References 47 and 145), typically of the MF and ABC families of drug exporters (Table 1). A common determinant of macrolide-specific (14- and 15-membered macrolides only) resistance/efflux (M resistance phenotype) is the MF family Mef(A) exporter first identified in Streptococcus pyogenes but widely distributed amongst Gram-positive bacteria and also present in Gram-negative organisms.145,146 A highly related (91% amino acid sequence identity) efflux system, Mef(E), has been described in Streptococcus pneumoniae, and while these determinants are still often referred to separately in the literature, they probably represent a common macrolide-specific efflux system that has been suggested be jointly dubbed Mef(A).147 Still, in light of differences in genetic context, distribution [mef(E) is disseminated in many more species] and impact on macrolide MICs [mef(A)-containing isolates of S. pneumoniae show higher MICs than do mef(E)-containing isolates], some researchers favour maintaining separate designations for these mef determinants.147a In the streptococci, Mef(A) is typically encoded by genes carried by mobile elements present in the chromosome,146 possibly associated with prophages.148 It represents a significant determinant of macrolide resistance in Streptococcus spp., particularly in S. pneumoniae149162 but also in S. pyogenes152,162,163 and other Streptococcus spp.,147,163,164 and appears to be common in commensal viridans group streptococci162,165 (see References 166 and 167 for reviews of macrolideresistance, including efflux, in streptococci). While the mef(A) gene has been infrequently reported in Gram-negative bacteria (e.g. Neisseria gonorrhoeae168), including anaerobes (e.g. Bacteroides spp.169), a recent study of randomly-selected Gram-negative commensal bacteria of 13 different genera obtained from healthy children revealed that fully 41% carried the mef(A) gene.170 Recently, too, the mef(A) gene was identified in Neisseria spp. isolated in the 1950 s and 1960 s, representing the oldest of any species to carry this gene.171 A chromosomal mef(A)-like gene (30% identical; 45% similarity at the amino acid level), cme, has recently reported in Clostridium difficile where it contributes to erythromycin resistance.172

Inducible resistance to both erythromycin and type B streptogramins (MSB resistance phenotype) reported in Staphylococcus spp. is also attributable to a putative efflux mechanism encoded by the plasmid-borne msr(A) gene.173 This ABC family protein contains the two prototypical ATP-binding domains but lacks any obvious membrane-spanning domains, raising questions about its ability to function as a drug exporter. Still, studies have demonstrated that Msr(A)-containing Staphylococcus spp. exclude/show reduced accumulation of erythromycin that was energy-dependent and abolished by classical ABC inhibitors like arsenate.173 One possibility is that Msr(A) associates with another protein that provides the necessary transmembrane domains for drug export.173 Msr(A) is implicated in the resistance of clinical isolates of S. aureus,174 MSSA in particular,175 but also coagulase-negative staphylococci.174

Several msr(A)-like elements have been described in Gram-positive bacteria including the plasmid-borne vga(A), a vag(A) variant, vga(A)v and vga(B) determinants of streptogramin A (e.g. dalfopristin) resistance found on mobile genetic elements in S. aureus,176179 and the chromosomal msr(C) determinant prevalent in E. faecalis and associated with modest resistance to macrolides (including 16-membered macrolides) and group B streptogramins.180182 A recent study has now confirmed the ability of vag(A) and vga(A)v to confer low-level lincosamide resistance in S. aureus and Staphylococcus epidermidis, and it has been suggested that the LSA phenotype occasionally found in staphylococcal isolates may be due to these elements.183 This same study also reported a novel finding that vga(B) confers only low-level resistance to group A streptogramins but substantially increases resistance levels to pristinamycin, a mixture of streptogramin A and streptogramin B compounds. Moreover, a chromosomal gene encoding a Vga(A)-like molecule has been reported in Listeria monocytogenes, Lmo0919, and shown, when cloned into a plasmid, to confer resistance to group A streptogramins and lincosamides in Staphylococcal spp.183 A msr(A)-like gene, now called msr(D),149 is invariably associated with the genetic element(s) that carry the mef(A/E) gene in S. pneumoniae,149,153 S. pyogenes102 and group A Streptococcus,184 though msr(D) (from S. pneumoniae) alone is sufficient for macrolide resistance.149 Interestingly, Msr(D) also promotes resistance to telithromycin, a ketolide, but not streptogramins, distinguishing it from Msr(A).149 msr(D) is also associated with mef(A) genes found in a variety of commensal Gram-negative bacteria.170

Efflux determinants of lincosamide resistance have also been reported, including the chromosomal lsa gene of E. faecalis responsible for the characteristic intrinsic resistance of this organism to lincosamides and streptogramins A (LSA resistance phenotype).173,185,186 Like Msr(A), this ABC family resistance protein lacks the usual transmembrane domains associated with ATP-dependent transporters and an efflux mechanism of resistance has yet to be proved. A related determinant (41% identical; 69% similar to Lsa) of low-level clindamycin resistance (cloned gene increased MIC of S. aureus 16-fold), Lsa(B), has been reported on a plasmid in Staphylococcus sciuri.187 An uncharacterized macrolide efflux mechanism distinct from mef(A) and msr(A/D) and suggested to export 14-, 15- and 16-membered macrolides (and to a limited extent, ketolides) has been reported in S. pyogenes strains inducibly resistant to MLS antimicrobials.188

Spontaneous lincomycin- and puromycin-resistant mutants of Bacillus subtilis showing elevated expression of a gene, lmrB, encoding a putative MF family multidrug exporter have been described.189,190 The lmrB gene occurs in an operon with lmrA, which encodes probable repressor protein. A like-named gene has also been reported as a determinant of efflux-mediated lincomycin resistance in Corynebacterium glutamicum.191 Other multidrug exporters that accommodate MLS antimicrobials include the LmrP (MF family) and LmrA (ABC family) exporters of Lactococcus lactis, which export macrolides, lincosamides and streptogramins,85 although this is of no clinical relevance, L. lactis being a non-pathogenic microorganism. The recently identified chromosome-encoded MF family multidrug exporter, MdeA, of S. aureus also accommodates macrolides (erythromycin), lincosamides (lincomycin) and streptogramins A (virginiamycin), and mutants overexpressing the mdeA gene show modestly increased (2-fold) resistance to lincomycin and virginiamycin.192 Moreover, homologues of this gene have been identified in Staphylococcus haemolyticus, Bacillus cereus and B. subtilis and all provide for modest (2- to 8-fold) increases in resistance to all three agents when expressed from plasmids.192

The ABC family MacAB-TolC system of E. coli is specific for macrolides (14- and 15-membered macrolides) and is unique in being the only known chromosome-encoded ABC type efflux system in Gram-negative bacteria that operates with MFP and OMF components (a plasmid-encoded ABC exporter associated with fluoroquinolone resistance in a Pseudomonas spp. operates with a MFP and, probably, OMF component193). Not surprisingly, given the broad substrate specificity of this family, many RND type multidrug exporters of Gram-negative bacteria accommodate macrolides74,77,145,194,195 and, where tested, lincosamides,196 probably explaining, at least in part, the general insusceptibility of many of these bacteria to these agents. Loss of AcrAB-TolC in E. coli had a modest (4-fold) impact on ketolide (telithromycin) resistance although treatment of E. coli or E. aerogenes with the efflux inhibitor Phe-Arg-ß-naphthylamide (PAßN) had a much more marked impact on ketolide (and macrolide) resistance (128- to 512-fold decrease) indicating that additional, presumed efflux mechanism(s) of macrolide and ketolide resistance occur in these enteric organisms.195 Expression of the RND family MtrCDE multidrug efflux system of N. gonorrhoeae has been reported in clinical isolates displaying reduced susceptibility to azithromycin and/or erythromycin,197,198 indicating that this multidrug transporter can be a determinant of acquired macrolide resistance in Neisseria. Studies on macrolide resistance in Haemophilus influenzae also implicated this organism's three-component RND family multidrug transporter, AcrAB-TolC, as a co-determinant of intrinsic and acquired macrolide resistance,199 including high-level macrolide resistance.200 A plasmid encoded resistance determinant showing substantial similarity (61–80% identity amongst the three components) to the RND family MexCD-OprJ multidrug efflux system of P. aeruginosa and providing resistance to the macrolides erythromycin and roxithromycin has been reported in an environmental Pseudomonas spp. This is the first example of a naturally-occurring plasmid-encoded RND family multidrug transporter.201 The MdfA and VceB multidrug exporters of E. coli and V. cholerae, respectively, also accommodate macrolides.74 Erythromycin resistance reversible by the efflux inhibitor PAßN has been reported in clinical Campylobacter spp. suggestive of an efflux mechanism of resistance,202 and the CmeABC RND-type pump of C. jejuni has been shown to accommodate erythromycin.203

Fluoroquinolones

Fluoroquinolones are an evolving class of antimicrobial204 that enjoys a broad spectrum of activity against Gram-positive, Gram-negative and mycobacterial pathogens.88,205208 Unlike most other efflux mechanisms, which are agent- or class-specific and encoded by genes present on mobile genetic elements, often plasmids, efflux determinants of fluoroquinolone resistance are almost invariably multidrug transporters encoded by endogenous chromosomal genes.74,209,210 In Gram-positive bacteria, the most significant efflux determinants of fluoroquinolone resistance are MF family efflux systems that are invariably homologues of the well-characterized NorA exporter found in S. aureus (Table 2) (see References 209 and 211 for more in-depth reviews of efflux-mediated fluoroquinolone resistance in Gram-positive bacteria). These exporters tend to provide modest resistance to older fluoroquinolones (e.g. norfloxacin, ciprofloxacin) but not newer agents of this class, although uncharacterized efflux mechanisms of broader range fluoroquinolone resistance have been reported in both S. aureus212 and S. pneumoniae.213 Recently, a second NorA-like (30% amino acid similarity) multidrug exporter of fluoroquinolones, NorB, was described in S. aureus, providing resistance to a broader range of fluoroquinolones that included sparfloxacin and moxifloxacin94 and may, in fact, explain the previously seen broad range fluoroquinolone efflux activity mentioned above. Unlike multidrug efflux determinants of fluoroquinolone resistance in Gram-negative bacteria, which export and provide resistance to multiple classes of clinically-relevant antimicrobials (see below), fluoroquinolone-exporting multidrug transporters of Gram-positive bacteria generally export fluoroquinolones as the lone clinically-relevant agent; most of their substrates not being classical antimicrobials.74,209 Unlike resistance attributable to mobile, agent-specific exporters, where resistant strains typically acquire the resistance genes, fluoroquinolone resistance owing to endogenous multidrug transporters typically arises from increased expression of the efflux genes. Enhanced expression of the norA gene has been reported in fluoroquinolone-resistant laboratory214,215 and clinical216,217 isolates of S. aureus, possibly owing to mutations on the norA promoter218 (see also Reference 209 for additional citations). Mutations in the 5' untranslated region of norA can be associated with enhanced stability of the mRNA, leading to increased steady-state levels of the message, effectively enhancing norA expression.219,220 Resistance to fluoroquinolones212,221,222 and non-fluorinated quinolones223 independent of NorA but attributable, at least in part, to an efflux mechanism has also been reported in S. aureus.

An efflux contribution to fluoroquinolone resistance has also been noted in S. pneumoniae,224,225 including clinical strains,150,226228 attributable in some instances to the MF family PmrA exporter (reviewed in Reference 209). Efflux-mediated fluoroquinolone resistance independent of PmrA has, however, also been reported in this organism.213 Highlighting the significance of efflux vis-à-vis fluoroquinolone resistance, efflux was shown to enhance survival of S. pneumoniae in a ciprofloxacin-treated mouse model of pneumonia.229 Efflux-mediated fluoroquinolone resistance has also been seen in viridans group streptococci.230,231 As in S. aureus, efflux-mediated resistance to fluoroquinolones in S. pneumoniae appears also to be limited to older fluoroquinolones.224,229,232 The Lde gene product of L. monocytogenes, showing 44% identity with PmrA, was shown to contribute to fluoroquinolone resistance of clinical isolates.233 Wild-type strains of enterococci have been shown to efflux fluoroquinolones,234 and a gene encoding a NorA homologue, emeA, has been identified and shown to contribute to intrinsic fluoroquinolone resistance in E. faecalis.235,235a Efflux is also implicated in resistance to ciprofloxacin and norfloxacin but not to newer fluoroquinolones in in vitro-isolated fluoroquinolone-resistant Bacillus anthracis,236,237 and fluoroquinolone efflux mechanisms have been described in other Bacillus spp. [e.g. B. subtilis where 3 MF family exporters capable of accommodating fluoroquinolones have been described (Table 2)]. A gene encoding a homologue of one of these, BmrA, has been identified in the B. anthracis genome,238 although its contribution, if any, to fluoroquinolone resistance remains to be tested.

In addition to MF family fluoroquinolone exporters, a limited number of ABC family fluoroquinolone/multidrug exporters have been reported in Gram-positive bacteria, including the EfrAB ciprofloxacin/norfloxacin exporter of E. faecalis and the well characterized LmrA pump of L. lactis, the model bacterial ABC type multidrug transporter (Table 2) (reviewed in References 40, 85 and 239). While LmrA is not relevant clinically, occurring as it does in a milk spoilage organism, it is noteworthy for its very broad range of substrates that include, in addition to fluoroquinolones, numerous clinically-relevant antimicrobials,85 and its functional similarly to P-glycoprotein, the mammalian multidrug transporter.239 An in vitro-selected ethidium bromide-resistant mutant of Mycoplasma hominis displaying a multidrug-resistant phenotype and reduced ciprofloxacin accumulation was shown to overexpress two genes encoding a putative ABC type efflux,240,241 another example, then, of a bacterial ABC exporter accommodating fluoroquinolones.

The MF family LfrA exporter was the first mycobacterial efflux determinant of fluoroquinolone resistance to be identified although numerous efflux determinants of fluoroquinolone resistance (low level) have now been described in the mycobacteria209 (see also Reference 95 for a review of mycobacterial efflux pumps, including those contributing to fluoroquinolone resistance) (Table 2). Most of these putative exporters are of the MF family, although a cloned SMR family exporter, Mmr, provided a modest (2-fold) increase in MIC to ciprofloxacin and norfloxacin,242 and a cloned ABC exporter, Rv2686c-2687c-2688c, increased MICs 2-fold (norfloxacin, sparfloxacin and moxifloxacin) to 8-fold (ciprofloxacin).243 But while efflux has been implicated in fluoroquinolone resistance in, for example, M. smegmatis,244 evidence for a contribution of known fluoroquinolone exporters to resistance in laboratory or clinical isolates is generally lacking—contributions to resistance are typically observed in strains harbouring genes cloned onto multicopy plasmids. Still, a recent report highlighting the enhanced expression of putative MF exporter, Rv1258, in a clinical isolate resistant to ofloxacin is suggestive of a role in resistance,245 although previous studies of this exporter confirmed a role in low-level tetracycline and aminoglycoside resistance only.129 In vitro-selected mutants of LfrA-deficient M. smegmatis showing a fluoroquinolone resistance/efflux phenotype have also been reported although the efflux determinant(s) remain to be identified.242

Efflux-mediated resistance to fluoroquinolones in Gram-negative bacteria, though only somewhat recently appreciated, was in evidence >20 years ago in P. aeruginosa, with examples of fluoroquinolone-resistant P. aeruginosa cross-resistant to other antimicrobial classes attributed (wrongly) to reduced permeability.246 While such mutants were later characterized by reduced fluoroquinolone accumulation,247249 and despite early indications of an efflux mechanism,248 the attendant changes in outer membrane protein profiles in such mutants led researchers to attribute resistance to outer membrane permeability defects. Clearly, however, fluoroquinolone-selected multidrug-resistant strains of P. aeruginosa owe their resistance to fluoroquinolones (and the other antimicrobials) to expression of endogenous, chromosome-encoded three-component multidrug efflux systems of the RND family (see References 250252 for reviews of RND family multidrug efflux systems in P. aeruginosa). Indeed, RND family exporters are commonly encountered determinants of fluoroquinolone resistance in laboratory and clinical isolates of this organism250254 and remain the most significant efflux determinants of fluoroquinolone resistance, not just in P. aeruginosa but in Gram-negative bacteria as a whole (reviewed in References 37, 74 and 210).

Efflux-mediated fluoroquinolone resistance (where the selecting agent in vitro or in vivo was a fluoroquinolone or a fluoroquinolone resistance phenotype in particular was highlighted) has been reported in a number of Gram-negative pathogenic bacteria including Aeromonas salmonicida,255 Campylobacter spp.,210,256,257 Citrobacter freundii,210,536 Enterobacter spp.,210,258 E. coli,196,210,259,260,537 Klebsiella spp.,76,210,261263 Morganella morganii,264 Proteus vulgaris,210 P. aeruginosa,74,210,265269 Salmonella spp.,75,270275 Serratia marcescens,276,276a Shigella dysenteriae,210 S. maltophilia,210,277 anaerobes such as Bacteroides spp.,74,210,278,279 and, possibly, N. gonorrhoeae.280 There are reports, too, of efflux-mediated resistance to nalidixic acid but not fluoroquinolones in Yersinia enterocolitica281 and A. baumannii.282 Moreover, AdeAB of A. baumannii is known to accommodate fluoroquinolones and has been shown to be unregulated in clinical isolates resistance to fluoroquinolones, though it appears to be important in these only for resistance to non-fluoroquinolones.283

Where identified, efflux is invariably determined by three-component efflux systems of the RND family7476,210,256,259,263,271273,276a,277 (Table 2) though not all RND family exporters accommodate and provide resistance to fluoroquinolones (e.g. the AmrAB-OprA efflux system of Burkholderia pseudomallei74) and some RND family transporters known to accommodate these agents have yet to be implicated as primary (selected for in vitro or in vivo by fluoroquinolones) determinants of fluoroquinolone resistance [e.g. RND family efflux systems in Acinetobacter baumannii, Burkholderia cepacia (cenocepacia) and Proteus mirabilis (Table 2)].74 In vitro-selected fluoroquinolone-resistant E. coli sometimes demonstrate a multiple antibiotic resistance (MAR) phenotype284 attributed to increased expression of the RND type AcrAB-TolC multidrug efflux system (reviewed in Reference 285). MAR strains,285 including clinical isolates resistant to fluoroquinolones,286 often carry mutations in the marRAB locus, and such mutations have now been described in laboratory-isolated, multidrug (including ciprofloxacin)-resistant E. coli O157:H7287,288 suggesting that the AcrAB-TolC homologue74 of this organism may similarly play a role in resistance to fluoroquinolones (and other agents) in this organism. Fluoroquinolone selection of multidrug-resistant S. maltophilia in vitro is also reminiscent of an efflux mechanism of the RND type.289

A chromosomally-derived determinant of a single component ABC family multidrug exporter capable of accommodating fluoroquinolones has been reported in V. cholerae290 although its significance if any regarding fluoroquinolone resistance remains unknown. Genes encoding an ABC family exporter associated with resistance to nalidixic acid and low-level ciprofloxacin resistance have also been identified on a large multiresistance plasmid in a Pseudomonas spp., encoding a probable three-component ABC-MFP-OMF drug exporter.193 There is a single reported example of an MF family exporter that accommodates fluoroquinolones, MdfA of E. coli,210,291 although there are no reports of resistance attributable to this efflux system. Intriguingly, whereas the MATE family of drug exporters is the least well characterized and includes the fewest number of characterized systems, those studied to date often accommodate fluoroquinolones, providing resistance at levels comparable to that seen, for example, for the RND family AcrAB-TolC efflux system of E. coli (Table 3). BLAST searches of available bacterial genome sequences also reveal that homologues of NorM from Vibrio parahaemolyticus, the prototypic MATE family multidrug exporter, are present in many Gram-negative (and some Gram-positive) organisms74 and, as such, these may be important contributors to fluoroquinolone resistance in bacteria.


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Table 3. MATE family pumps exporting fluoroquinolones

 
Aminoglycosides

Relatively few bacterial drug efflux systems are known to accommodate aminoglycosides [e.g. the AmrAB-OprA74 and BpeAB-OprB292 multidrug efflux systems of B. pseudomallei, the AcrAD-TolC multidrug efflux system of E. coli74 and the MexXY/OprM multidrug efflux system of P. aeruginosa74 (Table 1)], although there are numerous AcrD homologues in other Enterobacteriaceae74 suggesting that additional aminoglycoside-exporting efflux systems may be present in Gram-negative bacteria. The MexAB-OprM and EmrE pumps of P. aeruginosa have also been reported to provide a very modest contribution to intrinsic resistance to these agents though only in a low ionic-strength medium,293 making it unlikely that these will be significant determinants of aminoglycoside resistance in clinical isolates. Similarly, the LmrA multidrug exporter of L. lactis displays a weak ability to accommodate aminoglycosides, the cloned gene providing for modest increases in resistance to these agents.85 The majority of known aminoglycoside exporters are RND family efflux systems (EmrE is a SMR family exporter and LmrA is an ABC exporter), highlighting once again the significance of this family of multidrug pumps vis-à-vis export of and resistance to clinically important antimicrobials in Gram-negative bacteria.74 Still, only in P. aeruginosa is efflux a significant determinant of aminoglycoside resistance, with numerous reports of impermeability-type pan-aminoglycoside resistance in clinical isolates294305 characterized by reduced drug accumulation that is now attributable to efflux via MexXY/OprM269,306309 (see Reference 310 for a recent review of aminoglycoside resistance in P. aeruginosa, including efflux). In light of the demonstration that mexXY expression is inducible by aminoglycosides,137,311 MexXY/OprM-mediated aminoglycoside efflux also appears to explain the long-known phenomenon of adaptive aminoglycoside resistance in P. aeruginosa.311,312 Here, exposure of the organism to any aminoglycoside provides for enhanced pan-aminoglycoside resistance that is characterized by reduced drug accumulation but is, however, quickly lost in the absence of drug (adaptive resistance is reviewed in a recent comprehensive review of aminoglycoside resistance in P. aeruginosa310).

ß-Lactams

While many of the three-component RND family multidrug exporters of Gram-negative bacteria can accommodate ß-lactams74,313320 (see also Reference 321 for a review of ß-lactam resistance in bacteria including a discussion of efflux mechanisms) (Table 1), there are very few instances where these have been implicated in ß-lactam resistance in vitro or in vivo (i.e. selected by ß-lactams). In P. aeruginosa, overproduction of the MexAB-OprM system has been associated with clinical episodes of carbapenem (meropenem) resistance322 and there are reports of clinical ticarcillin-resistant P. aeruginosa that overproduce this efflux system.323,324 Resistance to imipenem that characterizes P. aeruginosa strains overproducing the MexEF-OprN multidrug efflux system80,250,252,325 is not, however, explained by efflux but rather by the concomitant decline in level of the outer membrane porin, OprD, in such mutants.326,327 OprD is the major portal for entry of carbapenems into this organism and its loss is the most common cause of carbapenem resistance in mutant strains.322,328,329 Overexpression of the MtrCDE multidrug efflux system of N. gonorrhoeae has also been highlighted as an important contributor to the high-level penicillin resistance of certain clinical isolates of this organism.330 A recent report, too, of high-level ampicillin resistance in a so-called ß-lactamase negative ampicillin-resistant (BLNAR) H. influenzae implicated the endogenous RND family exporter AcrAB-TolC as a co-determinant of this resistance,331 and efflux has been implicated in the cefuroxime resistance of clinical isolates of E. coli although the efflux system has yet to be identified.332 The broadly specific ABC type multidrug exporter LmrA of L. lactis shows a limited ability to accommodate ß-lactams but, as with its contribution to aminoglycoside resistance, this is noted when the pump is expressed from a multicopy vector.85

Others

Efflux-mediated resistance to fosmidomycin (targets enzymes of isoprenoid biosynthesis) in E. coli has been reported.333 The observation that low-level in vitro-selected fluoroquinolone-resistant S. aureus sometimes demonstrate a multidrug resistance phenotype that includes resistance to fusidic acid suggests that a multidrug exporter may accommodate and provide resistance to this agent in S. aureus.334 Many RND family pumps also accommodate organic solvents (reviewed in References 335 and 336) and, indeed, co-resistance to solvents and antibiotics has been used to imply the presence of RND family multidrug efflux system in resistant strains.337,338 Tributyltin, an antifouling agent found in marine paints, is exported by an RND family multidrug transporter of Pseudomonas stutzeri that also accommodates antibiotics.339 Three-component RND pumps are also known that export and provide resistance to heavy metals (reviewed in Reference 340).


    Efflux-mediated resistance to biocides
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 Abstract
 Introduction
 Efflux-mediated resistance to...
 Efflux-mediated resistance to...
 Evolution and natural function...
 Overcoming efflux-mediated...
 Concluding remarks
 References
 
Efflux determinants of biocide resistance display broad substrate specificity, accommodating a variety of structurally unrelated agents that can also include antibiotics (see References 25 and 341 for reviews of biocide resistance, including efflux mechanisms of resistance) (Table 4).


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Table 4. Efflux determinants of biocide resistance

 
Quaternary ammonium compounds

A number of efflux determinants of biocide resistance that accommodate quaternary ammonium compounds (QACs; e.g. benzalkonium chloride) have been described in Gram-positive bacteria, predominantly in Staphylococcus spp. (Table 4), including clinical isolates,342344 equine isolates,345 bovine isolates346 and food-associated isolates.347349 The majority of these efflux determinants are plasmid-encoded, SMR family exporters [e.g. Smr (QacC/D), QacE{Delta}1, QacG, QacH, QacJ] although QacA/B is a MF family efflux system, and resistance arises from plasmid acquisition.

Chromosomal efflux determinants of QAC resistance, though uncommon in Gram-positive bacteria, have been described and include the S. aureus NorA multidrug transporter implicated in fluoroquinolone resistance,192,343,350,351 the recently reported MF family MdeA and MATE family MepA multidrug efflux systems also present in S. aureus192,351a and the EmeA multidrug exporter of E. faecalis.235,235a An as yet unidentified putative multidrug transporter distinct from these but able to contribute to QAC resistance in S. aureus has also been reported.353 Efflux has also been implicated in QAC (i.e. benzalkonium chloride) resistance in L. monocytogenes354,355 although the efflux genes have yet to be identified.

Efflux systems able to accommodate biocides, including QACs, in Gram-negative bacteria (Table 4) are also multidrug transporters, with the exception of the Ag+-specific efflux systems356 (see below). In contrast to efflux-mediated biocide resistance in Gram-positive organisms, however, biocide exporters in Gram-negative bacteria are generally chromosomally-encoded (the exceptions being the qacE, qacE{Delta}1, qacF and qacG genes associated with QAC resistance). Many of the latter are associated with potentially mobile integron elements, perhaps explaining the broad distribution of qacE and especially qacE{Delta}1 (which is prevalent on class I integrons357359) in a wide variety of Gram-negative bacteria (Table 4). Still, the presence of the qacE determinants does not appear to correlate with resistance to QACs, at least in one study of Gram-negative bacteria with/without these genes360 suggesting that they may not be significant determinants of QAC resistance in these organisms. A number of the MATE (NorM of Neisseria spp., PmpM of P. aeruginosa) and RND (AcrAB-TolC, AcrEF-TolC and YhiUV-TolC pumps of E. coli; SdeXY pump of S. marcescens) family multidrug transporters implicated in antibiotic resistance have been shown to contribute to QAC resistance (Table 4) although since in many/most instances the contribution of Gram-negative multidrug transporters to QAC resistance has not been addressed, the numbers may be greater. The RND family exporter, MexCD-OprJ, a significant determinant of fluoroquinolone resistance in laboratory and clinical isolates,250,252 is inducible by QACs, although its contribution, if any, to QAC resistance has not been addressed.361 Recently, too, there have been a number of reports of strains of E. coli,362 including E. coli O157:H7363 adapted to e.g. benzalkonium chloride in vitro showing a multiple antibiotic-resistant phenotype reminiscent of mutants expressing the RND family AcrAB-TolC exporters of these bacteria. Moreover, the benzalkonium chloride-selected multidrug-resistant E. coli also showed enhanced efflux activity (measured using ethidium bromide as a model efflux compound) although the actual efflux determinant was not identified.362 In another study, several benzalkonium chloride-adapted S. enterica strains had the benzalkonium chloride resistance compromised by efflux inhibitors, consistent with an efflux mechanism of resistance, although, again, a specific mechanism was not identified.364 The SMR family EmrE multidrug exporter of E. coli also accommodates QACs.365,366 A Pseudomonas fluorescens isolate contaminating a batch solution of benzalkonium chloride and showing high-level resistance to multiple QACs that was compromised by CCCP treatment has been reported and is indicative of an efflux mechanism of resistance.367

Chlorhexidine

Chlorhexidine is a hand washing disinfectant extensively used in hospitals and as such it is not surprising to find nosocomial pathogens exhibiting reduced susceptibility to this agent.368 While the specific mechanism(s) of chlorhexidine resistance in most instances are unknown, a gene, cepA, encoding a putative efflux mechanism has been cloned from a chlorhexidine-resistant clinical isolate of K. pneumoniae and shown to increase chlorhexidine resistance in E. coli transformants.369 Attempts to address the contribution of CepA to the chlorhexidine resistance of the clinical K. pneumoniae isolate were, however, unsuccessful, owing to an inability to construct a cepA knockout strain. A cursory search of available bacterial genome sequences identifies a number of bacteria capable of producing CepA-like proteins including Shigella flexneri (accession number AAP18773 85% identity), E. coli K-12 (protein Yiip, accession number AAN83294 85% identity), E. coli O157:H7 (protein Yiip, accession number AAG59108 85% identity), S. enterica serovar Typhimurium (accession number AAL22901 83% identity), Salmonella typhi (accession number AAO71063 83% identity), Yersinia pestis (accession number AAM83655 75% identity), V. cholerae (accession number AAF96831 57% identity), Haemophilus ducreyi (accession number AAP96289 55% identity), P. aeruginosa (accession number AAG07350 50% identity) and Haemophilus somnus (accession number ZP_00132429, 47% identity) indicating that a chlorhexidine efflux mechanism might be somewhat widely distributed amongst Gram-negative pathogenic bacteria. The recently described MATE family MepA exporter of S. aureus also provides resistance to chlorhexidine.351a

Although a direct role for RND family exporters in chlorhexidine resistance has not been specifically studied, reports of benzalkonium chloride-363 and triclosan-363,370 adapted E. coli (including E. coli O157:H7) exhibiting a multidrug-resistant phenotype typical of overproduction of an RND family multidrug transporter and showing reduced susceptibility to chlorhexidine suggest that such pumps may, indeed, accommodate this biocide. Interestingly, too, like benzalkonium chloride, chlorhexidine has been shown to induce expression of the MexCD-OprJ multidrug efflux system of P. aeruginosa although, again, a role in chlorhexidine resistance was not examined.361 Still, chlorhexidine-adapted E. coli O157:H7 in one study did not exhibit a multidrug-resistant phenotype, suggesting that this biocide does not readily select for RND-type drug exporter-producing mutant strains, at least in vitro. QacA/B implicated in QAC resistance in Gram-positive bacteria also promotes reduced susceptibility to chlorhexidine (Table 4).

Triclosan

Triclosan is a biocide increasingly prevalent in household products and for which mechanisms of resistance are known in a variety of Gram-negative bacteria. Indeed, many of the RND family pumps associated with resistance to clinically important antibiotics are also able to accommodate triclosan [e.g. several of the three-component Mex pumps of P. aeruginosa,371 SdeXY of S. marcescens,372 SmeDEF of S. maltophilia,373 AcrAB-TolC of E. coli,374 CmeABC and CmeDEF of C. jejuni374a and AcrAB-TolC of S. enterica serovar Typhimurium (Table 4)] and triclosan readily selects for strains expressing/hyperexpressing these systems in vitro. RND/Mex efflux systems are, in fact, the major determinants of triclosan insusceptibility in P. aeruginosa.371 Moreover, recent studies showing ready selection, in vitro, of multiple antibiotic-resistant mutants of E. coli O157:H7363,370 and Salmonella spp.375 with triclosan are consistent with this biocide being accommodated by and selecting for multidrug efflux mechanism(s). An association between reduced triclosan susceptibility and increased resistance to multiple antibiotics in human and animal Campylobacter spp. isolates376 is also suggestive of the presence in Campylobacter spp. of an RND family multidrug exporter(s) that accommodates both triclosan and antibiotics.375 Indeed, the CmeABC and CmeDEF RND-type multidrug exporters of this organism are both able to accommodate and so provide resistance to triclosan.374a Growth in the presence of triclosan has also been shown to increase the frequency with which multidrug-resistant mutants of S. enterica could be selected in vitro.375 Again, while efflux was not examined, the phenotype (co-resistance to triclosan, antibiotics and cyclohexane) was typical of strains overproducing an RND family exporter (e.g. AcrAB-TolC). In contrast, chronic in vitro exposure of the dental pathogen Porphyromonas gingivalis to triclosan failed to select triclosan-resistant mutants despite the presence, in this organism, of an RND family exporter, XepCAB, whose contribution to antibiotic resistance has been documented.377 Still, while not all RND exporters may be significant determinants of triclosan resistance, the ability of most RND family pumps known to contribute to antibiotic resistance in clinical strains (Tables 1 and 2) to also contribute to triclosan resistance has not been tested. Thus, it is not yet clear the extent to which these might contribute to triclosan resistance in Gram-negative pathogens. The recent demonstration that triclosan treatment up-regulates putative efflux genes in Mycobacterium spp. is suggestive of the presence of a triclosan efflux mechanism of resistance in these bacteria.378

Silver

Silver (Ag+) is a biocidal agent whose best known use is in topical creams where it is the preferred antimicrobial for the treatment of serious burns, although other uses are known and include Ag+-coated bandages and Ag+-impregnated polymers used in medical devices (e.g. in catheters and heart valves) to prevent biofilm formation.356 Ag+-resistance in bacteria is known, however, particularly in Gram-negative bacteria (e.g. Salmonella spp.) where two plasmid-encoded efflux mechanisms have been described (SilP, an ABC type transporter and SilABC, a three-component RND family transporter; reviewed in Reference 356). Interestingly, genes encoding homologues of the SilABC system have been identified in the chromosomes of E. coli K-12 and E. coli O157:H7 where they were shown to play a role in Ag+ resistance.356 Such determinants are also found on large multiresistance plasmids in S. marcescens379,380 and K. pneumoniae379,381 (in one instance present on a large virulence plasmid of a clinical isolate381). Moreover, a recent hybridization study documented the presence of silABC homologous DNA in a variety of unnamed enteric bacteria of clinical origin.356

Others

Resistance to pine oil found in household cleaners has also been linked to the expression of RND family multidrug efflux systems, with in vitro-isolated pine oil-resistant E. coli showing overproduction of the AcrAB-TolC efflux system.382 The NorB MF family multidrug exporter of S. aureus implicated in fluoroquinolone resistance has been shown to contribute to reduced cetrimide susceptibility94 as have NorA and MepA (Table 4).

Biocide-antibiotic cross-resistance

While there is some debate in the literature regarding the real risks of selecting for biocide-resistant organism outside the laboratory, at biocide concentrations typically used383 (and, thus, the significance of resistance mechanisms, efflux or whatever), there is considerably more debate concerning the risk of biocide selection of antibiotic-resistant organisms.384389 This is particularly true given the existence of multidrug efflux systems that accommodate both classes of antimicrobial (e.g. NorA, NorB, MepA and MdeA systems of S. aureus; Mex systems of P. aeruginosa; AcrAB-TolC of E. coli; SdeXY of S. marcescens; SmeDEF of S. maltophilia)373 (Table 4). Studies of clinical S. aureus isolates showing reduced susceptibility to the QAC benzalkonium chloride have, for example, demonstrated enhanced expression of the NorA multidrug exporter in some of these, with an attendant increase in fluoroquinolone resistance.343,351 Moreover, QAC-resistant S. aureus selected in vitro often showed cross-resistance to fluoroquinolones as a result of increased norA expression in these and, indeed, QACs seemed to more effectively select for NorA-expressing mutants than did fluoroquinolones.350 Recently, too, a second MF family multidrug transporter that accommodates both QACs and antibiotics, MdeA, has been identified in S. aureus and again QAC-resistant laboratory isolates overproducing this protein showed a modest cross-resist to several antibiotics.192 Finally, a mutant overexpressing NorB was shown to be co-resistant to fluoroquinolones and cetrimide although cetrimide was not the selective agent.94

Several in vitro studies have shown that triclosan readily selects for multiple antibiotic-resistant P. aeruginosa,390,391 S. maltophilia373 and E. coli374 expressing these multidrug efflux systems, and for multidrug-resistant Salmonella spp.,376 E. coli K-12362 and E. coli O157:H7363 where multidrug efflux mechanisms are implicated. The correlation, too, between triclosan and multidrug resistance in human and animal isolates of Campylobacter spp.375 and Salmonella spp.337 also suggests that a common, presumed RND family efflux, mechanism exists in these organisms for triclosan and antibiotics, with an attendant risk that triclosan can select for antibiotic resistance in these organisms. Certainly, the RND family AcrAB-TolC and Cme multidrug exporters of S. enterica serovar Typhimurium and C. jejuni, respectively, provide resistance to both antibiotics and triclosan (Table 4). While not all three-component RND family multidrug efflux systems appear to accommodate and provide for resistance to triclosan (e.g. the MexXY/OprM system of P. aeruginosa) many of the known and predicted RND family exporters that are so widespread in Gram-negative bacteria74 will probably accommodate triclosan and so permit triclosan selection of multidrug resistance. Still, there are as yet no reports of biocide selection of antibiotic-resistant organisms outside the laboratory (a recent examination of resistance phenotypes of bacteria isolated from homes that employed/did not employ biocide-containing products found no correlation between biocide use and antimicrobial resistance392). The fact that household products that often needlessly contain biocide can be used ‘improperly’ by consumers and that diluted products and/or residues might allow for growth of multidrug efflux-hyperexpressing strains that are concomitantly multidrug-resistant cannot be ignored. What occurs in the laboratory can ultimately be replicated in the wild.


    Evolution and natural function of drug efflux systems
 Top
 Abstract
 Introduction
 Efflux-mediated resistance to...
 Efflux-mediated resistance to...
 Evolution and natural function...
 Overcoming efflux-mediated...
 Concluding remarks
 References
 
Drug specific

The bulk of bacterial drug/class-specific export systems described to date are encoded by mobile genetic elements that probably acquired the efflux genes from other (microbial?) sources, possibly antibiotic-producing microorganisms where they functioned as self-protection mechanisms. Probable efflux determinants of tetracycline resistance have, for example, been identified in tetracycline-producing Streptomyces spp.9193 and a possible chloramphenicol exporter has been reported in the chloramphenicol producer, Streptomyces venezuelae.393 Several polyketide/MLS-type antibiotic-producing actinomycetes have been show to carry known/putative efflux mechanisms of self protection, including both MF,394400 and, especially, ABC-type exporters (reviewed in Reference 401). A permeability (possibly efflux) mechanism has also been implicated in the demonstrated resistance of Streptomyces antibioticus to oleandomycin, the macrolide antibiotic that it produces.402 Still, given the prevalence of antimicrobial producers in natural (e.g. soil) environments, one cannot completely exclude the possibility that drug-specific efflux mechanisms arose from earlier non-drug exporters403 via mutation and selective pressure.

Multidrug

Despite their ability to accommodate a range of clinically-relevant antimicrobial agents and their significance vis-à-vis antimicrobial resistance in the clinic, multidrug transporters, particularly of the RND family, probably have as their major and intended function something other than export of noxious environmental agents. There is much debate in the literature regarding the natural function of these bacterial multidrug efflux systems, with evidence for induction of the systems by agents known to be exported by those same systems providing support for proposed roles in protection against noxious exogenous substances.404 The MexXY-OprM system of P. aeruginosa, for example, is both induced by and exports several antibiotics, including gentamicin, erythromycin and tetracycline137 and, as such, efflux of these agents may be its intended function. Still, it is also possible that the action of these agents on their ribosome targets induces expression of the MexXY-OprM efflux system as a result of accumulation of cellular products whose export is carried out by this efflux system and, indeed, a recent study indicates that drug–ribosome interaction is essential for drug induction of mexXY expression (K. Jeannot, M. L. Sobel, F. El Garch, K. Poole and P. Plesiat, unpublished work). Moreover, in contrast to other drug-inducible multidrug efflux systems (e.g. QacA, an MF family exporter that exports a variety of agents though no clinically relevant antibiotics) where drug binding to the cognate regulator (i.e. QacR) alleviates repression,404 providing support for QacA as an intended determinant of drug efflux, MexXY antimicrobial substrates that induce mexXY expression do not interact with or directly modulate the activity of the MexZ repressor of mexXY expression.405 The MexCD-OprJ efflux system of this same organism is also inducible by a number of its non-antibiotic substrates (e.g. the dyes rhodamine 6G, acriflavine and ethidium bromide) though not by any clinically-relevant antibiotics.406 This efflux system is, however, inducible by the antiseptics benzalkonium chloride and chlorhexidine361 although it is not clear if these are MexCD-OprJ substrates and if MexCD-OprJ expression provides for any increase in antiseptic resistance. As with MexXY, it is possible that MexCD-OprJ induction occurs as a result of some impact of these antiseptics on the cell.

The inducibility of the E. coli AcrAB system by toxic fatty acids407 and by bile,408 and the demonstrated role of AcrAB in the export of and resistance to bile salts409 is consistent with a role for AcrAB in protecting the cell from the action of these agents in the gut.407 Clearly, however, there is no evidence for antibiotics being the intended substrates. Similarly, a protective function has been attributed to the MtrCDE system which provides for resistance to faecal lipids in rectal isolates of N. gonorrhoeae410 and, probably, bile salts known to bathe mucous membranes.411 The CmeABC RND family multidrug efflux system also affords resistance to bile salts and, as a result, is necessary for colonization of chicken intestinal tracts.411a The bile inducibility of acrAB and the attendant AcrAB-dependent bile resistance seen in S. enterica serovar Typhimurium also supports a role for this system in bile export and resistance.412 Bile induction of acrAB has also been noted in V. cholerae, suggestive of a role for it in export of/resistance to this agent, and while a contribution of AcrAB to bile resistance was not directly assessed, energy inhibitors that compromised an apparent drug efflux activity in this bacterium also compromised bile resistance.413 Still, the ability to accommodate bile salts is not, in and of itself, proof of a protective function for any efflux system inasmuch as many of the so-called multidrug efflux systems of E. coli accommodate and provide resistance to bile salts.366,414 It would appear unlikely that a given organism would devote several systems to the export of bile salts and so these agents may be just one of many perhaps unintended substrates for these highly accommodating, broadly-specific efflux systems.

An early review implicated by-products of metabolism as probable substrates for multidrug efflux systems415 and reports of the up-regulation of the E. coli AcrAB efflux system in strains with mutations in central biosynthetic pathways, possibly as a result of accumulation of pathway intermediates,416 certainly support this. Additional examples of RND family exporters accommodating non-antimicrobial substrates include IefABC, an Agrobacterium tumefaciens exporter of isoflavanoids417 and the AcrAB efflux system of Erwinia amylovora that accommodates antimicrobials but is, in fact, important for virulence, plant colonization and resistance to plant (apple) phytoalexins, which actually induce acrAB expression in this organism.418 Clearly, then, details of efflux gene regulation and in particular the identification of compounds that directly influence efflux gene expression can be enlightening vis-à-vis the intended or natural function of multidrug efflux transporters. Relatively few of the so-far-described multidrug efflux systems are, apparently, regulated by two-component regulatory systems that typically mediate adaptive responses to environmental stimuli,419 though the nature of these stimuli remains elusive. These pumps include the RND family multidrug exporters SmeABC (regulated by SmeRS) of S. maltophilia,420 MdtABC (regulated by BaeSR)414,421 and multiple other RND family export systems (regulated by EvgAS)422 of E. coli, and AdeABC (regulated by AdeRS) of A. baumannii.423 The E. coli RND family AcrD aminoglycoside exporter and the MdtABC exporter of novobiocin and bile salts are both up-regulated in response to zinc.424

A clear indication that antimicrobial export may not be the intended function of many RND family drug exporters comes from the observation that, for example, P. aeruginosa carries genes for upwards of 11 RND family pumps, of which seven have been shown to accommodate many of the same antimicrobials, though each appears to be independently regulated by linked regulatory genes250 but not (with the exception of MexXY, see above) in response to antibiotics. The implication, certainly, is that each has a distinct function independent of a common role in antimicrobial efflux. The MexAB-OprM multidrug efflux system of P. aeruginosa serves as an excellent model of this family of pumps and, in particular, for studying regulation of tripartite RND-type exporters. Mutations in now three different regulatory (repressor) loci, mexR (nalB) (several citations in References 251 and 252), nalC269,425 and most recently nalD269,426 are associated with increased expression of this efflux system in laboratory and clinical isolates, highlighting the complexity of mexAB-oprM regulation in this organism and its probable role in multiple processes. Still, the identities of natural inducers capable of up-regulating mexAB-oprM (by modulating the repressor activities of MexR, NalC or NalD?) remain elusive and so too is our understanding of its intended substrates and function(s) inside the cell. A recent study suggests that the MexAB-OprM efflux system of P. aeruginosa promotes the release of molecule(s) ultimately important for the virulence of this organism though the actual virulence-related factors exported were not identified.427 The observation that MexAB-OprM hyperexpression in nalB strains impairs fitness and virulence428 also suggests that this efflux system has a physiological role in P. aeruginosa independent of antimicrobial efflux and resistance. Consistent with this, mutants hyperexpressing MexAB-OprM were readily selected in vivo in a rat model of acute P. aeruginosa pneumonia in the absence of any antibiotic selection.429 The specific nature of the selective in vivo growth advantage provided by this efflux system is, however, unknown. Overexpression of the SmeDEF multidrug efflux system, of S. maltophilia has also been shown to compromise fitness.430

The MexEF-OprN efflux system in P. aeruginosa is an intriguing example of an RND family multidrug efflux system, being both positively regulated by a LysR family regulatory protein, MexT (most RND-type systems described to date are repressor controlled) and inversely regulated with the outer membrane protein OprD.326,327 OprD is a basic amino acid porin and a major portal of entry for carbapenem antibiotics, and carbapenem resistance in this organism is often attributable to reduced or lost OprD production.328 Studies of mexEF-oprN regulation have, however, been complicated by the fact that many so-called wild-type strains harbour an inactive mexT gene.431 In a recent study80 of a clinical isolate carrying a wild-type mexT gene, multidrug-resistant mutants hyperexpressing mexEF-oprN and down-regulated for OprD were shown to carry mutations in a gene, PA2491, an oxidoreductase/dehydrogenase homologue previously described by Köhler et al.326 Co-regulation of an oxidoreductase and efflux system might be explained by their having a common role in, for example, detoxification of cellular metabolites, reminiscent of glutathione-mediated detoxification and export of exogenous and endogenous toxic compounds in yeast.432 Still, the fact that loss of PA2491 activity specifically activates mexEF-oprN expression suggests that the constitutively expressed326 PA2491 (unlike mexEF-oprN) has the primary role in ‘detoxification’, and only under circumstances where the enzyme is unable to keep up with metabolite production, due either to mutational loss of PA2491 or, perhaps, an excess of metabolite production would MexEF-OprN be recruited. That enhanced mexEF-oprN expression in PA2491 (and earlier nfxC) mutants is coupled with reduced production of a basic amino acid/peptide porin, OprD, suggests that such metabolites might well be derived from basic amino acids/peptides, whose reduced uptake in such mutants would clearly limit the production of downstream metabolites that might be the substrates for MexEF-OprN and PA2491. There is, in fact, precedence for bacterial efflux of amino acids433436 and their metabolites,435 indicating that under certain circumstances their accumulation within the cell is detrimental to cell health. Intriguingly, one such exporter described in E. coli, RhtA, apparently accommodates multiple amino acids433 displaying a broad specificity somewhat reminiscent of multidrug efflux transporters. Still, overproduced RhtA failed to provide resistance to antimicrobials in E. coli,433 although this study was carried out in a strain expressing the major RND-family multidrug efflux system, AcrAB-TolC, whose activity might have masked a modest contribution of RhtA to antimicrobial resistance. In addition, OprD has been shown to be a non-specific portal for the uptake of other substances, including gluconate,437 and so one cannot rule out other compounds that enter P. aeruginosa via OprD (or their metabolites) being the intended substrates for PA2491 and MexEF-OprN.

Additional studies highlight the probable role of MexEF-OprN in processes distinct from drug efflux. Recent transcriptome analysis of P. aeruginosa after 12 h interaction with airway epithelial cell, for example, revealed a substantial (10- to 15-fold) increase in expression of both mexEF-oprN and PA2491, apparently in parallel with increased damage of the epithelial cells (and release of cell contents?) likely to result from prolonged interaction with this organism.438 Possibly, epithelial cell contents generate intracellular pools of metabolite substrates for MexEF-OprN/PA2491, either following uptake of epithelial cell contents as e.g. nutrients or as a result of physiological changes promoted by exposure to these cell contents (a stress response?). In any case, these data indicate that P. aeruginosa may well encounter circumstances in vivo where PA2491/MexEF-OprN are needed. The demonstration, too, that mutants overexpressing MexEF-OprN were readily recovered from an experimental model of rat pneumonia in the absence of antibiotic selection429 indicates some advantage to MexEF-OprN expression in vivo, independent of antimicrobial export. The recent observation that mutational loss of the VsqR quorum-sensing and virulence regulator compromises mexEF-oprN expression in cells under oxidative stress (i.e. H2O2) suggests that this system may normally be induced in response to this stressor.439 Finally, hyperexpression of MexEF-OprN has been shown to compromise virulence440 possibly as a result of its negative impact on the type III secretion system charged with delivery of P. aeruginosa toxins into cells of infected tissues.441 Still, the apparent ability of this efflux system to compromise production of a cell-to-cell signalling molecule (the pseudomonas quinolone signal; PQS) needed for expression of various virulence genes could also explain its adverse impact on virulence.440 Indeed, a recent study of a PA2491 mutant overproducing mexEF-oprN revealed a negative impact on expression of the PQS genes, which in this study compromised biofilm development.441a That MexEF-OprN hyperexpression compromises expression of type III secretion genes was explained, however, by its export of intracellular signalling molecules needed to activate these genes, although given the apparent connection between expression of certain metabolic genes and type III secretion, it could not be ruled out that hyperexpression of this efflux system somehow impacted cellular metabolism.441 Similarly, MexCD-OprJ-hyperexpressing strains showed similar defects regarding type III secretion.441 Clearly, these studies highlight a role for MexEF-OprN (and MexCD-OprJ) independent of its drug-exporting capability.


    Overcoming efflux-mediated resistance
 Top
 Abstract
 Introduction
 Efflux-mediated resistance to...
 Efflux-mediated resistance to...
 Evolution and natural function...
 Overcoming efflux-mediated...
 Concluding remarks
 References
 
Bypassing efflux

Given the significance of efflux mechanisms, particularly multidrug efflux mechanisms of the RND family as regards antimicrobial resistance in important human pathogens, there is a need to address efflux in designing/developing new antimicrobials and in using existing agents. The value of newer fluoroquinolones in treating, for example, infections caused by S. aureus and, to some extent, S. pneumoniae is that they appear to be less well accommodated by the MF family NorA and PmrA pumps than are/were older agents (e.g. norfloxacin, ciprofloxacin). NorA expression has, certainly, minimal influence on the activity of/resistance to newer fluoroquinolone agents like gatifloxacin,442 nadifloxacin,217 garenoxacin,443 gemifloxacin,444 piperazinyl-linked fluoroquinolone dimers445 and a novel quinolone, WCK771,446,447 apparently because they are poor substrates for this efflux system. Similarly, garenoxacin is not significantly compromised if at all by efflux mechanisms that markedly increase norfloxacin and/or ciprofloxacin MICs in S. pneumoniae.448,449

The ketolide subclass of macrolides are emerging as an effective alternative to macrolides in treating S. pneumoniae450453 or S. pyogenes,454,455 being active against strains expressing the MefA efflux mechanism, presumably because they are not well exported by this efflux system. A recent study does confirm, however, the presence of a modest telithromycin efflux activity in mefA-containing S. pyogenes although it was not clear whether efflux was due to mefA or the msr-like gene also present in these strains.456 Ketolides like telithromycin also appear to be much poorer substrates for the AcrAB-TolC multidrug efflux systems in E. coli195,196 and E. aerogenes195 than lincosamides (e.g. clindamycin) and macrolides (e.g. clarithromycin). The combination of an A and B type streptogramin in quinupristin/dalfopristin works against streptogramin-exporting mutant strains of S. aureus in part because no one efflux mechanism accommodates both A and B type streptogramins in this organism [the ABC family exporter VgaA/B promotes resistance to type A streptogramins (e.g. dalfopristin) whereas the ABC-type exporter MsrA accommodates B streptogramins only (e.g. quinupristin), see Table 1].143,457 Newer 15-membered macrolide agents have been reported with activity against several Gram-negative animal respiratory pathogens (Mannheimia (Pasteurella) haemolytica, Pasteurella multocida, Haemophilus somnus and Actinobacillus pleuropneumoniae) and significantly, perhaps, these appear to be poor substrates for the AcrAB efflux systems in these organisms.458

The success/utility of the glycylcyclines (e.g. tigecycline) stems, too, from their being poor substrates for efflux via the Tet efflux determinants in both Gram-positive and Gram-negative bacteria.459 Again, however, RND family efflux systems in Gram-negative bacteria (E. coli,460 P. aeruginosa,461 P. mirabilis,462 K. pneumoniae463 and Morganella morganii464) are able, to some extent at least, to accommodate tigecycline and can promote reduced susceptibility to this antimicrobial. The recently described MATE family MepA pump of S. aureus also accomodates tigecycline.464a The activities of other newer antimicrobials (e.g. the oxazolidinone linezolid) are also reduced by RND family efflux systems (AcrAB-TolC and AcrEF-TolC of E. coli196) presumably because they are substrates for these broadly-specific exporters. Indeed, recent studies confirm that even novel experimental antimicrobials are substrates for RND-type pumps in, for example, E. coli465 and N. gonorrhoeae.466

Efflux inhibitors

In many instances, efflux is being targeted directly in an attempt to overcome resistance (reviewed in References 467 and 468). The literature is ripe with reports of compounds that reduce efflux-mediated fluoroquinolone resistance and, thereby, potentiate fluoroquinolone activity in S. aureus469,470 by interfering with the activity of the NorA multidrug exporter of this organism.470476 Inhibitors of Tet-mediated tetracycline efflux have also been investigated.477 Plant extracts with some ability to potentiate tetracycline and erythromycin activity against TetK- and MsrA-expressing S. aureus, respectively, have also been reported.478,478a,479 Broad-spectrum efflux pump inhibitors (e.g. PAßN) active against RND pumps in a variety of Gram-negative bacteria including P. aeruginosa, E. coli, H. influenzae, E. aerogenes, K. pneumoniae and Campylobacter spp.76,195,202,480482 though possibly not those in S. maltophilia,282,483 A. baumanni282 and B. pseudomallei292 have been reported. In some of these instances, however, it is possible that the outer membrane barrier is impeding inhibitor uptake and, thus, access to the efflux systems. Additional inhibitors active against RND pumps in P. aeruginosa,484490 the AcrAB-TolC and AcrEF-TolC pumps in E. coli491 and AcrAB-TolC in E. aerogenes492495 and K. pneumoniae495 have also been described.


    Concluding remarks
 Top
 Abstract
 Introduction
 Efflux-mediated resistance to...
 Efflux-mediated resistance to...
 Evolution and natural function...
 Overcoming efflux-mediated...
 Concluding remarks
 References
 
Efflux is a significant determinant of antimicrobial resistance, provided both by readily-acquired exogenous genes for drug-specific resistance and reduced biocide susceptibility and by chromosomal genes that contribute to intrinsic and/or acquired multidrug resistance, the latter following mutational hyperexpression of the efflux genes. Given the mobility of the former and the broad distribution of the latter, efflux mechanisms of antimicrobial resistance are widespread in bacteria and clearly compromise effective antimicrobial chemotherapy of bacterial infectious disease. While drug-specific exporters and multidrug exporters of the MF family in Gram-positive bacteria and RND and, increasingly MATE families in Gram-negative bacteria are commonly highlighted for their contribution to resistance to specific agents (tetracycline and MLS antimicrobials for drug-specific pumps and fluoroquinolones for the multidrug pumps), the association of the former with genetic elements harbouring multiple resistance genes and the broad substrate specificity of the latter indicate that efflux is, in fact, generally significant as determinants/co-determinants of multidrug resistance in bacteria. This is particularly worrisome with respect to the widely-distributed (in Gram-negative bacteria) RND pumps whose broad antimicrobial substrate profiles risk compromising the activities and, thus, use, not only of existing antimicrobials but experimental and yet-to-be-discovered agents as well.496 Indeed, a recent study intended to marry screens for novel antimicrobials with concomitant identification of their bacterial targets inadvertently discovered that the vast majority of antimicrobial ‘hits’ were substrates for the E. coli AcrAB-TolC RND family pump.465 There is always the risk, too, that the presence of these pumps in test/indicator organisms will compromise whole-cell screens for novel antimicrobials, with possibly effective agents ‘missed’ owing to their exclusion from the test organisms.497 Clearly, then, efflux must be addressed when screening and developing novel agents and in maintaining the utility of existing agents, at least in Gram-negative bacteria.496 Ongoing efforts at developing efflux inhibitors and the possible development of new agents that are less affected by efflux are clearly important approaches. These will undoubtedly be aided by the 3D structures of single-component ABC498500 and SMR501,502 and three-component RND-type41,503508 multidrug transporters as well as the details of pump assembly (for tripartite RND-type exporters; e.g. AcrAB-TolC509513 and MexAB-OprM514,515) and substrate recognition by multidrug exporters,499,516519 which may well inform structure-based development of agents able to bypass efflux (i.e. poor efflux substrates) or act as efflux inhibitors.38,520


    Acknowledgements
 
My own studies on efflux-mediated multidrug resistance in P. aeruginosa are supported by an operating grant from the Canadian Cystic Fibrosis Foundation.


    References
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 Abstract
 Introduction
 Efflux-mediated resistance to...
 Efflux-mediated resistance to...
 Evolution and natural function...
 Overcoming efflux-mediated...
 Concluding remarks
 References
 
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