Bacterial Multidrug Efflux Pumps at the Frontline of Antimicrobial Resistance: An Overview.

Multidrug efflux pumps function at the frontline to protect bacteria against antimicrobials by decreasing the intracellular concentration of drugs. This protective barrier consists of a series of transporter proteins, which are located in the bacterial cell membrane and periplasm and remove diverse extraneous substrates, including antimicrobials, organic solvents, toxic heavy metals, etc., from bacterial cells. This review systematically and comprehensively summarizes the functions of multiple efflux pumps families and discusses their potential applications. The biological functions of efflux pumps including their promotion of multidrug resistance, biofilm formation, quorum sensing, and survival and pathogenicity of bacteria are elucidated. The potential applications of efflux pump-related genes/proteins for the detection of antibiotic residues and antimicrobial resistance are also analyzed. Last but not least, efflux pump inhibitors, especially those of plant origin, are discussed.

1. Introduction

Bacteria evolve mechanisms of defense against harmful external substances that threaten their survival. Transporters are located on the bacterial cell membrane and play important roles in decreasing the concentration of endogenous and extraneous substances and promoting the growth of bacteria. It should be noticed that some transporters are also involved in the biodegradation of environmental toxic compounds [1]. Therefore, bacterial efflux pumps not only work as a functional transporter but also defend from environmental stress to maintain bacteria survival. Although some efflux pumps have specific substrates, some transporters can pump out multiple different kinds of antimicrobials, inducing multidrug resistance [2]. Sometimes, the extrusion of organic solvents or other substrates leads to the overexpression of transporters, thus creating co-selection of antimicrobial resistance features [3]. The overexpression of efflux pumps would also impact bacterial pathogenicity, involving biofilms and quorum sensing (QS) [4,5]. The efflux pumps export not only antimicrobials but also virulence determinants, including adhesins, toxins, or other proteins that are important for colonization in host cells [6].

Many studies have described efflux pumps-mediated resistance mechanisms, and more and more new efflux transporters and related proteins have been discovered. In recent years, additional functions of transporters were investigated, which involved bacterial virulence and self-protection against environmental pollutants. Nevertheless, the exact mechanisms and active domains of efflux pump transporters have not been clearly elucidated. There are many influencing factors on the inner and outer bacterial membrane, stimulating pump activity and promoting structure alterations of the transporters in the fluid membrane environment.

This article briefly summarizes the functions of efflux pumps in common Gram-positive and Gram-negative bacteria. Applications of efflux pump-related regulator proteins for detecting antimicrobial resistance and antibiotic residues, as well as newly discovered plant-derived efflux pump inhibitors, are discussed.

2. Functions of Efflux Pumps

Efflux transporters are mainly grouped into the following superfamilies: ATP-Binding Cassette (ABC) superfamily, Multidrug and Toxic Compound Extrusion (MATE) superfamily, Major Facilitator Superfamily (MFS), Resistance Nodulation and Cell Division (RND) superfamily, and Small Multidrug Resistance (SMR) superfamily. In 2015, a novel transporter family was identified, known as the Proteobacterial Antimicrobial Compound Efflux (PACE) superfamily [7]. The discovery and identification of bacterial multidrug-resistant (MDR) efflux pumps is still ongoing [8]. One of the differences among efflux pumps is their source of energy. The ABC family members function as efflux pumps through the hydrolysis of ATP for energy supply [9], while other transporters including the MATE, MFS, RND, and SMR superfamilies utilize the proton-motive force provided by H+ or the electrochemical gradient of Na+ to supply energy and then extrude multiple compounds [10]. In addition, there are also differences in the composition of efflux transporters. The RND-type multidrug efflux proteins use a tripartite complex as the structural basis of the pump, including an outer-membrane canal protein (OMP), an inner-membrane transporter, and a membrane fusion protein (MFP) that connect the above two components to pump out drugs and other harmful compounds [11,12].

2.1. Efflux Pump-Mediated Antimicrobial Resistance

The resistance mechanisms to antimicrobials are based on changes in drug targets, structural modifications or degradation of drugs, decreased permeability of outer membrane proteins to prevent the drugs from entering the cells, and enhanced efflux transporters to reduce intracellular drug concentrations. It was generally believed that efflux pumps and outer membrane proteins did not have a synergistic effect on reducing intracellular drug concentrations. However, a recent study found an interaction between active efflux pumps and the permeability barrier of the outer membrane in Burkholderia thailandensis [13]. In fact, the overexpression of efflux pumps plays an essential role in the acquisition of antimicrobial resistance, even of multidrug resistance. Understanding the molecular structure of efflux pumps and their crucial drug binding sites is critical for the development of efflux pump inhibitors. The structures of different efflux pump families have been reviewed in detail and will be briefly discussed below [14,15]. In the following, the main focus is on the effects of efflux pumps on biofilm formation, bacterial virulence and invasion, and oxidative stress responses, in addition to the acquisition of antimicrobial resistance.

2.1.1. ABC Superfamily

In Gram-negative microorganisms, the most extensively studied ABC-type transporters is the tripartite complexes MacAB-TolC efflux pump, which actively extrudes substrates including macrolides and polypeptide virulence factors powered by the ATPase MacB and participates in the secretion of enterotoxin TⅡ in Escherichia coli [16,17,18]. Rough-core lipopolysaccharide (LPS) or similar glycolipids is also regarded as a physiological substrate of the MacAB-TolC pump [18].The inner membrane protein MacB works as a homodimer complex that contains an N-terminal nucleotide binding domain that binds ATP and a C-terminal cytoplasmic tail [18]. The membrane fusion protein MacA binds the lipopolysaccharide core specifically and is activated by ATPase [18]. The tripartite-complex structure composed of the outer membrane protein TolC, the inner membrane protein MacB, and the periplasmic protein MacA provides a structural site for substrate transport [17]. The latest research shows that the lack of the MacAB efflux pump in Serratia marcescens increases the susceptibility to aminoglycosides and polymyxins, decreases the swimming motility and the ability of biofilm formation, even causes the loss of the ability to cope with superoxide stress [19]. In addition, MacAB also confers resistance to several penicillin-type antibiotics and As(III) in Agrobacterium tumefaciens 5A [20].

In Gram-positive microorganisms, the ABC transporter consists of a single transmembrane protein, such as EfrAB in Enterococcus faecalis, LmrA in Lactococcus lactis [21], Msr in Streptococcus, and PatA/B in Streptococcus pneumoniae [22,23]. The MDR pump EfrAB is a heterodimeric transporter that extrudes gentamicin, streptomycin, and chloramphenicol [24]. The expression of EfrAB is highly induced by sub-inhibitory concentration(sub-MIC) of these antibiotics and down-regulated by sub-MIC of EDTA (3 mM) [24]. The LmrA protein functions as a homodimer, comprising a nucleotide-binding domain and six alpha-helix transmembrane domains, which primarily recognize and transport macrolides and lincosamides [25]. The Msr protein harbors two nucleotide-binding domains with no transmembrane domain, conferring resistance to macrolides in Streptococcus and cooperating with the Mef transport family [26,27]. Nearly the whole family of hydrophilic fluoroquinolones, including ciprofloxacin and norfloxacin, is the substrate of PatA/B efflux pumps [28].

2.1.2. MATE Superfamily

All members of the MATE family present 12 alpha-helical transmembrane regions powered by electrochemical ion gradients [29]. MATE transporters are classified into NorM, DinF (DNA damage-inducible protein F), and eukaryotic subfamilies according to amino-acid sequence similarity [30]. The substrates of MATE family transporters are diverse and have different chemical structures, but fluoroquinolones are the substrates recognized by almost all transporters [31].

Among the Gram-negative bacteria, the most studied pump is the NorM efflux pump in Neisseria gonorrhoeae and Vibrio cholera [32]. The NorM efflux pump exports substrates including antimicrobial cationic compounds (quaternary ammonium compounds) and antimicrobials such as ciprofloxacin and solithromycin in N. gonorrhoeae [33]. NorM has the ability to extrude intracellular reactive oxygen species, therefore reducing the damage of oxidative stress [34]. Mutations of the DinF transport system confer increased susceptibility to moxifloxacin, ciprofloxacin, and levofloxacin in pneumococci [35]. The competence-induced protein A is encoded by cinA, recA, and dinF, which form an operon that is induced by quinolones through the SOS response [35].

2.1.3. MFS Superfamily

The MFS family is the largest characterized family of transporters, especially in Gram-positive microorganisms. Most members of this superfamily are MDR transporters and are constituted by 12 or 14 transmembrane segments [29]. The pumps Lde in Listeria monocytogenes [36] and NorA in Staphylococcus aureus [37] specifically extrude hydrophilic fluoroquinolones (e.g., ciprofloxacin and norfloxacin), while Mef in S. pneumoniae pumps out macrolides [38]. It has been found that the transcriptional start of mef(E)/mel genes locates in the mef(E)/mel promoter, and the attenuation of transcription will regulate mef-mediated macrolide resistance [39]. In addition, Msr of ABC family and Mef of MFS family enhanced macrolides extrusion synergistically, which increased resistance to 14-, 15-member ring macrolides [40].

In addition to extruding drugs, MFS transporter proteins also play important roles in other biological pathways. For example, MdrT and MdrM contribute to promoting the host immune response by activating IFN-β production of the type I interferon response and to maintaining cell wall stability [41]. Tet38, a chromosomally encoded MFS efflux pump, has an impact on multiple steps of the host cell invasion process of S. aureus, including adhesion, internalization, and trafficking in epithelial cells, and the subsequent step of the S. aureus infection in epithelial cells including bacterial viability and trafficking in phagolysosomes [41]. The AbaQ MFS transporter is also involved in Acinetobacter baumannii motility and virulence, as the loss of the abaQ gene decreases bacterial motility and virulence [41]. Additionally, the inactivation of genes encoding the RND, MATE, SMR, and ABC efflux pumps also reduces bacterial motility and virulence in comparison with the parental strain [42].

2.1.4. RND Superfamily

RND efflux pumps are generally present in Gram-negative bacteria, which expel an extensive spectrum of antibiotics and toxic compounds They include AcrAB-TolC in E. coli, AdeABC in A. baumannii [43], CmeABC in Campylobacter jejuni [44], MexAB-OprM in Pseudomonas aeruginosa [45], MtrCDE in N. gonorrhea [46], OqxAB in Klebsiella pneumoniae and Salmonella enterica [47,48], SmeABC in Stenotrophomonas maltophilia, and TtgABC in Pseudomonas putida [49,50]. The Inner transporters such as AcrB, AdeB, CmeB, MexB, TtgB, SmeB, and MtrD are responsible for specific substrate binding and the transportation of different classes of drugs, playing vital roles in clinically relevant resistance [51]. For instance, mutations of the acrB gene would cause the failure of ciprofloxacin therapy [52]. The expression of the above-mentioned efflux pumps is also regulated by transcriptional regulatory proteins belonging to the TetR family, including AcrR [53], CmeR [54], NalC/NalD [55,56], TtgR [57], SmeT [58], and MtrR [59], as well as MexR belonging to MarR family [60]. Table 1 shows the efflux pumps regulated by the TetR family and summarizes the residues that have been proved to be important for the binding of the activating molecule by mutational analysis. Amino acid residues in efflux pumps may be important sites for substrates binding, and the substitution of amino acid residues may affect the affinity of the substrates. For example, Val 141, Phe 626, Tyr 330, and Phe 180 in the substrate-binding domain of the OqxB transporter protein are crucial for binding and transporting different substrates [61]. Additionally, mutations of important amino acid residues are associated with efflux pump-mediated multidrug resistance. The latest research shows that changing the glycine residue 311 to the acidic amino acid glutamic acid (G311E) in the MATE family protein DTX6 markedly increases Arabidopsis plants’ resistance to paraquat and diquat [62].

Table 1

Efflux pumps regulated by the TetR family.

Microorganisms	Efflux Pump	Regulators	Crucial Amino Acid Residues	Substrates	References
C. jejuni	CmeABC	CmeR	-	Multidrug	[54]
E. coli	AcrBTetA	AcrRTetR	Gly-616His-64, Thr-103, Arg-104, Pro-105	MacrolideTetracyclines and Mg2+ complex	[65,66]
L. monocytogenes	FepA	FepR	-	Fluoroquinolones	[67]
N. gonorrhoeae	MtrCDE	MtrR	-	Hydrophobic antibiotics	[59]
V. cholerae	NorM	-	Glu-124, Glu190, Asp-155, Gly-184, Gly-187, Lys-185, Pro-189, Cys-196, and Tyr-384	Norfloxacin	[68]
P. aeruginosa	MexAB-OprM	MexR	-	Novobiocin	[55,56]
P. putida	TtgABC	TtgR	Ser-77, Glu-78, Asn-110, His-114	Tetracyclines, Chloramphenicol	[57]
S. altophilia	SmeDEF	SmeT	His-67, Ser-96, His-167	Tetracyclines, Chloramphenicol,Quinolones	[58]

The multidrug resistance efflux pumps AcrAB-TolC, MexAB-OprM, CmeABC, MtrCDE of the RND family are involved in the survival and pathogenicity of bacteria [6]. Several studies have found that AcrAB-tolC efflux pump in S. Typhimurium and E. coli affect bacterial adhesion and invasion in host cells and colonization and persistence in animals [6]. Bacteria produce pore-forming toxins, degrading enzymes, and virulence factors of the secretory system to exert their pathogenicity and evade the attack from the host immune system. The outer membrane protein TolC can transport not only drugs but also hemolysin and colicin V [63,64].

Heavy metal ions are poisonous or inhibit the growth and survival of bacteria at specific concentrations. Due to the widespread application of heavy metal ions in antimicrobials such as antiseptic disinfectants, bacteria have to take measures to deal with the stress of heavy metals. In E. coli, the RND superfamily plays an important role in the resistance to antibiotics and heavy metals. There are seven known RND proteins; AcrB, AcrD, AcrF, MdtB, MdtC, and YhiV are multidrug efflux pumps of the hydrophobic and amphiphilic efflux RND (HAE-RND) protein family, while CusA is a member of the heavy-metal efflux RND (HME-RND) family that exports Cu(I) and Ag(I) [69]. CusA together with CusB membrane fusion protein and CusC channel protein constitute the complete tripartite CusCBA efflux complexes [70]. Long et al. predicted the mechanisms of metal ions export [70]. Firstly, Cu(I) is combined with the chaperone CusF and then is delivered to three-methionine metal-binding sites (M49, M64, and M66) at the long N-terminal tail of CusB; secondly, the metal ion is transferred to the three-methionine cluster (M573, M623, and M672) inside the periplasmic cleft of CusA and then is released into the central funnel of CusA; finally, the metal ion enters the CusC channel for its final extrusion [70]. Bacteria protect themselves from toxic components of organic pollutants by efflux pump biodegradation. TtgABC, a tripartite RND efflux pump, confers toluene tolerance in P. putida [71].

The stress caused by the abuse of antimicrobials increases the occurrence of functional mutations in the RND family, possibly enhancing its efflux function. The substrate specificity of efflux pumps correlates with differences of amino acid residues in the drug-binding pocket, and bacteria become less sensitive to antimicrobials after the substitution of these amino acid residues. Numerous examples of mutations in the RND efflux pump which have been identified from clinical and environmental isolates and laboratory-evolved strains affect antimicrobial resistance and are fully described in this article [72]. The acquisition of a functional mutation in an efflux pump may be an adaptive response of bacteria to antimicrobials and undoubtedly increases difficulties in the clinical treatment of bacterial infections.

2.1.5. SMR Superfamily

The SMR family members consist of short polypeptide (100–150 amino acids) and span the cytoplasmic membrane as four transmembrane α-helices [73]. Those proteins with short hydrophilic loops permit to solubilization a broad spectrum of drugs, such as disinfecting agents including quaternary ammonium compounds, toxic lipophilic compounds including DNA intercalating dyes, toxic metabolites including nicotine intermediates, and polyamine compounds like spermidine [73,74]. In Gram-negative bacteria, the AbeS in A. baumannii transports sterilant ethidium, acriflavine, and benzalkonium, which also significantly increase resistance to amikacin [75,76]. The clinical strains of K. pneumoniae possesses the activated pump KpnEF and resists to benzalkonium chloride, chlorhexidine, and some other antiseptics [77]. The EmrE protein, present in both E. coli and P. aeruginosa, recognizes and mediates the extrusion of toxic polyaromatic compounds [78,79]. Specifically, the Qac protein is associated with resistance to some antiseptics and antibiotics [80]. The qacA/B genes is more frequently observed in S. aureus and E. faecalis, while the qacE gene is widely spread in Enterobacteriaceae and Pseudomonas spp. [80].

Genes encoding SMR proteins often occur on multidrug resistance plasmids and mobile genetic elements called integrons, which increase the risk of horizontal transmission of resistance [74]. Overexpression of efflux pumps induced by the exposure to QAC facilitates the horizontal transfer of mobile genetic elements carrying FQ resistance determinants (qnr, aac(60)-Ib-cr, oqxAB, qepAB) in Class 1 integrons (QacED1) [81]. Co-transfer of disinfectant-resistant genes and antibiotics-resistant genes among different species seriously influences the bactericidal effects of disinfectants and antibiotics.

2.1.6. PACE Superfamily

The PACE transporter family has been identified in the last 5 years, and the Acel protein in A. baumannii is the first transporter protein found in the PACE family [82]. This transporter contributes to extruding biosynthetic biocides (e.g., chlorhexidine, acriflavine, proflavine, dequalinium, and benzalkonium) [7]. It has been found that genomes encoding the PACE transporter family are highly conserved among bacterial species, which suggests that PACE transporter-related genes are acquired perpendicularly and maintain their characteristic in host species [83].

AceI (Acinetobacter chlorhexidine efflux protein I) is similar to members of the SMR family in size and in the predicted secondary structure. AceI contains two tandem bacterial transmembrane pairs. BTP-domain proteins homologous to AceI have also been discovered in many pathogens such as Burkholderia, Enterobacter, Klebsiella, Pseudomonas, and Salmonella species [83,84,85]. The structure of the AceI protein is in equilibrium between a monomer and a dimer. Increases in chlorhexidine concentration and pH promote the formation of an acetylated dimer, and the binding of chlorhexidine to the transcriptional protein AceR increases aceI transcription, thus extruding chlorhexidine [82].

Table 2 and Table 3 summarize the major efflux pumps and their specific substrates in Gram-positive and Gram-negative bacteria, respectively.

Table 2

Efflux pumps present in Gram-negative bacteria.

Efflux Pump Family	Efflux Pump	Regulator	Organisms	Substrates (Class)	Resistance to Specific Antibiotics a	References
ABC	MacAB-TolC	PhoPQ	E. coli,N. gonorrhoeae,S. maltophilia	Macrolides	EM	[18,86]
MATE	NorM	/	N. gonorrhoeae	Fluoroquinolones, EB, Rhodamine 6G	NF, CP	[29,33]
MFS	EmrAB-TolC	EmrR	E. coli	Cotrimoxazole	/	[40]
	MdfA, MdtM	/		Tigecycline, chloramphenicol	DC, CM	[86,87,88]
	QepA	QepR		Fluoroquinolones	FQ	[89]
	TetA	TetR		Tigecycline	TC	[66]
RND	AcrAB-TolC	AcrR	E. coli,K. pneumoniae,S. enterica	β-lactams, Fluoroquinolones	KF, CM, FQ, P	[90,91]
	AdeABC	AadR, AadS	A. baumannii	Aminoglycosides, Erythromycin, Chloramphenicol, Fluoroquinolones, Tetracyclines, Trimethoprim, some β-lactams, Bile salts	AZI	[43]
	CmeABC	CmeR	C. jejuni	β-lactams, Tetracyclines, Quinolones	TC	[44]
	MexAB-OprM	NalC/NalD	P. aeruginosa	Quinolones	CM, CP, TC, SM	[45]
	MtrCDE	MtrR, MtrR	N. gonorrhoeae	Fluoroquinolones	CP, RF	[46]
	OqxAB	OqxR	E. coli,K. pneumoniae,S. enterica	Chloramphenicol, Fluoroquinolones	CM, NT, NF, CP, LEV	[47,48]
	SmeDEF	SmeT	S. maltophilia	Aminoglycosides, TrimethoprimTetracyclines, Chloramphenicol	GM, CZ, IMP, MP, CAR, TC	[49]
	TtgABC	TtgR	P. putida	Chloramphenicol	CM, TC	[50]
SMR	AbeS	/	A. baumannii	Ethidium, Acriflavine, Benzalkonium	EM, NO	[72,73]
	EmrE	/	E. coli,P. aeruginosa	Quaternary ammonium compounds	Quaternary ammonium compounds	[75,76]
	KpnEF	/	K. pneumoniae	Benzalkonium chloride, Chlorhexidine	CT, EM, RF, TC, SM	[74]

a ACR, acriflavine; ADM, adriamycin; AG, aminoglycosides; AZI, azithromycin; CAR, carbenicillin; CLI, clindamycin; CM, chloramphenicol; CP, ciprofloxacin; CR, clarithromycin; CT, Colistin; CZ, cefoperazone; DA, dalfopristin; DAU, daunomycin; DC, doxycycline; DTM, distamycin; EB, ethidium bromide; EM, erythromycin; FQ, fluoroquinolones; FU, fusidic acid; IMP, imipenem; KF, cephalosporins; GM, gentamicin; LEV, levofloxacin; ML, macrolides; MP, meropenem; NF, norfloxacin; NO, novobiocin; NT, nitrofurantoin; OF, ofloxacin; P, penicillins; RF, rifampicin; ROX, roxithromycin; SM, streptomycin; TC, tetracycline; TM, trimethoprim. “/” mean no transcription regulators found.

Table 3

Efflux pumps present in Gram-positive bacteria.

Efflux Pump Family	Efflux Pump	Regulator	Organisms	Substrates	Resistance to Specific Antibiotics a	Reference
ABC	EfrAB	/	E. faecalis	acriflavine, ethidium bromide, safranin O, DAPI, daunomycin, doxorubicin, novobiocin, arbekacin, doxycycline and norfloxacin	GM, SM, CM	[24]
	LmrA	/	L. lactis	Macrolides, Lincosamides, Streptogramins	DAU, ADM	[21,25]
	Msr	/	Streptococcus	Macrolides	ML	[22,26,27]
	PatA/PatB	/	S. pneumoniaee	Fluoroquinolones	FQ	[23,28]
MATE	FepA	FepR	L. monocytogenes	Fluoroquinolones	NF, CP	[64]
	MepA	MepR	S. aureus	Fluoroquinolones, Tigecycline, Pentamidine	DT	[92,93]
MFS	Lde	/	L. monocytogenes	Fluoroquinolones	ACR, EB	[36]
	NorA, NorB, NorC	MgrA, NorG, ArlRS	S. aureus	Fluoroquinolones	NF, CP	[37]
	Mef	/	S. pneumoniae	Macrolides	EM, AZI, ROX, CR	[38,39,40]
SMR	Qac	QacR	S. aureus,Enterococcus spp.,E. faecalis	Quaternary ammonium compounds	Quaternary ammonium compounds	[77]

a ACR, acriflavine; ADM, adriamycin; AZI, azithromycin; CP, ciprofloxacin; CR, clarithromycin; CM, chloramphenicol; DA, dalfopristin; DAU, daunomycin; DT, dirithromycin; EB, ethidium bromide; EM, erythromycin; GM, gentamicin; FQ, fluoroquinolones; ML, macrolides; NF, norfloxacin; ROX, roxithromycin; SM, streptomycin.

2.2. Efflux Pumps Affect Biofilm Formation and Quorum Sensing (QS)

Biofilms, a microbial community attached to a surface, contribute to bacterial resistance and tolerance. Interestingly, the function of biofilms is directly or indirectly influenced by efflux pumps [94,95]. For instance, sub-inhibitory concentration of tigecycline directly restrain biofilm formation by downregulating the adeG efflux gene in A. baumannii [96]. QS is formed among cells, promotes the mutual communication of cells, and participates in bacterial activities through signal transduction. QS cooperation with biofilms improves the viability of bacteria by sensing changes in environmental signals. For example, the MexAB-OprM efflux pump extrudes acylated homoserine lactones with the contribution of QS, and overexpression of the Mex pump in P. aeruginosa results in the release of QS signals [6]. It has been found that QS among bacteria will be impeded if efflux pumps activity is hindered by inhibitors [97]. Similarly, the proper concentration of efflux pump inhibitors prevents biofilm formation, as observed for inhibitors of the NorA efflux pump in S. aureus [98].

The proteomic profiles of two A. baumannii strains grown in planktonic stationary phase or in mature solid–liquid biofilm were compared using proteomics, and the MacAB-TolC efflux pump was found to play an essential role in biofilm formation [99]. The efflux pump, which helps pathogen adapt to bad conditions occurring in mature biofilms, is involved in envelope stress responses that maintain membrane rigidity and confer resistance to high osmotic stress [99]. The MFS transporter proteins Pmt and AbaF are involved in A. baumannii biofilm formation. Pmt extrudes extracellular DNA and plays an essential role in forming the biofilm structure, while AbaF releases biofilm materials [41].

The RND-type MDR efflux system is well studied. It has been found that a number of RND transporters contribute to alterations of the membrane involved in bacterial functions, especially biofilm formation [100]. This was shown for A. baumannii efflux pump AdeB and its regulatory protein AdeRS. Deletion of adeAB genes or inhibition of the expression of these genes leads to the reduction or prevention of biofilm formation and of the QS system [101,102]. There is a positive correlation between biofilm formation and the mRNA levels of the efflux pump genes adeB, adeG, and adeI, which is altered by sub-MICs of colistin or polymyxin B [103]. The pump MexGHI of P. aeruginosa transport phenazine, which is required for biofilm morphogenesis [104]. The QS system has been shown to regulate the expression of RND efflux pumps. Conversely, the RND transporters can also influence QS via translocation of quorum signals [105]. It was shown that virulence and quorum sensing molecules of P. aeruginosa would be lost and reduced when the efflux gene mexI is mutated [106]. The ABC-type efflux system also plays a role in resistance to antifungal agents in fungi, particularly in Candida species. Similar to the RND pumps, it is involved in the secretion of quorum-sensing molecules and affects biofilms’ behavior [107].

3. Regulation of Efflux Pumps and Application of Efflux Pump-Related Genes/Proteins

3.1. Regulation Mechanisms of Efflux Pumps

In addition to exposure to antimicrobials or disinfectants, efflux pump expression is also regulated by a variety of regulatory systems and proteins. Currently, single regulatory proteins for the MDR efflux pump are mainly classified into four categories, i.e., AraC, MarR, MerR, and TetR [108,109,110]. These regulatory proteins contain DNA-binding domains and ligand-binding domains. For instance, the TetR family is a substrate-dependent transcriptional repressor and controls the expression of the efflux-related tet genes, which confer resistance to tetracyclines [111]. FepA is a novel type of MATE efflux pump, whose overexpression results in resistance to fluoroquinolones. It is regulated by FepR, a TetR-type repressor, which increases the MICs of norfloxacin and ciprofloxacin in L. monocytogenes [67]. The expression of the AceI protein, a transporter protein of the PACE family, is regulated by the LysR-type transcriptional regulator AceR [82]. There are extensively studies reporting that the transcriptional regulatory protein MepR, a MarR-type repressor, inactivates the MepA protein, whose substrates include biocides, fluoroquinolones, and tigecycline [92,93]. These regulators primarily respond to several types of signals including superoxide and peroxide (e.g., H2O2) [112], antimicrobials (e.g., antibiotics), toxic reagents (e.g., methylglyoxal), and biocides (e.g., triclosan) [113,114].

Another type of efflux pumps regulation is based on the two-component regulatory system, consisting of a cognate response regulator and a sensor histidine protein kinase, such as SoxRS, AdeRS, BaeRS, SmeRS, MacRS, ArlRS, EvgSA, BaeSR, CpxAR [115,116,117,118,119,120,121]. Each sensor detects a specific intracellular signal (e.g., antibiotics) and then induces histidine auto-phosphorylation, thus transferring the phosphate group to the proper aspartic acid and activating the response regulator [122]. For instance, it is known that AcrAB in E. coli is controlled by a series of different regulatory systems, including MarA, Rob, SoxS, AcrR, EnvR, MprA, PhoP, RpoE [123]. These regulatory systems are activated by external environmental signals, such as pH, the concentrations of antimicrobials, divalent metal ions, organic solvents, the growth phase, and oxidative stress [123].

3.2. Determination of Antimicrobial Resistance Based on Efflux Pump Gene Expression

Microorganisms harbor MDR efflux pumps resulting in inherent or acquired resistance to antimicrobial agents. The resistance-related genes encode the constituent proteins or regulatory proteins of efflux pumps, which function as the first line of defense against drugs, maintaining the survival of the bacteria [124]. Many efflux pump genes can be used for the rapid detection of antimicrobial resistance, which can be efficiently verified by PCR identification and MIC determination. The RND family of efflux pumps recognizes a large number of substrates, extruding the majority of drugs and increasing antimicrobial resistance. Examples are the Acr pump in E. coli, the Ade pump in A. baumannii, and the Mex pump in P. aeruginosa [76,125]. According to whether the efflux gene is expressed or not, we can determine antibiotic resistance and guide clinical therapy [126]. For instance, in the carbapenem-resistant A. baumannii isolate, the expression of the efflux genes adeB, adeG, and adeJ was increased by different folds [127]. Similarly, a stimulated expression of adeB and adeJ was also found in bacteria resistant to tetracyclines [128]. However, there is a slight relationship between substrates concentration and velocity of maximal transportation. For example, although cefaloridine can be strongly excreted by AcrB, it still possesses antibacterial activity in the presence of AcrB, which explains that the effective antibacterial dose of cefaloridine is much lower than the concentration required for the efflux [129].

In addition, some single-substrate efflux pumps are associated with high level of resistance and MICs. These single-substrate efflux proteins include the macrolide-specific efflux pumps MacAB in E. coli and Mef in S. pneumoniae [38], the hydrophilic fluoroquinolones efflux pump OqxAB in E. coli and S. enterica [130], the tetracycline-mediated efflux pump TetA/TetO in E. coli [131], etc.

3.3. Detection of Antibiotic Residues Based on Efflux Pump Proteins

Presently, efflux pump-related proteins are still under research for antibiotic residue detection, and only two regulatory proteins are used for the screening of antibiotics residues, including TetR and TtgR (belongs to TetR family). Receptor proteins involved in gene transcriptional regulation have been proven to be a powerful tool for detecting low antibiotic concentrations [132]. Hyerim et al. established a bioreporter method based on TetR and tetR promoter to detect doxycycline, using a green fluorescence protein gene as reporter gene, and 5nM doxycycline would induce high expression levels of green fluorescence protein [133]. Weber et al. developed an in vitro indirect enzyme-linked immunosorbent assay using TetR-tetO to accurately and rapidly detect tetracycline antibiotics, with the detection limits of doxycycline and tetracycline of 0.1 and 1.9 ng/mL, respectively [134]. Moreover, Espinosa-Urgel et al. established a novel microbial biosensor based on TtgR to detect drug concentrations around 22 µM with high fluorescence intensity [135]. It was demonstrated that green fluorescence protein-fused TtgR, a TtgABC efflux pump transcription regulator in P. putida, is most sensitive to ceftazidime, ciprofloxacin, and tetracyclines [135].

4. Efflux Pump Inhibitors (EPIs) in Antimicrobial Therapy

Efflux pumps act as a new target for antimicrobial combination therapy, enabling synthetic or plant-derived EPIs to assist antibiotics therapy against bacterial infections [136,137]. Multi-drug resistance is largely mediated by efflux pumps; hence, the development of efflux pump inhibitors is necessary to curb antimicrobial resistance. There are various ways to block or bypass the action of the efflux pumps, including decreasing the binding affinity of antibiotics to the transporter by modifying the drug’s chemical structure, increasing the permeability of the outer membrane to increase the intracellular drug concentrations, inhibiting or knocking out efflux pump-related genes, impairing the ATP energy supply, or designing substances that can compete with antimicrobials for the action site of efflux pumps to competitively inhibit the efflux activity [138].

A number of inhibitors have been discovered by computational analysis or artificial extraction from plants. Through high-throughput virtual screening of natural compound collections against NorM—a MATE transporter from N. gonorrhea—authors found that Terminalia chebula, a compound from an Indian medicinal plant, destroyed the binding of Na+ and ligands and turned the NorM transporter into a closed state [139]. Phenylalanyl–arginine β-naphthylamide, an inhibitor of AcrB in E. coli, has been shown to inhibit the extrusion of drugs via binding to the hydrophobic pocket of AcrB [140]. It can be combined with carolacton, a secondary metabolite in myxobacteria, for potential use in antimicrobial chemotherapy against AcrAB-TolC [141]. Tannic acid also acts as an inhibitor of S. aureus multidrug efflux pump, Tet, Msr, and others [142]. It has been found to significantly reduce the MIC of drugs such as tetracycline and erythromycin [142]. However, EPIs combined with antibiotics to assist MDR therapy in the clinic is an obviously potential challenge, depending on the intrinsic permeability properties of the bacterial outer membranes. Yang et al. demonstrated that the coupled use of tobramycin and EPI promoted the binding of EPI with tetracycline and acted on MDR P. aeruginosa [143]. They also explored the effect of tobramycin–EPI conjugates in relation to with fluroquinolone, rifampicin, and fosfomycin, showing that they effectively reduced the MIC80 of these drugs and exerted a strong joint effect [144]. Adamson et al. showed that the combination of EPI (trimethoprim and sertraline) and levofloxacin against the overexpression of MexAB-OprM pumps in P. aeruginosa produced advanced benefit compared with the monotherapy with levofloxacin [145]. Prasch et al. conducted similar research and demonstrated that when the above efflux pump inhibitors are co-administered with antibiotics, the antibiotic therapeutic dose can be reduced [146].

In addition to the EPIs mentioned above, plant-derived EPIs have also been researched (Table 4). Based on various extraction mechanisms, more than 20 different potential plant-derived EPIs have been reported [147,148]. In addition to good anti-inflammatory and antibacterial effects, compounds extracted from vegetal including berberine, Artesunate, and Curcumin inhibit the efflux pump activity of the Gram-negative bacilli E. coli and P. aeruginosa [149,150,151]. Vegetables (e.g., Momordica balsamina), seeds (e.g., milk thistle seeds), spices (e.g., pepper and cumin), essential oils derived from aromatic plants (e.g., trans-cinnamaldehyde and eugenol), etc., are excellent sources of EPIs [147,152,153,154,155,156]. Flavonoids, particularly flavonolignans, were found to have great application prospects in the fight against multidrug resistance by inhibiting bacterial ABC transporters and other efflux pumps [157].

Table 4

Plant-derived EPIs.

Bioactive Compounds	Bacterial Species	Pharmacological Activity	References
Berberine	P. aeruginosa	Inhibited the multidrug efflux system MexXY-OprM	[149]
Artesunate	E. coli	Inhibited the multidrug efflux pump system AcrAB-TolC	[151]
Curcumin	P. aeruginosa	Inhibited the expression of efflux pump	[150]
plant-derived flavonoids such as skullcapflavone II and nobiletin	Mycobacterial Species	Inhibited the activity of the efflux pump and decreased the rifampicin-resistance level	[153]
Extracts of milk thistle seeds and reserpine	Salmonella Typhi	Inhibited an efflux transporter STY4874	[154]
Hypericum olympicum L. cf. uniflorum-derived natural product	S. aureus	Inhibited NorA multidrug efflux pump activity	[147]
diterpene isolated from Chamaecyparis lawsoniana: ferruginol	Methicillin-resistant S. aureus (MRSA)	Inhibited the TetK pump	[155]
quinine isolated from Cinchona tree’s bark		Inhibited the activity of the efflux pump
piperine isolated from the Piperaceae family
harmaline isolated from Perganum harmala
4′,5′-O-dicaffeoylquinic acid isolated from wormwood (Artemisia absinthium)
triterpenoids from Momordica balsamina
carnosic acid from Rosmarinus officinalis
coumarins from Mesua ferrea
clerodane diterpene from Polyalthia longifolia		Downregulation of MFS and MATE family efflux genes such as norA, norB, norC, mdeA, and mepA
cumin spice (Cuminum cyminum)		inhibited LmrS drug transport (a proton-driven multidrug efflux pump in MRSA)
trans-cinnamaldehyde and eugenol	A. baumannii	downregulated the expression of efflux pump-related gene adeABC

5. Conclusions and Perspectives

This work reviewed functional studies of various efflux pump families whose substrates include heavy metals, disinfectants, preservatives, toxins, and virulence factors, in addition to conventional antibiotics. Apart from mediating multidrug resistance, efflux pumps also confer resistance to heavy metals and disinfectants, and even cross-resistance to them. This suggests that efflux pumps have much more complex actions than we initially thought. Efflux pumps play important roles in biological processes such as biofilm formation, quorum sensing, bacterial adhesion to host cells, and invasiveness. This further highlights the importance of developing efflux pump inhibitors. Many discovered efflux pump inhibitors are in clinical trials, though some have even been excluded due to their excessive toxicity. Therefore, there is an urgent need to find safe, green, and harmless efflux pump inhibitors. Plant-derived extracts are interesting candidates. Overall, the functional diversity of efflux pumps remains to be discovered, and the development and utilization of efflux pump inhibitors still require further exploration.

According to preceding research, proteins of the TetR family can be used for the detection of antibiotic residues involved in drug resistance. In addition to high-affinity transcriptional regulatory proteins reported above, there are other potential proteins that recognize a single substrate and have a specific drug binding domain. As shown in Table 3, regulatory and constitutive proteins have specific drug binding sites that can be used for the detection of antibiotics residues involved in the binding. Therefore, efflux pump-related proteins can also be used to detect antibiotic residues, especially transcriptional regulators, though only TetR family proteins are currently applied in practice. It is imperatively demanded that researchers discover more appropriate proteins with high binding specificity for antibiotics. Moreover, this article mentions the connections between the formation of biofilms and the expression of efflux pumps genes, which offers new perspectives to widen fundamental research. This article illustrated the functions of efflux genes and relevant transporters, a topic that requires constant exploration to provide guidance for clinical applications.