Staphylococcus aureus antimicrobial efflux pumps and their inhibitors: recent developments

1.   Introduction

The morbidity and mortality rates of Staphylococcus aureus are alarming [1]. First characterized, isolated, and named by Ogston [2], S. aureus has been well documented as a causative agent in many infections [3]. In particular, methicillin-resistant S. aureus (MRSA) and multidrug-resistant variants have been exceptionally troublesome, and clinical case estimations are predicted to worsen with time [3]–[7]. As a pathogen, S. aureus has developed a series of antimicrobial resistance mechanisms to ensure its survival [8], and multidrug-resistant strains are known to compromise the clinical efficacy of chemotherapy against infection, posing a considerable public health concern [9]. Bacteriological machinery that confers antimicrobial and multidrug resistance can serve as potential targets for modulation to restore the efficacy of antimicrobial agents compromised by such resistance determinants in S. aureus [10],[11].

2.   Mechanisms of antimicrobial resistance in S. aureus

Of the various virulence factors associated with a clinical infection, antimicrobial resistance mechanisms possessed by S. aureus represent critical factors in determining clinical outcomes regarding morbidity and mortality [12]. S. aureus has amassed an impressive arsenal of antimicrobial resistance systems [13].

One of the first such resistance mechanisms involves the enzymatic degradation of the penicillin-derivative methicillin, a physiological characteristic associated with poor clinical outcomes in hospitalized patients with infection [14]. The production of the enzyme β-lactamase was demonstrated to hydrolyze the β-lactam ring, the active site of the β-lactam class of antimicrobial agents [15]. More recently, extended-spectrum β-lactamases (ESBLs) have been reported [16]. These ESBLs enjoy broad spectra for structurally-distinct molecular substrates and at significantly high levels of enzymatic activities [17]. Furthermore, ESBL-encoding determinants are transferable between bacterial species, especially to and from S. aureus clinical isolates [18].

A related antimicrobial resistance mechanism involves the well-characterized alterations in the cell wall [19], such as that reported in vancomycin-resistant S. aureus (VRSA), and those mediated by modulation of cell wall peptidoglycan synthetic enzymes, such as the well-studied glycosyltransferases [20]. These pathogens have been problematic in food processing industries, necessitating the development of new molecular detection methods for monitoring resistance determinants as they move through these environments [21].

Another bacterial resistance mechanism of S. aureus involves modifying the antimicrobial target [22]. One well-studied example of this type of resistance mechanism encompasses alterations of DNA gyrase, a target of the fluoroquinolone antimicrobials [23]. Similarly, alterations in the A subunit of the RNA polymerase enzyme confer resistance to the nucleic acid synthesis inhibitor class of compounds called rifamycins, such as rifampicin [24],[25].

The antimicrobial resistance system that involves target protection represents another class of resistance mechanisms utilized by S. aureus [26]. One noteworthy antimicrobial resistance apparatus includes protecting the ribosome, a target of protein synthesis inhibitors such as tetracycline [27]. The Tet(M) and Tet(O) proteins bind the 30S subunit of the prokaryotic ribosome, preventing the action of the antibiotic on translational inhibition and permitting protein synthesis and bacterial growth [28]–[30].

More recently, the development of biofilms has provided a novel means of antimicrobial tolerance and persistence in S. aureus and other pathogens [11],[31]. In particular, biofilm formation has been effectively measured using bioluminescent markers for biomass and physiological analyses, while chemical dyes, such as crystal violet, safranin, and resazurin, have been utilized to assess biofilm structure [32]. Such new methods for assessing biofilm activity and integrity undoubtedly continue to be of clinical relevance in directing avenues for chemotherapy of infectious diseases.

Antimicrobial efflux is a prominent resistance mechanism in clinical isolates of S. aureus [3],[10],[33], (Figure 1). These exporters reside in the membrane and frequently extrude multiple structurally-distinct antimicrobial agents [34]. Some of these efflux pumps are energized by ATP hydrolysis in a primary active transport process discussed extensively elsewhere [35],[36]. Other antimicrobial efflux pump systems are driven by electrochemically-based ion motive forces, such as those held by proton- or sodium-gradients, processes termed secondary active transport [37], and they represent bacterial systems that constitute suitable targets for modulation [10]. Because of their extensive presence in S. aureus, this review primarily considers recent developments concerning superfamilies of bacterial secondary active efflux pump systems.

Figure 1.  Staphylococcus aureus efflux pumps. Important drug efflux pumps of S. aureus belonging to MFS, MATE, SMR, and ABC families are encoded on the chromosome and plasmids.

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3.   MATE superfamily of multidrug efflux pumps in S. aureus

The MATE (Multidrug and Toxic Compound Extrusion) family of proteins possess 12 transmembrane helices comprising 400–500 amino acids similar to the MFS family of efflux pumps [38] and are widely distributed in bacteria, plants, and animals, although their functions are poorly understood. In plants and animals, MATE proteins are presumed to play essential roles in detoxifying cellular metabolites and excretion of xenobiotics [39]. In bacteria, the MATE proteins use the energy derived from H⁺ or Na⁺ electrochemical gradients to transport antimicrobial compounds outside the cell, thereby lowering their intracellular concentrations [40]. Bacteria carrying MATE efflux proteins can resist diverse compounds, including antibiotics (aminoglycosides, fluoroquinolones), DNA-binding dyes such as ethidium bromide and acridine orange, and anticancer drugs. These efflux pumps were initially placed under the MFS family of proteins due to their structural and functional similarities. Based on the amino acid sequence similarity, the MATE family of proteins is broadly grouped into three groups represented by the prototype efflux pumps like Na⁺-dependent NorM of Vibrio parahaemolyticus, H⁺-dependent efflux pumps such as YdhE of Escherichia coli, and DinF of Pyrococcus furiosus (PfMATE), and the eukaryotic subfamilies [41]. The crystal structures of Na⁺-dependent NorM from V. cholerae (NorM-VC) and Neisseria gonorrhoeae (NorM-NG) [42] and the H⁺-dependent pumps DinF from Pyrococcus furiosus (PfMATE) and Bacillus halodurans (DinF-BH) [43],[44] have been determined, forming the basis for the elucidation of molecular mechanisms underlying the efflux behavior of MATE proteins [40],[45].

The staphylococcal efflux pump MepA belongs to the chromosomally-encoded MATE family of efflux proteins and is the only efflux pump under this group reported from S. aureus so far [46],[47]. The gene encoding MepA is located on an operon mepRAB (multidrug export protein), which also has a gene coding for a transcriptional regulator protein MepR that binds to the promoter regions of both mepA and mepR, and the overexpression of mepR resulted in the reversal of MDR phenotype of S. aureus due to the transcriptional inhibition of mepA [46],[48]. The predicted secondary structure of MepA has 12 transmembrane helices formed by 451 amino acids [48]. Due to the lack of homology with proteins of known function, the fundamental role of MepA in Staphylococcus physiology is largely obscure. At the amino acid level, MepA shows 26% identity with CdeA of Clostridium difficile and 21% identity with NorM of Vibrio parahaemolyticus, both belonging to the MATE family of proteins with fluoroquinolones as efflux substrates [48]–[50]. Multiple compounds act as substrates for MepA, including fluoroquinolones (norfloxacin, ciprofloxacin, moxifloxacin), tigecycline, benzalkonium, cetrimide, chlorhexidine, and ethidium bromide [46]. The low level of identity of MepA with NorM is also evident from its low affinity for fluoroquinolones compared to NorM. Efflux pump inhibitors (EPIs) such as reserpine, paroxetine, and certain phenothiazines and thioxanthenes inhibit the efflux activity of MepA [48],[51]. Using site-directed mutagenesis and in silico modeling, Schindler et al. [52] predicted a substrate transport pathway involving helices 1, 2, 4, 7, 8, and 10 that form a large central cavity, with amino acid residues Ser-81, Ala-161, Met-291, and Ala-302 within the cavity assuming essential roles in substrate binding and the efflux activity [52]. More recently, cells harboring MepA exposed to the monoterpene estragole had lowered MICs for ethidium bromide and ciprofloxacin [53]. In the same study, estragole showed a similar reduction in the MIC for norfloxacin in S. aureus expressing NorA [53]. Another report showed that the synthetic compounds 1,8-naphthyridines sulfonamides synergized with ciprofloxacin or ethidium bromide in cells of S. aureus harboring MepA as measured by MIC assays [54]. Fluorescence emission analysis of ethidium bromide transport showed inhibition of drug efflux in MepA-containing cells with 2,3,4-trifluoro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide [54]. An evaluation of the molecular docking properties demonstrates that the efflux pump inhibition effect directly affects MepA [54]. A monocyclic monoterpene phytochemical, called limonene, was evaluated in S. aureus with MepA for efflux activity, demonstrating a direct inhibitory effect on drug transport [55]. In the same study, molecular docking analysis showed interactions of limonene with multiple amino acid residues of MepA [55]. The new work indicates that limonene is a suitable efflux pump inhibitor and suggests that it may be a suitable platform for developing new derivatives to enhance inhibitory modulation [55].

4.   SMR superfamily of multidrug efflux pumps of S. aureus

The SMR (small multidrug resistance proteins) superfamily of efflux proteins in S. aureus, such as the QacC (Smr/Ebr), QacJ, QacG, and QacH, are plasmid-encoded, while SepA is encoded on the chromosome [3]. Small membrane proteins represent the SMR family of efflux proteins with 100–150 amino acid residues forming four transmembrane helices, and these are distinctly different from their MFS counterparts, QacA and QacB, with little or no sequence homology [56]. The plasmid-encoded Smr/Ebr (Staphylococcal multidrug resistance/Ethidium bromide resistance) protein was the first efflux pump discovered in S. aureus responsible for ethidium bromide resistance, which was subsequently renamed QacC [57],[58]. QacC extrudes diverse biocides such as quaternary ammonium compounds, DNA-intercalating dyes, and phosphonium ions but differs from QacA/B in their inability to efflux acriflavine [57],[58]. QacC has 107 amino acid residues in its 4 TMS, which form dimers across bacterial membranes, creating a pore-like structure that allows the substrate to pass through [59]. Using site-directed mutagenesis, Grinius and Goldberg [60] showed that a Glu-13 residue located on a hydrophobic domain of QacC is crucial for the drug/H⁺ antiport activity, while Glu-24 is predicted to be responsible for drug specificity. Among the qac family of efflux protein conferring genes, qacC is highly conserved and is located on conjugative, rolling-circle replicating (RCR) plasmids with a novel gene transfer mechanism responsible for spreading the qacC gene [61],[62]. The gene gets transferred between rolling-circle plasmids of variable backgrounds without the assistance of insertion sequences or other similar gene mobility mechanisms [62]. QacG was discovered as a 107 amino acid long efflux pump with 69.2% identity to QacC and encoded on a 2.3 kb pST94 resistance plasmid [63].

The QacH protein was first reported by Heir and colleagues in Staphylococcus saprophyticus as a 107 amino acid protein encoded on a 2.4-kb plasmid (p2H6) with 78% and 70% identity with Smr and the QacG proteins, respectively [64]. Homologous proteins of QacH in Gram-negative bacteria include QacE and EmrE, with about 40% similarity [65]. Bjorland and colleagues reported a 2.65 kb rolling circle plasmid pNVH01 in equine Staphylococcus species harboring the gene encoding a 107 amino acid efflux protein QacJ [66]. The QacJ protein is homologous with Smr/QacC (72.5% identity), QacG (82.6%), and QacH (73.4%). The pNVH01 plasmid carrying the qacJ gene is widely distributed in coagulase-positive and -negative Staphylococcus species that exhibit resistance to a wide range of biocides.

A comparison of amino acid sequences of over sixty SMR efflux proteins revealed the highly hydrophobic nature of these sequences with a highly conserved glutamate at position 14 across all sequences and TMS-specific motifs [67]. Paulsen et al. [68] performed qacC-phoA and qacC-lacZ fusions and mutational analysis to understand the functional roles of conserved amino acids in the QacC protein. This study attributed an essential role for Cys-42 in substrate recognition. Two other amino acid residues, Tyr-59 and Trp-62, were also proposed to have important functional roles in the efflux activity of QacC.

5.   MFS multidrug efflux pumps of S. aureus

The major facilitator superfamily of solute transporters is one of the leading known constellations of related integral membrane proteins [69],[70]. The transporters of this superfamily are known to share related primary sequences, highly conserved sequence motifs, and protein structures [71]. In terms of secondary structure, transport proteins of the MFS typically possess 12 or 14 membrane-spanning α-helices (Figure 2) [72]. The N- and C-termini of these transport systems reside on the cytoplasmic side of the membrane [73].

The members of the MFS differ in terms of the energetics that drive solute transport (i.e., passive or secondary active transport), the structurally diverse nature of their substrates (e.g., sugars, amino acids, antimicrobial agents, and ions), and directions of solute transport across the membrane (i.e., symport, uniport or antiport) [6],[74]

Nevertheless, the similarities in sequences and primary and secondary structures predict that the transporters of the MFS undergo transport across the membrane by a shared catalytic mechanism [74],[75]. A unifying principle that ties these seemingly disparate properties, i.e., similarities in sequence but differences in substrate profiles, modes of energetics, and transport direction, lies in discovering highly conserved amino acid sequence motifs [71],[73]–[77].

Figure 2.  Major facilitator superfamily transporter predicted secondary structures. (A) The predicted two-dimensional structures in the membrane of (A) SdrM [59] and (B) NorA [78] are shown as generated by Protter [79].

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Based on three-dimensional structural studies, transporters of the MFS are known to harbor two asymmetric domains consisting of a C-terminal bundle composed of helices 7 through 14 and N-terminal bundles characterized by helices 1 through 6, respectively [72]. Members of less well-characterized MFS transporters are also thought to harbor 12 or 14 membrane-spanning segments (Figure 3). Thus, studies of newly discovered transporters of the MFS benefit from the structural insights. The MFS transporters contain exposed cavities that alternately orient their substrate binding sites to either side of the membrane during solute transport [80]. These transport proteins are thought to operate by forming so-called inverted topological repeats composed of three-helix units repeated in tandem along the length of the transporter and functionally connected to a multi-helical hinge system to carry out conformational changes during transport [81],[82]. Interestingly, these systems, i.e., alternating access, the inverted topology units, and the molecular hinge, appear to be unified by conserved amino acid sequence motifs that have been demonstrated to be essential for bacterial multidrug resistance [83].

Figure 3.  Predicted three-dimensional structures of major facilitator superfamily transporters. On the left is the predicted structure for SdrM (Q99S97, SDRM_STAAN) from S. aureus, a multidrug efflux pump with 14 predicted transmembrane domains [59]. The predicted structure for NorB from S. aureus is on the right with 12 putative membrane-spanning segments (A0A6B5H8J6_STAAU) [84].

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One of the first drug efflux pump systems to be characterized in S. aureus was reported to export tetracycline actively and was demonstrated to be extra-chromosomally encoded on plasmid-based mobile genetic elements [85]. These plasmid-encoded determinants are TetA(K) and TetA(L) [86],[87]. Shortly afterward, the genome-encoded NorA efflux pump was discovered [78]. Initially shown to export norfloxacin, a fluoroquinolone, the NorA transporter was demonstrated to transport multiple structurally different antimicrobial agents, becoming a well-known multidrug efflux pump of central importance [88],[89]. Related determinants encoded NorB, NorC, and NorD [90]–[92]. Another notable multidrug efflux pump from S. aureus is the plasmid-encoded QacA, known for its export of a variety of seemingly unrelated variety of antimicrobial substrates [93]. Related transporters from S. aureus were denoted QacB, QacC, QacG, and QacJ, all plasmid-based [56]. The MdeA transporter was reported to be encoded as a genomic element and harbor multiple substrates for transport [94],[95]. More recently, the chromosomally based LmrS from S. aureus was discovered by our laboratory and shown to actively export a large variety of structurally-distinct antimicrobials [96]. Other MFS transporters from S. aureus include SdrM, Sav1866, Tet(38), and MepA [10],[69],[97].

Recently, the functional roles of acidic residues were evaluated in QacA, where Asp-34 and Asp-411 were shown to recognize substrate, whereas Glu-407 could bind substrate and participate in protonation during transport catalysis [98]. These residues, conserved amongst other MFS drug transporters, may serve as suitable targets for novel efflux pump inhibition. Highly conserved amino acid sequence motifs are crucial for structural stability, transport, and modulation of MFS symporters and antiport-based efflux pumps [83],[99]. In addition to previously known motifs A, B, and C, Shang et al. recently discovered conserved motifs, called Motif-1 and Motif-2, with influences on transporter stability and binding of ethidium bromide [100]. Molecular physiological studies involving conserved amino acid sequence motifs and multidrug efflux pump modulation are needed and show strong promise toward reestablishing the efficacy of antimicrobial action against pathogenic strains of S. aureus [10],[99],[101].

Studies from our laboratory showed that an extract of cumin spice from Cuminum cyminum, cumin seed oil, and a principal bioactive agent called cuminaldehyde inhibited the growth of E. coli host cells harboring the LmrS multidrug efflux pump from S. aureus [96],[102], (Table 1). We also demonstrated that cumin extract inhibited the ethidium bromide transport activities of LmrS, such as efflux and accumulation [102]. Interestingly, TetR21, a member of the TetR family of repressors, suppressed the gene expression levels of lmrS and the gene encoding Tet(38) [103]. A recent study by Nava et al. showed that calcium ion (Ca²⁺) enhanced the ethidium bromide efflux activity of LmrS [104]. Furthermore, these investigators demonstrated that when in the presence of antibiotics, a Ca²⁺ mediated transient was generated in cells of S. aureus, which then positively modulated LmrS after inducing a physiological process that could aid bacterial survival in harsh pH conditions [104]. This new work points to Ca²⁺ as a potential regulator of antimicrobial transport activity and controlling gene expression programs [104].

Thiazol and a well-known group of thiazolidinedione derivatives had been shown to interact with NorA of S. aureus elements using molecular docking systems [105]. These compounds demonstrated a synergistic relationship with norfloxacin in host cells harboring NorA [105]. Capsaicin, a known efflux pump inhibitor [106], was conjugated to newly developed molecules of 1,3,4-oxadiazole and shown to increase the antimicrobial activity of ciprofloxacin and reduce the transport of ethidium bromide by NorA, thus, pointing to these conjugates as potentially novel efflux pump inhibitors with promising potency [107], (Table 1). Molecular docking simulations were performed on a predicted NorA structure in which substrate ciprofloxacin and putative NorA-inhibitors derived from capsaicin were shown to make contacts with amino acid residues lining the hydrophobic cavities of the substrate-binding core and the inward and outward-facing drug binding sites [108]. In this study, specific residues formed distinct interactions between NorA and ciprofloxacin, capsaicin, and the novel putative efflux pump inhibitor CID-44330438 [108]. Interestingly, residues common to all three molecules included Phe-47 and Trp-293 [108], indicating shared elements in the solute transport and modulatory systems of antimicrobial efflux at transmembrane helix one, a known drug-binding site region of MFS transporters [109]–[111], and helix ten where Trp-293 resides [78].

Another naturally occurring plant compound, a terpene-based agent called eugenol, and several compounds derived from it showed reductions in the MICs for efflux substrates ethidium bromide and norfloxacin by NorA [112]. Though efflux by NorA was not directly measured in this study, the investigators demonstrated synergy with these transport substrates and eugenol or isoeugenol [112]. Further, molecular docking simulations showed a close association between many amino acid residues in a predicted model of NorA and 4-allyl-2,6-dimetoxyphenol or allylbenzene [112]. These eugenol derivatives show promise as efflux pump inhibitors and synergistic modulators involving NorA. Other terpene-based compounds called carvacrol and thymol were effective antibacterial agents for S. aureus cells that contained NorA [113]. Molecular simulation docking studies showed that these compounds made close contact with many aliphatic or aromatic residues in the interior pocket of NorA, suggesting they dictate a suitable target for modulation of transport activity [113].

Another study of the terpene-based agents combined α-terpinene with essential oil from Chenopodium ambrosioides was conducted on Tet(K), showing a reduced MIC of tetracycline and ethidium bromide [114]. Quercetin, a flavonoid-based compound known to modulate Tet(K) and NorA [76],[115],[116], was recently demonstrated to enhance the antimicrobial actions of the antimicrobials erythromycin, tetracycline, and norfloxacin in host cells of S. aureus through its stabilizing interaction with Ser-138 of NorA [117]. Similarly, new chalcone derivatives, known efflux pump inhibitors of NorA of the MFS and MepA proteins from the MATE family [118]–[121], were recently shown to synergize with norfloxacin and ethidium bromide in cells harboring NorA or MepA [122]. Along these same lines, synergy was observed between ethidium bromide, norfloxacin, and the so-called 1,8-naphthyridine sulfonamides, suggesting the latter could be an efflux pump inhibitor of new interest [123]. More recently, a compound from various citrus fruits and vegetables, a phenolic-based compound called ferulic acid and esterified derivatives, showed reductions in the MICs for NorA substrates and demonstrated synergy between them [124]. In another study, propyl ferulate conferred a reduction in the Tet(K)-mediated MICs of ethidium bromide [125]. Lapachol and norlachol agents were used as a platform to synthesize various hydroxylamine derivatives, which showed lowered MIC values for ethidium bromide and norfloxacin [126]. Although direct measurement of drug efflux activities via the NorA or Tet(K) pumps was not demonstrated experimentally, the ferulic acids and hydroxylamines from lapachol and norlachol represent new chemical classes of interest for inhibiting S. aureus growth clinically [125],[126].

Another group of modulators involving NorA and Tet(K) efflux pumps is represented by the sesquiterpene α-bisabolol from plants like Matricaria chamomilla L [127]. Recently, α-bisabolol was used to form a so-called inclusion complex with a β-cyclodextrin to enhance water solubility and provide greater bioavailability [127]. Several inclusion complexes reduced the MICs for norfloxacin in cells with NorA and tetracycline in host bacteria with Tet(K) [127].

An antibiotic called elaiophylin produced by species of the Streptomyces genus, such as S. hygroscopicus was recently shown to inhibit ethidium bromide transport by NorA, and molecular docking analysis showed strong binding affinities with residues Tyr-57, Ile-258, Ser-262, Pro-384 of NorA [128]. Analogs of the putative NorA modulator dihydroquinazoline showed reduced gene expression of the norA determinant in an over-expressing strain of S. aureus [129]. Furthermore, the novel analogs demonstrated synergistic action with the transport substrate ethidium bromide and, importantly, a significantly reduced amount of intracellularly located S. aureus cells within host-monocytes in culture [129].

Recently, a group of purified silymarin flavonolignans that had previously been observed to reverse multidrug resistance in S. aureus showed non-competitive inhibition of efflux pump activities in NorA and MepA multidrug transporters [130]. One of these flavonolignan-based compound derivatives, 2,3-dehydrosilybin B, repressed the gene expression programs for various antimicrobial transporters from distinct superfamilies [130]. The flavonolignans show quorum sensing attenuation properties, further pointing to these compounds as potential multidrug resistance modulators [130]. Further, modulating agents that can affect antimicrobial transport across the membrane and regulate gene expression of drug resistance or virulence factors will continue to be of particular interest. A recent investigation reported on the genomic nature of genetic elements for antimicrobial resistance and virulence factors for over 100 S. aureus isolates from lower animals (dogs, cats, and cows) [131]. In addition to sharing genes for superantigens, the new study showed that all such isolates shared genes encoding antimicrobial efflux pumps LmrS [96], Tet(38) [97],[132], NorA [78], and MepA [48], and regulators of gene expression, such as MrgA (previously designated NorR) [133], and the ArlRS system [131].

Thus, studies of gene sharing amongst bacterial pathogens will shed light on developing novel strategies for reducing the conditions that foster the emergence of pathogens as they move through animal and human populations. Because the multidrug efflux pumps of the MFS are widespread and relatively well understood, they can serve as effective targets for transport inhibitors or modulation of gene expression programs in cells of S. aureus [134]. Future strategies for restoring the efficacy of antimicrobial agents against clinical pathogens of S. aureus entail a deeper understanding of the physiological mechanism of antimicrobial efflux systems and their relationship to the biochemistry of efflux pump inhibitors [135],[136].

Table 1.  MFS antimicrobial efflux pump substrates and inhibitors.

Efflux pump	MW (kDa)	TMS	Substrates	Inhibitors	References
LmrS	47.7	14	Linezolid (oxazolidinone), Phenicols (chloramphenicol, florfenicol), erythromycin, trimethoprim, lincomycin, kanamycin, fusidic acid, QACs (tetraphenylphosphonium), Dyes (ethidium bromide), Detergents (sodium dodecyl sulfate)	Cumin seed oil, Cumin aldehyde, Reserpine	[96],[102]
NorA	42.3	12	Hydrophilic fluoroquinolones (norfloxacin, ciprofloxacin), QACs (benzalkonium), Dyes (ethidium bromide, Hoechst 33342), Biocides (acriflavine, cetrimide, benzalkonium chloride)	Verapamil, Capsaicin, Capsaicin 1,3,4-oxadiazole conjugates, Piperine and piperine analogs (SK-20, SK-56, SK-29), Chalcone, Baicalein, Caffeic acid, Coumarin, Boeravinone B, Benzophenanthridin, 15-copaenol, Caffeoylquininc acids, Genistein, Dimethyl octaol, Nerol, Estragole, Indirubin, Kaempferol Rhamnoside, Tannic acid, Phyllanthin, Curcumin, Osthol, Orizabins, Murucoidins, Biricodar (VX-710), Timcodar (VX-853), Ginsenoside, Dithiazole thione derivative (DTT10), Pieric acid amides derivatives, Phyllanthin, α-Bisabolol, 1,8-naphthyridines sulfonamides Brachydins (BR-A, BR-B), Berberine, Berberine INF55 (5-nitro-2-phenyl-1H-indole) and analogs, Diterpenes (ferruginol), 2-phenyl-4(1H)-quinolone and 2-phenyl-4-hydroxyquinoline derivatives, Menadione, Crysoplenol, Crysoplenetin, Sarothrin (5,7,4′-trihydroxy-3,6,8-trimethoxyflavone), Olympicin A, Reserpine, Aldonitrones (Z)-N-benzylidene2-(tert-butoxy carbonyl amino)-1-(5-iodo-1H-indol-3-yl) ethan), Indole analogue (compound 13 and 14), Sophoraflavanone G, Diosmetin, Tiliroside (kaempferol-3-O-β-D-(6″-E-p-coumaroyl), Chrysoeriol, Penduletin, Galangin, Carvacrol, Thymol	[78],[88],[89],[106],[107],[117],[118],[123],[127],[137]–[165]
NorB	49	12	Hydrophilic and hydrophobic fluoroquinolones (norfloxacin and ciprofloxacin, moxifloxacin, sparfloxacin), Biocides (tetraphenylphosphonium, cetrimide), Dye (ethidium bromide), tetracycline, QACs (tetraphenylphosphonium, cetrimide)	CuFe2O4@Ag, Clerodane diterene 16α-hydroxycleroda-3,13 (14)-Z-dien-15,16-olid 6	[150],[166],[167]
NorC	48.9	12–14	Hydrophilic and hydrophobic fluoroquinolones (ciprofloxacin, norfloxacin, moxifloxacin, garenoxacin, sparfloxacin ), Dye (rhodamine)	Nanobody (single-domain camelid antibody), Clerodane diterpene 16α-hydroxycleroda-3,13 (14)-Z-dien-15,16-olid	[167]–[169]
MdeA	52	14	QACs (benzalkonium chloride, dequalinium, tetraphenylphosphonium), Dye (ethidium bromide, Hoechst 33342, acriflavine, and rhodamine 6G), Hydrophilic fluoroquinolones (virginiamycin, novobiocin, mupirocin, fusidic acid, norfloxacin, ciprofloxacin), Anthracyclines (doxorubicin, daunorubicin), Macrolides	Piperine, Clerodane diterpene 16α-hydroxycleroda-3,13 (14)-Z-dien-15,16-olid, Osthol, Imperatorin, Tangeretin	[95],[167],[170],[171]
SdrM	56.4	14	Dyes (acriflavine, ethidium bromide), Fluoroquinolone (norfloxacin)	carbonyl cyanide m-chlorophenylhydrazone	[59]
QacA	55	14	QACs (tetraphenylphosphonium, benzalkonium chloride, dequalinium), Biguanidines (chlorhexidine), Diamides (pentamidine), Dyes (ethidium bromide, rhodamine, acriflavine), Cetyltrimethylammonium bromide (Ct), Tetraphenylarsonium chloride (Guanylhydrazones), Propamidine isethionate, Diamidinodiphenylamine dihydrochloride	Silybin, Volkensiflavone, Morelloflavone, Verapamil, Reserpine, Hydantoin PI8a	[93],[161],[172]–[177]
QacB	55	14	QACs (tetraphenylphosphonium, benzalkonium chloride, diamidinodiphenylamine dihydrochloride, cetrimide), Dyes (ethidium bromide, rhodamine, acriflavine), Biguanidines (chlorhexidine),	Silybin, Volkensiflavone, Morelloflavone, Hydantoin PI8a	[161],[175],[177]
Tet38	48	14	Tetracycline, Unsaturated fatty acids (palmitoleic and undecanoic acid), Fosfomycin, Tunicamycin, Congo red	Minocycline, glycerol-3-phosphate (G3P)	[97],[132],[168],[178]
TetA(K)	50.7	14	Tetracyclines	Nocardamines, Essentials oil, Isopimarane diterpenes Osthol, 5,7-Diacetoxy-8-(3-methyl-2-butenyl)-coumari, 3-(2-Methyl but-3-en-2-yl), Xanthyletin	[165],[179]–[181]
Tet63	-	14	Tetracycline and Doxycycline	Tigecycline	[182]
FexA	49.3	14	Florfenicol, Chloramphenicol		[183]

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6.   Conclusions and future directions

Morbidity and mortality rates reported by clinical studies of multidrug-resistant S. aureus isolates are of tremendous concern from a public health standpoint [10],[11],[184],[185]. Addressing the concern will involve continued studies of the molecular mechanisms that confer multiple antimicrobial resistance in these pathogens [3],[186]. Antimicrobial transporters are known to dictate multidrug resistance and represent promising targets for inhibition to restore clinical efficacy against infection [135]. Towards this, investigators have garnered a great deal of mechanistic and structural features of the drug transporters that can permit the development of suitable new modulators of resistance and infection [187]. Modulators that directly affect antimicrobial transport and show gene expression regulation are promising [131]. Comparative analyses of bacterial genomes can discover new targets for modulation [188]–[190].

While these efforts are promising, the field still lacks a detailed molecular understanding of antimicrobial translocation across the membrane [191]. Once the molecular pathways are delineated through dedicated transport systems and are definitively elucidated, efflux pump inhibitors can be designed with improved accuracy and, thus, improved antimicrobial efficacy for clinical treatment of infection [192],[193].

New developments regarding the nature of transport for multiple structurally disparate antimicrobial agents via specific efflux pumps are constantly being reported [194],[195]. However, we do not yet understand how multidrug efflux pumps dictate the specific translation of certain substrates while keeping others out and preventing unwanted leakages through the pumps in the cells of pathogens, especially for S. aureus. We anticipate that molecular analyses of the transport systems that confer single- versus multiple-drug transport yield new advances for improved chemotherapies. The molecular mechanisms that dictate passive versus active transport are not yet clearly understood. For example, we do not yet understand how these various antimicrobial transporters mediate ion-selectively in antiporters while not requiring such specificities in passive transporters. Along these lines, it remains poorly understood how multidrug pumps prevent unwanted translocation of ions which could collapse ion-motive forces that drive secondary active transporters of clinically relevant chemotherapeutics. Thus, we foresee that studies of the energetic mechanisms that drive the activities of the multidrug efflux pumps and how these systems related to the accumulation of antimicrobial agents to one side of the bacterial membrane will shed light on the nature of molecular configurations that will make effective targets for new modulators [10],[134],[196].

Lastly, the highly conserved nature of the members within each of the known transporter superfamilies predicts that such related members operate by mechanisms shared between them. Thus, elucidating molecular mechanisms that are believed to be commonly shared by members within each of the transporter superfamilies represents the Holy Grail of multidrug resistance in pathogenic microorganisms. We anticipate that once the detailed molecular mechanisms of antimicrobial transport are understood, treating severe infection by S. aureus will provide efficacious clinical outcomes.