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Development of Efflux Pumps and Inhibitors (EPIs) in A. baumanii

Lidan Xing, Prince Amoah Barnie, Zhaoliang Su* and Huaxi Xu*

Department of Immunology, Institute of Laboratory Medicine, Jiangsu University, Zhenjiang, PR China

*Corresponding Authors:
Xu Huaxi
Department of Immunology
Jiangsu University, Xuefu Road 301
Zhenjiang, China
Tel: +86-511-887910
Fax: 86-511-88791739
E-mail: [email protected]
 
Su Zhaoliang
Department of Immunology
Jiangsu University
Xuefu Road 301, Zhenjiang, China
Tel: +86-511-887910
Fax: 86-511-88791739
E-mail: [email protected]

Received Date: November 06, 2013; Accepted Date: December 16, 2013; Published Date: January 07, 2014

Citation: Xing L, Barnie PA, Su Z, Xu H (2014) Development of Efflux Pumps and Inhibitors (EPIs) in A. baumanii. Clin Microbial 3:135. doi: 10.4172/2327-5073.1000135

Copyright: © 2014 Xing L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Over the past three decades, Acinetobacter baumannii has emerged as an important nosocomial pathogen worldwide. Certain strains of A baumannii are now resistant to many common antimicrobial agents, including fluoroquinolone, and multidrug resistance (MDR) is often responsible for the failure of antibiotic therapy. Reasearchers have paid close attention to the emergency of MDR-AB, and PDR (pandrug-resistance)-AB, even XDR (extreme-drug resistance)-AB isolated from clinical patients, mediated primarily through the production of β-lactamases, aminoglycoside-modifying enzymes (AMEs), mutation in gene for DNA gyrase subunit A (gyrA) and DNA topoisomerase (parC), as well as efflux pumps. In this article, we discussed the roles of various efflux pumps expressed in Acinetobacter baumanii and efflux pump inhibitors (EPIs), aimed at providing an overview of the mechanism to antibiotics in Acinetobacter baumanii.

Keywords

Acinetobacter baumanii; Efflux Pumps; RND; adeABC; Efflux Pump Inhibitors (EPIs)

Introduction

Acinetobacter baumanii is a Gram-negative, non-fermenting, oxidase-negative and non-motile coccobacillus, [1]. It can survive in water, soil, surface of skin and hospital environment. It cause a series of opportunistic infections such as epidemic pneumonia, skin and soft tissue infections, bacteraemia, urinary tract infections and septicemia app:addword:septicemia in immunocompromised patients, especially in the intensive care unit [2]. With the overuse of broad-spectrum antibiotics, A. baumannii has become the mayor pathogen in nosocomial infections [3]. To make matters worse, therapeutic outcomes in patients are severely affected due to the emergency of acquired multidrug resistance. These multidrug resistant strains of A. baumannii employ resistant mechanisms such as the use of chromosomally encoded cephalosporinase, degradation of enzymes, modification or protection of the drug target sites, decreased permeability of the membrane and the active efflux of antibiotics [3,4]. Resistant mechanisms in A. baumannii have aroused researchers’ widespread attention in last decades.

Recent studies show that over-expression of efflux system is an efficient mechanism for drug resistance. Various chromosomally encoded efflux systems, both intrinsic and acquired, have been characterized as contributing to MDR in A. baumannii. The best characterized are the AdeABC, first identified in an MDR clinical isolate BM4454 [5], and adeDE in Acinetobacter Genomic DNA group 3 [6]. This review is focused on efflux mediated resistance in A. baumannii, especially on the resistance-nodulation-cell division (RND) family and EPIs.

Efflux Superfamilies in Acinetobacter baumannii

Efflux system, composed of an inner membrane protein (IM, such as RND efflux) linked by a major fusion protein (MFP) to an outer membrane factor (OMF), are able to extrude a wide range of substrates often unrelated in structure [7], these substrates include aminoglycosides, quinolones, tetracycline, and minocyline. According to the homology of amino acid sequence, five superfamilies of efflux systems are closely related to drug resistance: RND family, the major facilitator superfamily (MFS), multidrug and toxic compound extrusion (MATE) families, the small multidrug resistance (SMR) and the ATPbinding cassette (ABC) transporters [8] (Table 1). Among the efflux systems, RND pumps are the most prevalent systems in gram-negative bacteria [9-11].

Efflux Pumps Energy resource Substrates
RND adeABC Proton motive force Aminoglycosides, tetracyclines, erythromycins, fluoroquinolones, β-lactams, chloramphenicol, trimethoprim, cefotaxime, minocyline,some toxicant and dyes such as ethidium bromide
adeDE Ceftazidime, amikacin, ciprofloxacin, chloramphenicol, erythromycin, ethidium bromide, meropenem, rifomycin, tetracycline
adeIJK Similar to adeABC, include β-lactams, tetracyclines, chloramphenicol, erythromycins, fluoroquinolones, trimethoprim, and sodium dodecyl sulfate (SDS) but not ethidium bromide
adeFGH Chloramphenicol, clindamycin, tigecycline, fluoroquinolones, trimethoprim, sulfamethoxazole and varies dyes such as ethidium bromide, sarranine
adeXYZ β-Lactams, ciprofloxacin, tetracycline, rifampin, chloramphenicol
MFS Tet Proton motive force TetA-tetracycline only; TetB- tetracycline and minocycline; TetM- tetracyclines
cmlA Chloramphenicol
CraA Chloramphenicol
AmvA Mainly dyes, disinfectants, detergents; and only antibiotic-erythromycin
SmvA Erythromycin, methyl viologen, quaternary ammonium salt
MATE abeM Proton motive force Fluoroquinolones, aminoglycosides, chloramphenicol, acriflavine,daunorubicin, rhodamine6G, ethidium bromide and so on
SMR abeS Proton motive force Dyes, detergents; chloramphenicol, fluoroquinolones, erythromycin, and novobiocin
ABC ABCB1  ATP hydrolysis Cancer cells

Table 1: Efflux pump and its substrates.

RND superfamilies

Efflux pumps of the RND superfamily play a significant role in producing multidrug resistance in gram-negative bacteria [12], such as AcrB in Escherichia coli, MexB in Pseudomonas aeruginosa [3], and AdeABC, AdeDE, AdeIJK, AdeFGH in Acinetobacter baumannii [6,7,13-16]. Some efflux pumps of the RND family have been shown to play an important role in the colonization and persistence of bacteria in the host thereby maintaining the bacterium cell in steady state as well as expelling toxic compounds from the bacterium cell [17].

AdeABC

AdeABC, the major and the first identified efflux pump in RND, it is a kind of ATP-dependent multidrug transporter. In this efflux pumps, antimicrobials can be expelled from cells by utilizing the proton motive force. [4]. The adeABC operon encodes AdeA (Membrane Fusion Proteins, MFP), AdeB (Inner Membrane Efflux Transporters, IM) and AdeC (Outer Membrane Proteins, OMP). These three genes are contiguous in the genome. Adjacent to them are two-component regulatory systems, adeR and adeS, which specify proteins homologous to sensors and regulators of two-component systems, respectively (Figure 1). AdeB, which consist of 12 transmembrane fragments, has the capacity to recognize antibiotics and extrude them out of cell, play a significant role in the efflux pump. Thus the adeB has been proposed to be an epidemiological tool for detecting clinical MDR strains. It has been shown that adeC is not essential for the MDR strains conferred by the pump, because an mutant displays similar resistance to the substrates of AdeABC to that of the parental strain when adeC is inactivated [18], but AdeC can expel antibiotics out of cell, because it is located in the epicyte and has porin as well. AdeA, which stabilize OMP, allows antibiotics to pass the inner and the outer membranes of the bacteria without accumulating within the periplasm [13].

clinical-microbiology-Structure-system

Figure 1: Structure of adeABC system.

AdeABC is very common in both resistant and susceptible A. baumannii strains. Alexander et al. [19], show the adeA, adeB, adeR and adeS are present in 80% of 116 strains of Acinetobacter, while adeC is present in 40% [19,20] also found that 34 carbapenem resistant A. baumannii expressed all the 5 genes, whereas other 2 strains are defective of adeC [20]. Expression of adeABC is tightly regulated by the two-component regulatory system, adeR-adeS. These adeS and adeR are sensor kinase and response regulator, respectively [10,18]. They are located upstream of adeABC and are transcribed in the opposite direction [8]. Mutations in AdeRS have been shown confer to overexpression of AdeABC, whereas inactivation of adeR-adeS is responsible for suscepbility in A. baumannii [18], it is known that adeR-S acts as a transcriptional activator in the pump. Mutation in adeR (Pro116Leu) and adeS (Thr153Met or Gly30Asp) will cause overexpression of AdeABC [18], and an insertion of an ISAba1 upstream from the operon can also lead to overexpression of AdeABC, thereby enhancing efflux effect [14,21]. It has been reported that the AdeABCoverexpressing strains confer resistance to aminoglycosides, and decrease susceptibility to fluoroquinolones, cefotaxime, tetracycline, chloramphenicol, erythromycin, trimethoprim, minocyline [13] and tigecycline [14,22]. Recent data suggest that this system decreased netilmicin susceptibility of A. baumannii [19]. However, the role of AdeABC in carbapenem resistance strains raises many controversies. According to [23], overexpression of AdeABC leads to significantly highlevel resistant to carbapenems, especially to imipenem, meropenem, and other class D carbapenemases [23]. However, in OXA-23-producing strains, overexpression of AdeABC is associated with resistant to meropenem but not to imipenem following inactivation of the pump [20]. Moreover, the overexpression of the AdeABC efflux pump is suggested to confer high-level resistance to carbapenems in conjunction with carbapenem-hydrolyzing oxacillinases in clinical isolates [24,25]. Thus, researchers agreed that overexpression of AdeABC contributes to carbapenem resistance, but other efflux mechanisms may probably also be involved. In addition, NaCl, in physiological concentrations, can induce significant tolerance to aminoglycosides, carbapenems, quinolones, and colistin among isolated clinical strains of MDR A. baumannii, resulting in broad-spectrum tolerance to antibiotics [26]. Several reports further suggest that AdeABC exhibits a wide substrate range that includes biocides, dyes and disinfectants, and other chemical substrates in A. baumannii [5,13,18].

AdeDE

The adeDE efflux pump has been reported to confer resistance to ceftazidime, amikacin, ciprofloxacin, chloramphenicol, erythromycin, rifampin, meropenem, tetracycline in Acinetobacter genomic DNA group 3(GDG3)[6,27] and imipenem in A. baumannii [28]. It however differs from AdeABC, AdeDE increases the host resistance to ceftazidime and rifampicin while AdeABC protects the host from cefotaxime. Unlike what was found for AdeABC efflux systems, the structural gene for the outer membrane proteins (OMP) is not found in downstream of the adeDE gene cluster [6].

Chu proved that 70% out of 83 GDG3 isolates were positive for adeE, while all the 59 GDG2 (A. baumannii) isolates were negative, and found that adeE was not only expressed in GDG3, but also exist in GDG13TU and GDG17 isolates [27]. Lin found that adeE was only detected in adeSR-adeABC/adeIJK/int1-negative isolates that were relatively susceptible to some antibiotics, and also proved that adeE did not coexist with adeIJK and adeABC. In addition, mRNA expression of adeE was not observed in strains that were positive for adeE [29]. Thus, different distribution of RND genes may likely be used as indicators for differentiating Acinetobacter species. But recent studies suggest that adeE coexists with adeB in a small proportion of A. baumannii isolates [28].

AdeIJK

AdeIJK is the second identified efflux pump in RND families. AdeIJK encode a three-component RND efflux system consisting of AdeI, AdeJ and AdeK, which is similar to MFPs, inner membrane efflux transporters: RND proteins and OMFs, as in the adeABC efflux [30]. As opposed to AdeABC, AdeIJK is intrinsic present in all strains of A. baumannii. It contributes to resistance to β-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, pyronine, safranin, imipenem and sodium dodecyl sulfate (SDS) but not to ethidium bromide [28,30]. Several reports have shown that the levels of adeIJK overexpression are lower than those of adeABC, suggesting a threshold for toxicity for the host and that adeIJK is tightly regulated [8,16,31].

There are no ORFs coding for regulatory proteins in the vicinity of the adeIJK genes, therefore expression of adeIJK seems not to be specifically regulated but controlled at integrated level. Recent study showed that AdeN, a TetR-Type regulator, regulates the expression of AdeIJK in A. baumannii and also represses expression of the adeIJK (16). Moreover, some strains can express adeIJK, adeABC and int1 simultaneously in MDR-AB [29].

AdeFGH

AdeFGH, encoded by the adeFGH operon, also confers multidrug resistance when overexpressed [15]. Expression of adeFGH is regulated by LysR-type transcriptional regulator (LTTR), named adeL, which locate upstream from the operon and transcribed in the opposite direction [8]. Mutation in adeL may lead to the overexpression of adeFGH [32]. It is responsible for high-level resistance to fluoroquinolones, tetracyclines, tigecycline, chloramphenicol, trimethoprim, sulfamethoxazole and various dyes such as EB, sarranine but has little effect when compared to β-lactams, erythromycin, rifampin and aminoglycosides [15,33].

AdeFGH is also very popular in A. baumannii. PAβN is found to inhibit the ability of the AdeFGH pump to efflux chloramphenicol, trimethoprim and clindamycin in the A. baumannii strains [34].

AdeXYZ

AdeXYZ, chromosomally encoded efflux systems, shares more than 97% identity with AdeIJK [30]. AdeXYZ is found in Acinetobacter GDG3, Acinetobacter GDG13TU and Acinetobacter GDG 17 [27]. AdeX, adeY, adeZ are similar to MFP, RND, OMP in RND efflux system. The identity of the theoretical peptides to AdeX, AdeY and AdeZ are 80, 89 and 87, respectively [27]. In addition, AdeXYZ and AdeDE are predominantly in GDG3 while AdeABC efflux system is specific for GDG2 [27]. Until now, the function of adeXYZ in antimicrobial resistance is not well understood.

MFS

MFS belong to prevalent efflux system. Tet, cmlA, CraA, AmvA and SmvA are the most clinically relevant pumps in MFS superfamily in A. baumannii. TetA confers resistance to tetracycline alone while TetB is involved in tetracycline and minocycline resistance, but these two efflux pumps have no effect on tigecycline [35]. Researchers have found no strain carry TetA and TetB simultaneously in 79 tetracyclineresistant A. baumannii clones [35]. TetA, TetB and TetM were all resisitant to tetracyclines [36]. Another MFS resistant gene cmlA, found in MDR A. baumannii strain AYE, which is resistant to β-lactams (except imipenem, piperacillin-tazobactam, ticarcillin-clavulanate), aminoglycosides, fluoroquinolones, chloramphenicol, tetracycline, and rifampin, extrude chloramphenicol [37]. CraA is also reported to display intrinsic resistance to chloramphenicol [38]. Together with other efflux genes, CraA is overexpressed in response to NaCl exposure [26]. AmvA, a member of the MFS efflux pumps, mediates dyes, disinfectants and detergents resistance in A. baumannii [8,39]. Moreover, SmvA confers resistance in A. baumannii with erythromycin, methyl viologen and quaternary ammonium compounds [3].

MATE

MATE families have two energy sources: the PMF (proton motive force) and the sodium ion gradient [40]. A member of the MATE family, AbeM, a H+-coupled multidrug efflux pump extrudes fluoroquinolones, aminoglycosides, chloramphenicol, trimethoprim, erythrocin, ethidium bromide, and dyes [41]. AbeM protein, which consists of 448 amino acid residues, contains numerous hydrophobic amino acid residues and 12 hydrophobic regions. The substrates for the AbeM efflux pump are not widely known as compared to adeABC [41]. Some studies have shown that expression of abeM is not related to resistance to cephalosporins or aztreonam [42]. To date the contribution to antimicrobial resistance of this system in clinical isolates remains unknown.

SMR

abeS, a chromosomally encoded SMR efflux pump, exhibits efflux effect in relation to chloramphenicol, fluoroquinolones, erythromycin, novobiocin, dyes and detergents in A. baumannii [43]. abeS, a 330-bp gene, encodes a 109-amino-acid protein, and displays 100% identity to the product of ORF ABAYE1181. It exhibits 51.8% identity to E. coli EmrE found in the A. baumannii AYE genome [37]. Subsequently, abeS is confirmed to be an energy-dependent efflux pump, and its extruding energy source is proton (H+) [43].

ABC

ATP-binding cassette (ABC) transporters mediate multidrug resistance though ATP-dependent drug efflux pumps. It can energize the transport of variety of compounds across biological membranes via ATP hydrolysis and confer cellular resistance to a broad spectrum of drug substrates [44]. ABCB1, is a 170 kD transmembrane glycoprotein from the super family of ATP-binding cassette and one of the most widely studied transporters that enable cancer cells to develop drug resistance [45]. The major mechanism of efflux is dependent on energy generated for protein transport through the hydrolysis of ATP [46]. ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), serves as an ATP-dependent efflux pump for a variety of chemicals, including many antimicrobial agents [45].

The Development of Efflux Pump Inhibitors (EPIs)

Resistant to antibiotics is increasingly becoming a worldwide severe problem in recent decades. Efflux system plays significant roles in antibiotic resistance in A. baumannii and many other Gramnegative bacteria. Subsequently, efflux pump inhibitors were developed to decrease the resistant effects employed by efflux system. PAβN and CCCP were the best studied synthetic inhibitors while reserpine and verapamil are the most used EPIs in clinical practice in recent years. EPIs can increase the intracellular concentration of antibiotics expelled by efflux pumps, and then maintain the initial sensitivity to antibiotics associated with overexpression of efflux pumps [47] and decrease the frequency of resistant mutant strains [48]. EPIs can hinder efflux channel, block-up energy resources of efflux pumps and make bacterium cells sensitive to the antibiotics.

PAβN

PAβN (phenylalanine-arginine β-naphthylamide, also called MC- 207 110), is a well studied EPI and has been described as a broadspectrum efflux pump inhibitor. PAβN has no effect on bacteria cells itself, unless used synergistically with β-lactam [49], fluoroquinolones and macrocyclic antibiotics. It is known to reverse resistance in Pseudomonas aeruqinosa, Staphylococcus aureus, Enterococcus and Gram-negative bacteria through increasing the level of accumulation of efflux pump substrates within the cell [50,51]. Many studies suggest that PAβN reduce the minimal inhibitory concentrations (MICs) of several β-lactam antibiotics such as chloramphenicol, rifaximin, tigecycline, clarithromycin against E. coli, E. aerogenes, or P. aeruginosa [49,52-56]. Again, PaβN is able to enhance the permeability of cell membrane to increase antibiotic levels in the bacterium cells [51].

CCCP

CCCP (Carbonyl cyanide-m-chlorophenylhydrazone) is a strong uncoupler that disturbs electrochemistry gradient to inhibit RND, MATE, MFS and SMR, whose driving force come from proton motive [57]. Thereby increasing intracellular concentrations of antibiotics. CCCP has become a necessary tool for studying efflux systems MICs. It is known that with CCCP, MICs of fluoroquinolone, chloramphenicol, gentamicin, amikacin, ceftazidime, cefoperazone/sulbactam and imipenem in A. baumannii decreases relatively as compared to without CCCP [58-64].

Reserpine and verapamil

Reserpine and verapamil can block-up substrates’ channel to play its role. Alkaloid reserpine is the first identified inhibitor of NorA, but it is documented to be toxic to the host cell if used to inhibit the activity of NorA [65]. Compared to CCCP, reserpine decreases the MICs of nalidixic acid, ciprofloxacin and norfloxacin, and the reserpine affects isolates which are not affected by PAβN. This therefore suggests that the two inhibitors may possibly act on different efflux pumps [66].

Others

NMP(1-(1-naphthylmethyl)-piperazine), can partially reverse MDR in A. baumannii [58], it has stronger effect than PAβN when higher concentration (100 mg/L) was used, but its effect is limited at a lower concentration (25 mg/L) [54]. In addition, NMP is capable of inhibiting the overexpression of RND pumps in Escherichia [54]. Omeprazole and lansoprazole are proton pump inhibitors, which can suppress the activity of H+/K+-ATP enzyme. Omeprazole is effective in Helicobator pylori and Staphylococcus aureus [67], but it’s function in A. baumannii is not clearly been documented.

Interestingly, researchers have found a natural EPI extracted from a plant which is used in Chinese medicine, named 5΄-methoxyhydnocarpin (5΄-MHC), which inhibits the NorA MDR pump of a human pathogenic Staphylococcus aureus when combined with antibiotics [68].

Conclusion

The positive rate of multidrug resistance A. baumannii is dramatically increased in recent decades all over the world, and the mechanism of resistance is complicated. Together with antibiotics, EPIs provide a new horizon to reverse bacterial resistance, but the studies are in preliminary stages. Its usage is known to be restricted to in vitro because the effective inhibitory concentration is much higher than the concentration used in clinical application, so it could be toxic if applied to patients. Investigators are recommended to research into these mechanisms explored by A. baumannii in other to produce effective and safe EPIs for clinical use.

Competing Financial Interests

The authors have declared that no competing interests exist.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31270947, 81072453, 31170849), the Natural Science Foundation of Colleges and Universities in Jiangsu Province and Innovation Fund for candidate of doctor in Jiangsu Province (Grant No. 09KJB310001 and CXZZ11_0593, respectively).

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