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REVIEW ARTICLE

Efflux pumps as antimicrobial resistance mechanisms

Keith Poole Department of Microbiology & Immunology, Queen's University, Kingston, ON, Canada
Pages 162-176 | Published online: 08 Jul 2009

Abstract

Antibiotic resistance continues to hamper antimicrobial chemotherapy of infectious disease, and while biocide resistance outside of the laboratory is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are not uncommon. Efflux mechanisms, both drug‐specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials in important human pathogens. Multidrug efflux mechanisms are generally chromosome‐encoded, with their expression typically resultant from mutations in regulatory genes, while drug‐specific efflux mechanisms are encoded by mobile genetic elements whose acquisition is sufficient for resistance. While it has been suggested that drug‐specific efflux systems originated from efflux determinants of self‐protection in antibiotic‐producing Actinomycetes, chromosomal multidrug efflux determinants, at least in Gram‐negative bacteria, are appreciated as having an intended housekeeping function unrelated to drug export and resistance. Thus, it will be important to elucidate the intended natural function of these efflux mechanisms in order, for example, to anticipate environmental conditions or circumstances that might promote their expression and, so, compromise antimicrobial chemotherapy. Given the clinical significance of antimicrobial exporters, it is clear that efflux must be considered in formulating strategies for treatment of drug‐resistant infections, both in the development of new agents, for example, less impacted by efflux or in targeting efflux directly with efflux inhibitors.

Introduction

More than 60 years on from the introduction of the first antibiotic, penicillin, into clinical practice and despite an available antimicrobial armamentarium that today numbers in the hundreds, bacterial infectious disease remains a major determinant of human morbidity and mortality. Resistance to antibiotics occurs typically as a result of drug inactivation/modification, target alteration (i.e. target site mutations) and reduced accumulation owing to decreased permeability and/or increased efflux Citation1–3. Last of the resistance mechanisms to be identified, efflux was first described as a mechanism of resistance to tetracycline in Escherichia coliCitation4,5. 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 (adenosine triphosphate)‐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 Citation6 for an in‐depth review of drug efflux families). Though not unique to Gram‐negative bacteria, RND family transporters are most commonly found in such organisms Citation7 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 family exporters (e.g. the macrolide‐specific MacAB‐TolC efflux system ()). Drug efflux systems can be drug‐/class‐specific as for the original Tet pump and the more recently described Mef exporters of macrolides (reviewed briefly in Citation8) 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 (). The genes for agent‐specific efflux mechanisms typically occur on mobile genetic elements (transposons, integrons, plasmids) whose acquisition from other organisms drives resistance Citation8. In contrast, multidrug efflux systems are almost invariably encoded by endogenous, chromosomal genes that are expressed either constitutively (where they contribute to intrinsic resistance) or following mutation (where they contribute to acquired resistance) Citation9. While there is some support for agent‐specific efflux systems originating from antibiotic‐producing micro‐organisms and, so, intended as antibiotic‐exporters Citation9,10, there are increasing indications that the chromosomal multidrug efflux systems are intended as other than antimicrobial resistance determinants and have some other primary export function in bacteria Citation9,Citation11, their clinical relevance notwithstanding.

Table I. Efflux mechanisms of antimicrobial resistancea.

Key messages

  • Given the ever‐growing list of efflux mechanisms, organisms harbouring them, and antimicrobials negatively impacted by them, this clearly important resistance mechanism warrants continued study and, in some instances, serious consideration as a therapeutic target.

  • Elucidation of the intended or natural function of multidrug efflux mechanisms is important for identifying and, so, anticipating environmental conditions or circumstances that might promote their expression and in so doing compromise antimicrobial chemotherapy.

  • Antimicrobial efflux can be inhibited directly, via inhibition of efflux pump activity, or indirectly, via interference with efflux gene expression or efflux pump assembly.

Tetracyclines

Tetracycline resistance mediated by the MF family TetA pump was first reported in 1980, and today there are more than 20 different Tet efflux proteins described in bacteria Citation12,13, mostly in Gram‐negative bacteria but also in Gram‐positive bacteria and the mycobacteria (http://faculty.washington.edu/marilynr/). Most of the >20 tet efflux determinants are found only in Gram‐negative organisms, although a few (e.g. Tet(L) and Tet(K)) are more commonly associated with Gram‐positive bacteria. tet genes are almost invariably encoded on mobile genetic elements (plasmids, transposons, integrons) and often with additional resistance genes, with their acquisition providing for resistance to tetracycline and additional agents Citation9,Citation12. Chromosomal tet efflux determinants have also been reported, in Staphylococcus aureus (tet38) Citation14 and Helicobacter pylori (HP1165) Citation15, with resistance resulting from mutational overproduction (tet38) or from an as yet undefined tetracycline‐dependent translational /post‐translational effect on pump activity (HP1165) Citation15. Although not previously seen in Neisseria, a tet efflux determinant (tet(B)) was very recently reported in tetracycline‐resistant Neisseria meningitidisCitation16,17. Tet‐mediated efflux is the predominant mechanism of tetracycline resistance in many Gram‐negative bacteria (e.g. Salmonella spp. Shigella spp., E. coli, Acinetobacter spp., and Chlamydia spp.), although it has yet to be reported as a mechanism of resistance in, for example, Campylobacter and is comparatively rare in Gram‐positive organisms Citation12,13, where ribosomal protection mechanisms of tetracycline resistance predominate Citation12,13,Citation18. 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 Citation8,Citation19. 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 while Gram‐positive tet genes are controlled by a ‘translational attenuation’ mechanism Citation8. Many of the RND family pumps found in Gram‐negative bacteria accommodate tetracyclines, although there are few reports of RND family efflux systems as primary determinants of tetracycline resistance, with only a few in vitro studies documenting, for example, tetracycline selection of RND pump‐producing resistant mutant strains (e.g. Pseudomonas aeruginosa mutants overproducing MexAB‐OprM and Stenotrophomonas maltophilia mutants producing SmeDEF Citation9).

Macrolides, lincosamides, ketolides, and streptogramins

A number of efflux mechanisms of resistance to the macrolide, lincosamide, ketolide, and streptogramin (MLKS) group of antibiotics are known (reviewed in Citation20), typically of the MF and ABC families of drug exporters (). Major determinants of macrolide‐specific (14‐ and 15‐membered macrolides only) resistance/efflux (M resistance phenotype) are the MF family Mef(A) and Mef(E) exporters first identified in Streptococcus pyogenes and Streptococcus pneumoniae, respectively, but now seen in a number of other Gram‐positive as well as Gram‐negative organisms, and including anaerobes Citation9,Citation21–24 (). These highly related systems (c. 90% sequence identity) were once thought to represent a common macrolide‐specific efflux system proposed to be jointly dubbed Mef(A) Citation25, but given differences in their genetic context (mef(E) is found in the conjugative transposon MEGA (macrolide efflux genetic assembly) element and mef(A) is found on the closely related conjugative transposons Tn1207.1 and Tn1207.3Citation26,27), microbial distribution (mef(E) is disseminated in many more species Citation23,Citation28), geographic distribution (mef(E) predominates in North America and Asia while mef(A) predominates in Europe Citation28), and impact on macrolide MICs (mef(A)‐containing isolates of S. pneumoniae show higher minimum inhibitory concentrations (MICs) than do mef(E)‐containing isolates) some researchers favour maintaining separate designations for these mef determinants Citation23. Recently, a third mef variant, mef(I), has been described in S. pneumoniae, showing c. 90% identity to mef(E) and mef(A) and not associated with MEGA or Tn1207Citation29. Mef is a major contributor to macrolide resistance in several Streptococcus spp. Citation9,Citation30–36 and is primarily responsible for the increasing rate of macrolide resistance seen in S. pneumoniae worldwide (e.g. Citation37,38).

Both mef(E) and mef(A) are part of an operon that includes a downstream gene, dubbed mel or msr(D), encoding an ABC family exporter homologue of Msr(A) Citation28,Citation39. Msr(A) contributes to inducible resistance to erythromycin and type B streptogramins (MSB resistance phenotype) in Staphylococcus spp. Citation40 although Mel/Msr(D) does not promote resistance to streptogramins Citation28. Despite the well known and characteristic genetic linkage of mef and mel/msr(D), until recently their respective importance vis‐à‐vis macrolide resistance has not been addressed. A study Citation28 in which the genes were individual cloned into a susceptible S. pneumoniae isolate, however, revealed that each could promote resistance on their own (and msr(D) also promoted a modest increase in ketolide resistance, presumably explaining the modest impact of mef‐containing elements on ketolide susceptibility and ketolide efflux activity in the streptococci Citation41). Intriguingly, another study in which the mef and mel genes were individually disrupted on the MEGA element of a macrolide‐resistant S. pneumoniae isolate demonstrated that loss of either substantially reduced resistance (though not to the same extent as when both were deleted; i.e. each, alone, could contribute to resistance), indicating that maximal resistance required both and, thus, Mef/Mel may well function as a dual efflux pump Citation39.

Originally seen only in staphylococci, where it is implicated in macrolide and streptogramin resistance in clinical isolates Citation9, msr(A) has recently been reported in Streptococcus spp., Enterococcus spp., Corynebacterium spp., Pseudomonas spp. (including P. aeruginosa), Enterobacter spp. Citation42, and Bacteroides fragilisCitation43. Related ABC family efflux determinants of streptogramin A resistance, Vga(A) and Vga(B), have also been reported in Staphylococcus spp. and are similarly plasmid‐/transposon‐encoded Citation40,Citation44. Unlike Msr(A), these provide low‐level resistance to lincosamides and do not accommodate macrolides Citation40,Citation44. Recently a Vga(A) variant, Vga(A)LC, was described in Staphylococcus haemolyticus that provided marked resistance to lincosamides Citation45. Originally thought to be streptogramin A‐specific, Vga(B) has now been shown to reduce susceptibility to pristinamycin, a mixture of A and B type streptogramins Citation44. The presence of vga(A) has also been correlated with low‐level quinupristin/dalfopristin resistance in Staphylococcus hominisCitation46, suggesting that vga determinants may well be able to accommodate, to some extent at least, both A and B type streptogramins. A chromosomal efflux determinant of intrinsic resistance to lincosamides, A type streptogramins Citation47, and quinupristin/dalfopristin (i.e. Synercid) Citation48 has also been identified in Enterococcus faecalis.

Efflux‐mediated resistance to MLKS antimicrobials is also present in Gram‐negative bacteria, most often mediated by 3‐component RND family exporters that appear to explain the intrinsic resistance of many Gram‐negative bacteria to these agents Citation9,Citation49 (). Efflux (by CmeABC and others) is, for example, a major contributor to intrinsic Citation50 and acquired (low‐ and high‐level) Citation51–53 macrolide resistance in Campylobacter spp. and is implicated in telithromycin resistance Citation51,52. Notably, high‐level macrolide and telithromycin resistance in Campylobacter often reflects a contribution of both efflux and target site mutations (i.e. mutations in the 23S rRNA and ribosomal protein genes) Citation51,Citation53. Efflux similarly plays a role in intermediate‐ and high‐level macrolide and telithromycin resistance in Haemophilus influenzae, again working cooperatively with target site mutations in promoting high‐level resistance Citation54.

Fluoroquinolones

Target site mutations are by far the most common means of resistance to the fluoroquinolone (FQ) class of antimicrobials Citation55–60, with mutations in the so‐called quinolone resistance determining region (QRDR) of type II topoisomerases commonly associated with high‐level resistance to these agents Citation60. Still, quinolone efflux has been reported in a number of bacteria Citation9,Citation61–68 (), and several efflux determinants of FQ resistance have been identified in Gram‐positive Citation55 and Gram‐negative Citation57–59,Citation69 bacteria, including anaerobes Citation56 (). These are typically related to the chromosome‐encoded MF family NorA multidrug transporter of Staphylococcus aureus (in Gram‐positive bacteria) Citation9,Citation55 and the RND family AcrAB‐TolC/MexAB‐OprM multidrug transporters common in enteric (e.g. E. coli, Salmonella spp.) and non‐fermenting (e.g. P. aeruginosa) Gram‐negative organisms Citation9,Citation70 (). FQ efflux has also been reported in the mycoplasma Citation71 and mycobacteria Citation72, although no clinical significance has yet been attributed to these efflux mechanisms. While RND family transporters accommodate a wide variety of clinically relevant antimicrobials, and their production is, thus, associated with reduced susceptibility to multiple antibiotics Citation66,Citation70, they are most frequently cited as determinants of FQ resistance Citation9, and, indeed, invariably all reports of in vitro‐/in vivo‐selected FQ resistance in Gram‐negative bacteria where efflux is involved is attributable to this group of efflux systems Citation9. ABC, MATE, and SMR family multidrug efflux systems able to accommodate FQs are also known Citation9 (), although their clinical significance, if any, is unknown.

In contrast to target site mutations that can provide high‐level resistance to FQs, efflux mechanisms tend to promote low‐level resistance to FQs Citation57. Still, clinical resistance to these (and other) agents is typically multifactorial, with many highly FQ‐resistant isolates harbouring both efflux and target site (i.e. topoisomerase II) mutations (e.g., P. aeruginosaCitation66,Citation73–75, Salmonella entericaCitation66,Citation76,77, E. coliCitation64, Proteus mirabilisCitation78, Bacteroides fragilisCitation79, Campylobacter spp. Citation66,Citation80, Enterococcus faecalisCitation81, Enterococcus faeciumCitation61, Vibrio choleraeCitation66, Klebsiella pneumoniaeCitation82, Vibrio fluvialisCitation63, and Citrobacter freundiiCitation66), with the former not only contributing to overall resistance but also possibly serving, during development/evolution of resistance in vivo, to protect cells long enough for target site mutations to develop Citation83. Even in the absence of evidence linking an observed efflux gene expression to resistance in a clinical isolate, then, it is quite possible that efflux played a role in the evolution of the currently observed resistance phenotype. In agreement with this, FQ‐selected (in a mouse thigh model) mutants of S. pneumoniae expressing an apparent efflux mechanism showed a minimal increase in resistance but much more readily yielded highly‐resistant strains than did the initial (i.e. non‐efflux) strain Citation84. Still, in vitro studies show that efflux mutations can precede or follow target site mutations in the progressive development of increased FQ resistance, depending on the organism and the selecting FQ Citation57. In any case, studies showing that highly FQ‐resistant target site mutants cannot be selected in vitro from efflux‐deficient mutants of P. aeruginosaCitation85, E. coliCitation86Salmonella enterica serovar Typhimurium Citation87, and Campylobacter spp. Citation80 clearly highlight the significant contribution of efflux to high‐level FQ resistance, as does the observation that loss of multidrug efflux (AcrAB in E. coli [Citation86or S. enterica serovar Typhimurium Citation88; MexAB‐OprM in P. aeruginosaCitation85; CmeABC in CampylobacterCitation50) undermines the resistance provided by target site mutations, in some instances rendering the mutants susceptible despite the presence of these target site mutations Citation85,86,Citation88.

Aminoglycosides

Relatively few bacterial drug efflux systems are known to accommodate aminoglycosides (e.g. the AmrAB‐OprA Citation66 and BpeAB‐OprB Citation89 multidrug efflux systems of Burkholderia pseudomallei, the AcrAD‐TolC multidrug efflux system of E. coliCitation66, the AdeABC multidrug efflux system of Acinetobacter baumanniiCitation90, and the MexXY/OprM multidrug efflux system of P. aeruginosaCitation66 ()), although there are numerous AcrD homologues in other Enterobacteriaceae Citation66 suggesting that additional aminoglycoside‐exporting efflux systems may be present in Gram‐negative bacteria. These aminoglycoside exporters are all RND family efflux systems, 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 Citation66. Still, only in P. aeruginosa is efflux a significant determinant of acquired aminoglycoside resistance, with numerous reports of impermeability‐type pan‐aminoglycoside resistance in clinical isolates characterized by reduced drug accumulation that is now attributable to efflux via MexXY/OprM Citation91. The aminoglycoside‐exporting pumps of B. pseudomallei do, however, contribute to the organism's intrinsic resistance to these agents Citation66,Citation89.

β‐lactams

The most common mechanism of acquired resistance to β‐lactam antimicrobials is β‐lactam destruction by β‐lactamase enzymes (chromosome‐encoded or expressed from acquired mobile genetic elements (plasmids, transposons, integrons)) Citation92. While many of the RND family multidrug efflux systems prevalent in Gram‐negative bacteria do accommodate these agents Citation9,Citation92 and, indeed, the RND family Mex pumps of P. aeruginosa are, for some β‐lactams, more effective determinants of resistance in vitro than this organism's chromosomally‐encoded AmpC β‐lactamase Citation93, there are very few reports of efflux mechanisms contributing to β‐lactam resistance in clinical or, indeed, in vitro‐selected resistant strains. Still, those β‐lactam‐exporting systems that have been described are invariably of the RND family (). The P. aeruginosa MexAB‐OprM RND family multidrug efflux system that accommodates a range of β‐lactams has, for example, been implicated in resistance to the penicillin ticarcillin Citation94,95 and the carbapenem meropenem Citation96,97 in clinical isolates. Expression of Mex efflux systems has also been linked to ertapenem (and aztreonam) resistance in clinical strains of P. aeruginosaCitation98, although direct evidence for an efflux contribution was lacking. An efflux contribution to carbapenem resistance has also been noted in Enterobacter cloacaeCitation99, and meropenem resistance in clinical isolates of Stenotrophomonas maltophilia correlates with elevated expression of the RND family SmeDEF multidrug efflux system Citation100 previously shown to accommodate β‐lactams Citation101. Overexpression of the MtrCDE multidrug efflux system of Neisseria gonorrhoeae has also been highlighted as an important contributor to the high‐level penicillin resistance of certain clinical isolates of this organism Citation102. A recent report, too, of high‐level ampicillin resistance in a so‐called β‐lactamase‐negative ampicillin‐resistant (BLNAR) H. influenzae implicated the endogenous RND family exporter AcrAB‐TolC as a co‐determinant of this resistance Citation103. Efflux has also been implicated in the cefuroxime resistance of clinical isolates of E. coliCitation104, cefoxitin resistance in B. fragilisCitation79, and, surprisingly, oxacillin resistance in methicillin‐resistant Staphylococcus aureus (MRSA) Citation105. Finally, the MexXY multidrug efflux determinant of aminoglycoside resistance has been shown to contribute to cefepime resistance in clinical isolates of P. aeruginosaCitation106.

Biocides

Efflux as a mechanism of reduced biocide (i.e. antiseptics, disinfectants and preservatives) susceptibility is well established, with a variety of plasmid‐ and chromosome‐encoded determinants of quaternary ammonium compound (QAC; e.g. benzalkonium chloride), triclosan and silver resistance reported in Gram‐positive and Gram‐negative bacteria Citation9,Citation107 (). Several plasmid‐encoded, SMR (e.g. Smr (also called QacC/D), QacE▵1, QacG, QacH, QacJ) and MF (QacA/B) family QAC exporters have been reported in Gram‐positive bacteria, predominantly S. aureus (and including MRSA Citation108,109), as well as Gram‐negative bacteria (SMR family; QacE, QacE▵1, QacF and QacG) Citation9. A number of chromosome‐encoded multidrug transporters also accommodate QACs (e.g. the NorA pump implicated in FQ resistance in S. aureus, and several RND family pumps found in Gram‐negative bacteria Citation9) (). While the level of resistance provided is generally quite modest, there are reports of bacteria contaminating QAC‐containing solutions where efflux is implicated Citation110. A number of RND family efflux systems are able to accommodate additional biocides, including silver, chlorhexidine, and, especially, triclosan Citation9 (), with the latter agent readily selecting for strains expressing/hyperexpressing these systems in vitroCitation9. Several of the RND family Mex efflux systems are, in fact, major determinants of triclosan insusceptibility in P. aeruginosaCitation111. Interestingly, the MexCD‐OprJ efflux system of P. aeruginosa is inducible by chlorhexidine (and benzalkonium chloride) Citation112 and contributes to chlorhexidine resistance (A. Campigotto, K. Poole, unpublished). In a recent report, Enterobacter gergoviae contaminating cosmetic formulations containing the preservative parabens apparently expressed an efflux mechanism of parabens resistance Citation113.

Given the ability of NorA and RND family efflux systems to accommodate both biocides and antimicrobials used to treat infectious diseases, the possibility exists (realized to date only in vitro) of biocides selecting for organisms expressing/overexpressing these efflux systems and, so, resistant to antibiotics Citation9. QAC‐resistant S. aureus selected in vitro often showed cross‐resistance to FQs as a result of increased norA expression in these, and, indeed, QACs seemed to more effectively select for NorA‐expressing mutants than did FQs Citation114. Several in vitro studies have shown that triclosan readily selects for multiple antibiotic‐resistant P. aeruginosa, S. maltophilia, and E. coli expressing these multidrug efflux systems, and for multidrug‐resistant Salmonella spp. and E. coli O157:H7 where multidrug efflux mechanisms are implicated (reviewed in Citation9,Citation115). The correlation between triclosan and multidrug resistance in human and animal isolates of Campylobacter spp. Citation116 and Salmonella spp. Citation117 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‐resistant Campylobacter and Salmonella. At present, however, there are no reports of biocide selection of antibiotic‐resistant organisms outside the laboratory, and, indeed, 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 resistance Citation118.

Additional agents

The RND family AcrAB‐TolC efflux system of H. influenzae is implicated in resistance to an experimental peptide deformylase inhibitor (LBM415) Citation119, and the homologous efflux system in E. coli has recently been identified as the most probable determinant of insusceptibility to a panel of experimental antimicrobials Citation120. Highlighting the broad substrate specificity of RND family pumps, recent studies have also confirmed that many of these accommodate and, so, explain the resistance of Gram‐negative bacteria to oxazolidinones (e.g. linezolid) Citation9,Citation121,122. Recently, too, reports of probable efflux contributions to metronidazole and isoniazid/ethambutol resistance in Helicobacter pyloriCitation123 and Mycobacterium tuberculosisCitation124, respectively, have surfaced. Finally, the intrinsic resistance of N. meningitidis to the polycationic antimicrobials, polymyxin B (PXB) and mammalian cationic antimicrobial peptides is known to be due in part to the RND family pump, MtrCDE Citation121.

Overcoming efflux‐mediated antimicrobial resistance

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. One approach is to develop agents less impacted by known efflux mechanisms Citation125,126. The value of newer fluoroquinolones, for example, in treating infections caused by S. aureus and, to some extent, S. pneumoniae is that they appear to be less well accommodated by the FQ‐exporting MF family NorA and PmrA pumps of these organisms than are/were older agents (e.g. norfloxacin, ciprofloxacin) Citation9,Citation125,126. Similarly, the ketolide subclass of macrolides is emerging as an effective alternative to macrolides in treating Streptococcus spp., being active against strains expressing the Mef efflux mechanisms Citation9,Citation125,Citation127, presumably because they are less well exported by this efflux system. Still, telithromycin efflux has been reported in the streptococci, and efflux mechanisms, including those of the RND family, tend to compromise ketolide activity in many Gram‐negative bacteria Citation9,Citation125. As well, the combination of an A and B type streptogramin in Synercid (i.e. quinupristin/dalfopristin) works against streptogramin‐exporting mutant strains of S. aureus apparently because no one efflux mechanism accommodates both A and B type streptogramins. Still, Vga(B) does, in fact, accommodate pristinamycin, a 70/30 mixture of A and B type streptogramins Citation44, and the presence of vga(A) has been correlated with low‐level quinupristin/dalfopristin resistance in Staphylococcus hominisCitation46. 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 Citation128. Again, however, RND family efflux systems in Gram‐negative bacteria are able, to some extent at least, to accommodate tigecycline and can promote reduced susceptibility to this antimicrobial Citation9 (). Given the broad distribution of RND family efflux systems in Gram‐negative bacteria and the broad substrate specificity of these pumps (as indicated above, recent studies confirm that even novel experimental antimicrobials are substrates for RND type pumps in Escherichia coliCitation120), it is questionable whether efflux avoidance will be practical in Gram‐negative organisms.

Given that a strategy based on efflux avoidance is unlikely to be successful in overcoming RND pump‐mediated resistance, RND pumps represent an obvious target for the development of efflux inhibitors, with such inhibitors likely necessary to deal with RND pump‐mediated antimicrobial resistance in Gram‐negative organisms Citation125,126,Citation129. Possible approaches include inhibiting efflux either directly or indirectly, via interference with efflux gene expression or, given that RND type pumps are multicomponent, by interfering with pump assembly Citation125. In targeting efflux, however, it is important to realize that efflux is often only one of several available mechanisms of resistance to a given antimicrobial. Thus, its inhibition will have a significant therapeutic effect only for those antibiotics and in those organisms where efflux is the major contributor to resistance. Resistance to FQs in many bacterial species and to macrolides in H. influenza, for example, can arise from both target site mutations and efflux via RND type pumps but in organisms harbouring both, loss of efflux dramatically reduces resistance to FQs and macrolides (see above). Thus, efflux inhibition is a viable strategy for countering RND pump‐mediated resistance to FQs and macrolides in these organisms. Another consideration is that a single bacterium may contain multiple RND systems able to confer resistance to a given antibiotic. P. aeruginosa and various Enterobacteriaceae, for example, have multiple pumps that can accommodate FQs Citation9. Consequently, only broad‐spectrum inhibitors able to interact with multiple transporters should be considered for combination therapy with FQs. In the case of aminoglycosides (e.g. tobramycin) used to prevent acute exacerbations of P. aeruginosa lung infections in patients with cystic fibrosis (CF), however, the major determinant of resistance is a single RND type efflux system, MexXY‐OprM Citation91. As such, a MexXY‐OprM‐specific inhibitor would be effective in overcoming aminoglycoside resistance in this organism.

The literature is ripe with reports of compounds that target MF (e.g. NorA) and RND family multidrug exporters and, so, enhance the antimicrobial susceptibility of resistant isolates expressing efflux mechanisms Citation57,Citation130,131. Importantly, many of the RND pump inhibitors are broad‐spectrum, active against multiple RND pumps, both within a given organism and in a variety of Gram‐negative bacteria (e.g. P. aeruginosa, E. coli, H. influenzae, Enterobacter aerogenes, Klebsiella pneumoniae and Campylobacter spp.) Citation9,Citation132. Still, none of these has been confirmed as being clinically useful, and there remain the obvious problems associated with preclinical and clinical development of a combination therapy (i.e. pump inhibitor + antibiotic).

Concluding remarks

Efflux as a mechanism of resistance, drug‐specific and multidrug, is well established in the antimicrobial literature, although clearly all the players, real and potential, have yet to be identified. There are, for example, many reports of efflux activity independent of known/identified efflux systems (), and a recent study of the reservoir of resistance determinants present in soil‐dwelling bacteria is suggestive of novel, possibly efflux, mechanisms of resistance to agents for which efflux is not yet identified as a resistance mechanism Citation133. Conversely, the clinical significance of many known efflux determinants has yet to be demonstrated—studies that rely on cloned genes and in vitro strain construction in identifying efflux determinants of resistance serve only to identify potential resistance mechanisms. Still, bacterial genomes are rich in efflux genes, and a great many of the corresponding efflux systems do accommodate antimicrobials (a comprehensive study of the efflux genes in E. coli revealed many to have at least some ability to promote (when cloned) resistance to at least some antimicrobials Citation134, and all nine of the RND family exporters that have been studied to date in P. aeruginosa show some ability to accommodate antimicrobials Citation9,Citation135). Given the focus, for many agents, on target site mutations (e.g. in FQ) as primary determinants of high‐level resistance it is likely, too, that an efflux contribution is often overlooked. Indeed, when one does look, one often finds both in FQ‐resistant clinical strains.

While AcrAB‐TolC and related pumps in Gram‐negative bacteria certainly contribute to intrinsic resistance, they also contribute to acquired resistance as a result of their increased production, usually as a result of mutations in regulatory genes that control efflux gene expression and, so, promote increased efflux gene expression Citation14,Citation65,Citation82,Citation103,Citation135–138. Still, in light of the probable roles of RND pumps in the export of other than antimicrobials and, so, their possible recruitment in response to specific cellular and/or environmental signals, non‐mutational upregulation of these efflux systems in the absence of antimicrobial selection is also a possibility. Indeed, strains of P. aeruginosa recovered from experimental animal models of pneumonia were shown to be expressing RND family efflux mechanisms that promoted resistance to FQs in the absence of FQ administration to the animals Citation139. The membrane‐damaging biocides chlorhexidine and benzalkonium chloride (and various other membrane‐damaging agents; A. Campigotto, S. Fraud, K. Poole, unpublished observations) have been shown to promote expression of the MexCD‐OprJ efflux system (and, so, multidrug resistance) in P. aeruginosa. Thus, hospital biocides and/or in vivo conditions (i.e. during an infection) that disrupt P. aeruginosa membranes may well promote antimicrobial resistance. Finally, it has recently been demonstrated Citation140,141 that oxidative stress (promoted by biocides peracetic acid and hydrogen peroxide) stimulates expression of the PA5471 gene required for MexXY expression Citation142, suggesting that oxidative stress may well induce MexXY expression and, so, promote aminoglycoside resistance. Significantly, P. aeruginosa encounters substantial oxidative stress in the lungs of CF patients Citation143, and efflux‐mediated aminoglycoside resistance (involving MexXY) is disproportionately represented amongst CF versus other clinical P. aeruginosa isolates Citation91. The recent observation that most CF isolates harbour mutations in the mexZ gene encoding a repressor of mexXY and, so, are likely to express this efflux system Citation144 is consistent with environmental conditions at the site of infection (oxidative stress) promoting/selecting for efflux gene expression and, so, antimicrobial resistance. Still, it was unclear in this latter study whether, and to what extent, patients would have been exposed to aminoglycosides and, so, whether aminoglycosides might have played a role in selection of the potentially MexXY‐expressing isolates. In any case, it is clearly vital to elucidate the details of ‘natural’ efflux gene regulation and to identify the growth/environmental circumstances that promote efflux gene expression so as to be able to predict when and where in a clinical setting these efflux systems might be recruited in pathogenic bacteria and, so, compromise antimicrobial chemotherapy. Given the ever‐lengthening list of efflux mechanisms of antimicrobial resistance, the spread of existing mechanism amongst a growing number of bacteria and the increasing number of agents whose activity efflux is shown to compromise, to some extent at least, this clearly important resistance mechanism warrants continued study and, in some instances, serious consideration as a therapeutic target.

References

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