Role of Efflux Pumps on Antimicrobial Resistance in Pseudomonas aeruginosa

Antimicrobial resistance is an old and silent pandemic. Resistant organisms emerge in parallel with new antibiotics, leading to a major global public health crisis over time. Antibiotic resistance may be due to different mechanisms and against different classes of drugs. These mechanisms are usually found in the same organism, giving rise to multidrug-resistant (MDR) and extensively drug-resistant (XDR) bacteria. One resistance mechanism that is closely associated with the emergence of MDR and XDR bacteria is the efflux of drugs since the same pump can transport different classes of drugs. In Gram-negative bacteria, efflux pumps are present in two configurations: a transmembrane protein anchored in the inner membrane and a complex formed by three proteins. The tripartite complex has a transmembrane protein present in the inner membrane, a periplasmic protein, and a porin associated with the outer membrane. In Pseudomonas aeruginosa, one of the main pathogens associated with respiratory tract infections, four main sets of efflux pumps have been associated with antibiotic resistance: MexAB-OprM, MexXY, MexCD-OprJ, and MexEF-OprN. In this review, the function, structure, and regulation of these efflux pumps in P. aeruginosa and their actions as resistance mechanisms are discussed. Finally, a brief discussion on the potential of efflux pumps in P. aeruginosa as a target for new drugs is presented.


The Silent Pandemic of Antimicrobial-Resistant Bacteria
Antibiotics revolutionized medicine in the 1930s-1940s, allowing millions of lives to be saved due to their ability to end infections caused by pathogenic bacteria. The first antibiotic used to treat an infectious disease was arsphenamine, also known by the trade name Salvarsan. This synthetic molecule, discovered by Paul Ehrlich, was widely used for the treatment of syphilis [1]. In time, the first commercially available broad-spectrum antibiotic was penicillin, discovered by Ian Flemming in 1929 [2]. Penicillin was introduced and widely used in the 1940s, during World War II, and helped to save many soldiers suffering from war wounds. However, its indiscriminate use quickly led to the emergence of the first penicillin-resistant bacterial strains [3].
Antimicrobial resistance (AMR) is an old pandemic that has become a major public health crisis. According to the World Health Organization (WHO), the topic is considered one of the most important threats to the human species in the 21st century [4]. It is a natural phenomenon shaped by natural selection and evolution. However, in recent decades there has been an acceleration in the emergence of AMR bacteria due to selective pressures Protein transport systems received much attention in the 1990s, with structural, functional, and phylogenetic studies that aimed to uncover their distinct evolutionary origins [39,43,44,49,50]. These first studies, focused on the RND (super)family, enabled the identification of three common protein units, an inner transmembrane transporter, a periplasmic protein, and an outer membrane channel protein, which combine to form a trans-periplasmic channel [45,51,52] that allows the capture of substrates in the periplasm or cytoplasm [45]. After the assembly of the protein complex, the system stabilizes in two different stages: a resting stage, and a transport stage [53,54]. Driven by the proton motive force, the RND substrate efflux goes through cycles of site access, substrate binding, and extrusion, all mediated by a strictly coordinated rotational mechanism [55] that allows the system to work as a pump mechanism. As other families began to be discovered and more effort started to be put in the study of efflux pump mechanisms, different structures and driving forces began to be described, such as single protein mechanisms [56][57][58][59][60], ion gradients [56,57], and ATP [59] dependent pumps.

General Role of Efflux Pumps in Bacteria in a Natural Environment
Efflux pumps evolved as a way for bacteria to interact with the environment [100]. Their ubiquitous presence in nature [101] allowed bacteria to survive in their ecological niches [38], protecting them from toxic compounds produced by other species of bacteria or by the host, antimicrobial molecules, reactive oxygen species (ROS), and toxic byproducts of biochemical degradation pathways [38,43,52,[102][103][104]. Efflux pumps can be specific for only one substrate or can export a range of molecules [38,62,105]. This difference in substrate selection is directly associated with the physicochemical characteristics of the substrate and the molecular interactions between the substrate, protein binding site, and environment, possibly reflecting the ability of the efflux pumps to capture their substrates [106]. As such, some of the main substrates of efflux pumps cited in the literature are detergents [52,62,107,108], antibiotics [52,62,102,108,109], organic solvents [102,110], dyes [62,102,[107][108][109], disinfectants, aromatic hydrocarbons [109], cationic biocides [111], free fatty acids [112], lipophilic drugs, amphiphilic agents [107], antiseptics [62,108], anticancer drugs, uncouplers [108], and anti-virulence compounds [43].
The transcriptional regulation of efflux systems depends directly on the concentration of external signals [113], together with the action of internal transcription factors such as the TetR family proteins [114], LysR-type transcriptional regulators (LTTRs) [115], the multidrug regulator OstR [113], and activators MarA, Rob, and SoxS [112]. Cross-talking between efflux systems [113] and the need for chaperone-assisted folding increases its complexity, as observed in the downregulation of OmpF porin, which reduces cell permeability [52]. As observed, the presence of efflux pumps contributes to the maintenance of cell viability, virulence, and quorum sensing [43,102], assisting the bacterial cell in tolerating rapidly changing environments [101]. In this case, mechanisms of virulence and resistance evolved to fulfill different functions in the natural environment earlier than and not directly related to their role in multidrug resistance [43].

Efflux Pumps Involved in AMR in Bacteria
The first study that directly evaluated the action of efflux pumps on antibiotic resistance dates to the 1980s and revealed the action of plasmid-transferred energy-dependent efflux pumps on tetracycline resistance in Escherichia coli [46]. McMurry and colleagues demonstrated the ability of efflux pumps to shift the drug even against a concentration gradient [46], showing a strict relationship between the efflux pump together with outer membrane permeability and the acquisition of drug resistance [42,52].
Efflux systems have been found in comparable numbers among pathogenic and nonpathogenic bacteria. Its emergence earlier than the extensive exposure to antimicrobial agents suggested that their evolution as a resistance mechanism occurred independently of the use of antimicrobials [44]. The efflux system allows the bacteria to survive at low levels of drugs, conferring time to develop a more specialized mechanism of resistance [116]. When combined with the outer membrane barrier, the efflux system guarantees cellular protection from a range of compounds [106], favoring the dissemination of gene coding for such systems. Some isolates have genes for different efflux pumps in their genome, acting as genetic reservoirs [48,52]. This organization can also occur from gene duplication events [117] and gene cassettes [116].
Efflux pump specificity can follow a nonlinear behavior depending on the variety of compounds it can transport, leading to efficient extrusion even in cases of low substrate affinity for the binding site [106]. At the same time, such bonding can lead to the blocking of rotational movement and impediment of extrusion [53]. In this way, the structural changes observed can allow the binding of two or more substrate molecules in the same system, such as the extrusion of hydrophobic and hydrophilic compounds, expanding the specificity of the efflux pump [55].
The role of efflux pumps in AMR can be characterized as a byproduct of physiological functions that are beneficial to bacteria [38], and their efficiency depends on several factors, both internal and external to the bacterium. The presence of efflux pump genes can be considered the first step in the acquisition of resistance to antibiotics [118].

Efflux Pumps as a Mechanism of Antimicrobial Resistance in P. aeruginosa
P. aeruginosa has a naturally low susceptibility to antibiotics, explained by its extensive intrinsic resistome [119], low membrane permeability [120], and ability to form biofilms [16]. In its core genome, there are several antibiotic resistance genes, such as bla ampC , and the genes encoding the multidrug efflux pumps (Mex) MexXY, MexAB-OprM, MexCD-OprJ, and MexEF-OprN [121], all of which are part of the RND superfamily.
These efflux pumps are tripartite protein complexes, which are drug/proton antiporters that catalyze the extrusion of their specific substrates from the periplasm through the outer membrane [122]. They are composed of three different proteins: a periplasmic adaptor protein, commonly known as periplasmic membrane fusion protein (PMFP), such as MexA, MexX, MexC, or MexE; a resistance-nodulation-cell division transporter (RNDt), such as MexB, MexY, MexD, or MexF, and a channel-forming outer membrane factor (OMF), such as OprM, OprJ, or OprN [123]. Structural analyses of the MexAB-OprM efflux pump show that the PMFP forms a stable complex associated with the inner membrane and the RNDt, both of which are recruited by the OMF, which resides in the outer membrane [124,125] (Figure 1). P. aeruginosa has a naturally low susceptibility to antibiotics, explained by its extensive intrinsic resistome [119], low membrane permeability [120], and ability to form biofilms [16]. In its core genome, there are several antibiotic resistance genes, such as blaampC, and the genes encoding the multidrug efflux pumps (Mex) MexXY, MexAB-OprM, MexCD-OprJ, and MexEF-OprN [121], all of which are part of the RND superfamily.
These efflux pumps are tripartite protein complexes, which are drug/proton antiporters that catalyze the extrusion of their specific substrates from the periplasm through the outer membrane [122]. They are composed of three different proteins: a periplasmic adaptor protein, commonly known as periplasmic membrane fusion protein (PMFP), such as MexA, MexX, MexC, or MexE; a resistance-nodulation-cell division transporter (RNDt), such as MexB, MexY, MexD, or MexF, and a channel-forming outer membrane factor (OMF), such as OprM, OprJ, or OprN [123]. Structural analyses of the MexAB-OprM efflux pump show that the PMFP forms a stable complex associated with the inner membrane and the RNDt, both of which are recruited by the OMF, which resides in the outer membrane [124,125] (Figure 1).  As discussed before, efflux pumps are major factors in developing multidrug resistance in bacteria. The four multidrug efflux pumps in P. aeruginosa are involved in the extrusion of toxic molecules and contribute to reduced antibiotic susceptibility [126]. Furthermore, overexpression of these multidrug efflux pumps in P. aeruginosa is directly associated with resistance to most anti-pseudomonal drugs [80,127] and can potentially reduce the efficiency of new classes of drugs developed against P. aeruginosa and other Gram-negative pathogens, such as LpxC inhibitors [128,129] and cefiderocol [130]. These results highlight the importance of taking efflux mechanisms into consideration when developing antipseudomonal drugs.
Other efflux pumps identified in P. aeruginosa, such as MexJK and MexGHI-OpmD, have been mainly associated with quorum-sensing modulation and cell-to-cell communication [88,131] and will not be discussed further in this review.

MexAB-OprM
The operon MexAB-OprM was the first multidrug efflux pump reported in P. aeruginosa [78] and is considered the main contributor to antibiotic resistance, with MexA as the PMFP, MexB as the RNDt, and OprM as the OMF [132]. When the drug concentration increases near the pump, MexB goes through a conformational change and can eject active molecules toward a tunnel formed by MexA and OprM across the periplasm and outer membrane [133].
MexAB-OprM is constitutively expressed in wild-type strains, and its expression is controlled mainly by the repressor genes mexR [134], nalC [135], and nalD [136]. The use of antibiotics selects P. aeruginosa strains with increased MexAB-OprM expression [137,138]. Several mutations in repressor genes have been associated with this phenomenon [139], in particular mutations causing translational disruption, such as nonsense substitutions and frameshifts [138,140], disruption by insertion sequences [139,141], and non-synonymous substitutions that alter the molecular structure of the repressors [137,142]. However, there are commonly reported non-synonymous substitutions, such as V126E in mexR, which are not associated with an increase in efflux pump expression [143,144].
MexAB-OprM can export several antibiotics, including quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, novobiocin, and most β-lactams [79], and it has been shown to be a major factor in the resistance to the herbal antimicrobial compound carvacrol [81]. Overexpression of this efflux pump is associated with resistance to most antipseudomonal antibiotics, except for colistin [80], and carbapenemase-producing carbapenemresistant P. aeruginosa strains often show an increased mexAB-oprM expression, which could be contributing to its resistance to carbapenems [145]. Moreover, it has been shown that overexpression of MexAB-OprM and AmpC, a chromosomally encoded class C βlactamase that contributes to the resistance to many penicillins [146,147], has synergistic effects on the resistance of P. aeruginosa to most antipseudomonal β-lactams, except for ceftolozane/tazobactam, imipenem, and imipenem/relebactam [148].

MexXY
The MexXY efflux pump is formed by the PMFP MexX and the RNDt MexY and is the only multidrug efflux pump operon in P. aeruginosa that does not contain a coding sequence for an outer membrane factor. However, it does form a multidrug efflux pump together with OprM from the MexAB-OprM operon [94]. In some strains, such as P. aeruginosa PA7 [149], the MexXY operon contains a coding sequence for another outer membrane factor called OprA, which can also form a complex with MexXY [95,150].
Its expression is antimicrobial-inducible and is controlled mainly by the repressors MexZ [151], the two-component regulatory system ParRS [152], and by the aminoglycosideinducible anti-repressor ArmZ [153,154]. Several inactivating or single amino acid mutations in mexZ have been associated with an increased expression of MexXY [144,155,156], particularly in cystic fibrosis isolates [125,157], a phenomenon that has also been reported for parR [125,152,158] and parS [125,152].
MexXY is known to contribute to aminoglycoside resistance [159], and its overexpression is commonly observed in strains bearing aminoglycoside-modifying enzymes (AMEs), in which MexXY and the AME work in synergy to promote aminoglycoside resistance [156]. Moreover, it has been shown that MexXY is also associated with resistance to most antipseudomonal antibiotics, similarly to MexAB [80], and it is particularly important in cystic fibrosis lung isolates of P. aeruginosa that harbor a defective MexAB pump [160]. In the OprA-harboring strain P. aeruginosa PA7, MexXY is also able to expel and confer resistance to carbenicillin and sulbenicillin, but only when partnering with the OMF OprA [95]. Moreover, specific single amino acid substitutions in MexY have been linked to increased resistance to aminoglycosides, cefepime, and fluoroquinolones, highlighting the importance of the MexXY system for antibiotic resistance in P. aeruginosa [160].

MexCD-OprJ
The MexCD-OprJ operon encodes an efflux pump that is usually silent or expressed at a low level in P. aeruginosa [161], which normally does not contribute to this pathogen's natural resistance to antibiotics; however, it is associated with resistance to several classes of antibiotics when overexpressed [82,162]. Its expression is mainly regulated by the repressor nfxB, which is transcribed divergently from the mexCD-oprJ operon [82]. Several mutations in nfxB have been associated with an increased expression of MexCD-OprJ, such as nucleotide deletions [163], missense, and nonsense mutations [162].
MexCD-OprJ is mainly associated with resistance to fluoroquinolones such as levofloxacin and ciprofloxacin [80,161], but can also extrude other antimicrobial agents such as macrolides, novobiocin [79], tetracyclines, chloramphenicol [162,164,165], zwitterionic cephalosporins, such as cefepime and cefpirome [166], and the biocide chlorhexidine [83]. Additionally, mutations in mexD may change the substrate specificity of the efflux pump and have been associated with resistance to carbenicillin [167], ceftolozane-tazobactam, and ceftazidime-avibactam [163]. However, strains that overexpress MexCD-OprJ commonly show greater susceptibility to aminoglycosides and some β-lactams [166,168,169], while the overexpression of this efflux pump may lead to an increased susceptibility to complement-mediated killing [170]. Recently, Sanz-García and colleagues showed that exposure to subinhibitory concentrations of ciprofloxacin led to a selection of mutants with increased expression of MexCD and cross-resistance to other antibiotics [100].
Interestingly, variations of mexCD-oprJ genes have been found in mobile genetic elements in P. aeruginosa and several other bacteria [84,171]. A group of these mobile efflux pump operons have been called tmexCD-toprJ ("t" for transferable), and data suggest that they originated from the chromosome of a Pseudomonas spp. [85]. Several variations of the original tmexCD-toprJ have been identified since, either chromosomally or plasmid located, and have been directly associated with resistance to tetracyclines and reduced susceptibility to several other classes of antibiotics under laboratory conditions [84,85].

MexEF-OprN
As for MexCD-OprJ, the MexEF-OprN system is generally inactive under normal conditions but has been associated with resistance to chloramphenicol, quinolones, and trimethoprim when overexpressed [86]. The pump is regulated by MexT, a LysR-like transcriptional activator [172], and MexS, a putative oxidoreductase of unknown function [172,173], both encoded by genes located upstream of the mexEF-oprN operon (Figure 1). Interestingly, mexT is commonly found with inactivating mutations in wild-type P. aeruginosa strains, and overexpression of MexEF-OprN arises from the reversion of these mutations [174]. The mexS gene expression is also activated by MexT [172], and several mutations in mexS, commonly observed in clinical strains, are known to cause overexpression of MexEF-OprN [173,[175][176][177].
Several other genes have also been associated with the modulation of mexEF-oprN expression. Iftikhar et al. [178] reported that a P. aeruginosa mutant with a defective pvcB gene from the pvc paerucumarin synthesis operon showed significant repression in mRNA levels of mexEF-oprN, mexT, and mexS, suggesting the involvement of the pvc operon in the transcriptional regulation of mexEF-oprN. It has also been shown that mvaT and ampR, global expression regulators in P. aeruginosa, negatively regulate MexEF-OprN [179,180], and data suggest the existence of still unknown MexEF-OprN regulators [177].
Overexpression of MexEF-OprN is also associated with decreased expression of the porin OprD because of the MexEF-OprN activator MexT acting as an oprD repressor [172,181]. Inactivating mutations and downregulation of oprD have been associated with resistance to carbapenems [139,182] and colistin [183], which explains why strains overexpressing MexEF-OprN are commonly resistant to antibiotics not extruded by this efflux pump.

Efflux Pumps and Biofilm Formation
Bacterial biofilms consist of microbial communities structured and organized in a matrix of exopolysaccharides (EPS), produced and secreted by the organisms themselves, adhered to a surface [184]. Efflux pumps play an important role in bacterial biofilm formation. Most studies in this area show that efflux pump expression is upregulated in biofilms, conferring greater antibiotic resistance [16]. There is an important positive feedback relationship between biofilms and efflux pumps. Tests of efflux pump inhibitors showed a significant reduction in biofilm in different bacteria [185]. The mechanism that underpins the relationship between efflux pumps and biofilm formation differs between bacterial species, but three main mechanisms have been identified [186]. Firstly, biofilm formation requires several molecules to be transported to the extracellular environment, a function that depends partially on efflux pumps [187]. Secondly, intercellular communication for biofilm formation is called quorum sensing, a type of molecule-mediated communication that requires certain molecules to be released into the extracellular environment. Without these molecules, biofilms cannot obtain the structure of a mature biofilm, expand, or release planktonic cells [188]. Thirdly, some antibiotics act on the regulation and synthesis of elements for the biofilm; therefore, efflux pumps that expel antibiotics are related to the maintenance of the biofilm [189].
Acyl-homoserine lactones (AHL) are important molecules for biofilm production in P. aeruginosa, as they facilitate quorum sensing [190]. Pearson et al. showed that the inhibition of efflux pumps led to an increase in the intracellular concentration of AHL and a reduction in the extracellular concentration, suggesting a clear association between efflux pumps and elements in biofilm formation [191]. The MexAB-OprM pump plays an important role in P. aeruginosa quorum sensing by delivering AHLs to the extracellular environment [187]. Furthermore, the overexpression of efflux pumps on P. aeruginosa can improve biofilm formation, with effects including denser structure, as demonstrated by Sánchez et al. [192]. P. aeruginosa with mutations on nalB and nfxB overexpresses MexAB-OprM and MexCD-OprJ, respectively, resulting in a denser biofilm formation compared with wild-type P. aeruginosa.
The MexEF-OprN efflux pump drives the efflux of 4-hydroxy-2-heptylquinoline (Pseudomonas quinolone signal), which is used by P. aeruginosa to facilitate quorum sensing [193]. Interestingly, studies comparing the transcriptome of planktonic and sessile cells (basal cells of the biofilm) demonstrate an upregulation of several genes associated with efflux pumps such as mexAB-oprM and mexCD-oprJ [36,194,195]. The work by Harrington et al. evaluated the P. aeruginosa transcriptome in an ex vivo pig lung model and a sputum model, showing a repression in MexGHI-OmpD expression in the lung model-associated biofilm [196]. Thus, it is possible to conclude that pump efflux interferes with biofilms but also that biofilms interfere with efflux pump regulation.

Efflux Pumps as Targets for New Drugs
Since the role of efflux pumps as a mechanism of resistance to several antimicrobials became known, significant resources have been devoted to developing molecules that can inhibit this system. Studies in this area began by attempting to find antibiotic-like molecules expelled from the bacteria to inhibit these pumps, such as derivatives of tetracyclines. Despite several in vitro studies, these drugs have never been clinically tested, mostly for reasons of serum instability and toxicity [197]. The first efflux pump inhibitors studied were verapamil, 2,4-dinitrophenol, lansoprazole, protonophore carbonyl cyanide m-chlorophenylhydrazone, and Phe-Arg-ß-naphthylamide. The latter is a dipeptide, a class of molecules that has been studied for their efflux pump inhibitory properties [198][199][200][201][202]. In P. aeruginosa, Phe-Arg-ß-naphthylamide increases the activity of quinolones, decreasing their expulsion from the bacterial cell and lowering their minimum inhibitory concentrations [203]. After these, several other similar molecules were studied, such as MC-207,110, MC-04,112, MC-02,434, MC-510,051, MC-04,124, and MC-02,595 [204]. MC-04, 124 showed similar results to the other compounds in the series; however, the authors considered it to be a less toxic drug [205,206]. Peptides have also been successfully employed to inhibit SMR family efflux pumps. Mitchell and collaborators synthesized several peptides that targeted the TM4 region of P. aeruginosa SMR. The TM4 domain is important for homodimer assembly and for a functional SMR pump. They showed that the synthesized peptides were able not only to decrease the efflux via SMR but also to improve the biocidal activity of other compounds [207]. In addition to synthetic components, dozens of natural and herbal products have been tested for use as efflux pump inhibitors; while some have shown inhibitory activity, clinical studies have not yet been carried out [208].
Revisiting existing drugs with safe clinical use is an alternative to the development of new molecules. In vitro studies have shown that mefloquine, a well-known molecule widely used in the treatment of malaria [209], acts as an efflux pump inhibitor in P. aeruginosa [210]. Trimethoprim, a drug used in combination with sulfamethoxazole to treat various infections, also has efflux pump inhibitory activity. The use of trimethoprim together with ciprofloxacin has a synergistic effect, increasing sensitivity in P. aeruginosa to ciprofloxacin, although the mechanism is still unclear [211]. A further study with trimethoprim and sertraline, using Galleria mellonella as an in vivo infection model, showed the effectiveness of both molecules as synergists in levofloxacin treatment compared with levofloxacin alone. Significant increases in P. aeruginosa-infected G. mellonella survival were seen with treatment with levofloxacin and trimethoprim, or levofloxacin and sertraline, compared with monotherapies [212]. On the other hand, Laborda and collaborators tested a library of 1243 natural products for molecules capable of both increasing the expression of efflux pumps and providing them with a substrate without leading to an increase in antibiotic resistance in P. aeruginosa. The authors hypothesized that increasing the expression of these genes and the efflux of the inducing molecule could overcome the efflux of antibiotics while decreasing the efflux of virulent molecules to the outside. They found four compounds that were able to increase the expression of the mexCD-oprJ or mexAB-oprM genes while also decreasing the virulence of P. aeruginosa in tests with Caenorhabditis elegans: two coumarin-like compounds, one 1,4-benzodioxan-like compound, and one 4-chloroindole compound [213]. Additionally, using a drug library selection strategy, Tambat and collaborators found that the ethyl 4-bromopyrrole-2-carboxylate molecule can act synergistically with antibiotics, lowering their minimum inhibitory concentration in strains that overexpress RND family transporters [214]. Finally, comparative and structural studies between MexB and MexY from P. aeruginosa have shown that the presence of bulky tryptophan residues in the hydrophobic pit prevents binding of the efflux pump inhibitor ABI-PP [215].
Despite several potential candidates for efflux pump inhibitors in P. aeruginosa, obtaining an effective drug is still the subject of several studies. P. aeruginosa constitutively expresses several efflux pumps with different substrate specificities, different physicochemical properties and different efflux constants (K E ). Understanding these variables and how the compounds correlate with these different penetration mechanisms is crucial to effectively combat P. aeruginosa antibiotic resistance. It is also interesting to report that these efflux pump inhibitors can also affect biofilm production since there is a close relationship between the efflux of signaling molecules for biofilm formation and quorum sensing, as discussed earlier [216]. The action on quorum sensing molecules has been shown in transcriptome studies, where Phe-Arg-β-naphthylamide was used to reduce the expression of genes related to biofilms [217].

Concluding Remarks
P. aeruginosa is an opportunistic pathogen of extreme clinical importance that is responsible for most secondary respiratory tract infections in patients with cystic fibrosis or undergoing intubation. The emergence of MDR and XDR strains of P. aeruginosa is a serious aggravating factor to this problem. Efflux pumps are among the most important AMR mechanisms that lead to the emergence of MDR and XDR strains. These protein complexes evolved from a system of interaction with other bacteria, the environment, and hosts with a survival resource. Efflux pumps can export dozens of different compounds into the extracellular environment, such as quorum sensing molecules, virulence factors, and toxic compounds such as antibiotics. This primary mechanism of antibiotic resistance is responsible for reducing the intracellular concentration of the drug to a sub-inhibitory concentration at which other more robust and specific resistance mechanisms can evolve and be selected for. As such, efflux pumps in P. aeruginosa have received significant attention in relation to the development of new drugs. Although several genes have been associated with the transcriptional regulation of efflux pump genes, P. aeruginosa has a complex transcriptional regulatory network in which many genes can be affected by unique global expression modulators. In addition, putative transcription factors with no known targets have been identified through in silico approaches. Additional studies are needed to accurately identify and characterize all trans-acting elements and substrates involved in efflux pump regulation, which may be crucial for the design of new antipseudomonal drugs. Unfortunately, the big pharmaceutical companies have abandoned investments in the development of new drugs due to the high associated cost. In this scenario, it may be necessary to establish public-private partnerships between governments, pharmaceutical companies and the WHO, aiming at the discovery or development of new antibiotic molecules.