Mechanisms Efflux Pumps of Acinetobacter baumannii (MDR): Increasing Resistance to Antibiotics

Acinetobacter baumannii has greatly increased its degree of resistance to become multidrug resistant (MDR) over the past 30 years and is on the red line of the most widely replicated bacteria according to World Health Organization (WHO). The efflux pumps are the main cause for the increasing antibiotic resistance of A. baumannii originated from nosocomial infection. The progressive resistance of A. baumannii even on the recent drugs (tigecycline and fosfomycin) reduces to very effective antibiotic scale. With attention focused on MDR and pan-drug-resistant (PDR) in A. baumannii multiple works on efflux pumps chemical inhibitor (NMP, PAβN, omeprazole, verapamil, reserpine, CCCP) are still in progress. Certain inhibitors from plants (Biricodar and timcodar, Falvone, Mahonia, Dalea versicolor, Lycopus europaeus, and Rosmarinus officinalis) have the capability to have such compounds according to their very significant synergistic effect with antibiotics. In this review we focused on the growth of antibiotic resistance to explain the mechanism of efflux pumps into these different super families and a comprehensive understanding of the extrusion, regulation and physiology role of drug efflux pumps in the essential development of anti-resistivity drugs. We recapitulated the evolution of the work carried out in these fields during the last years and in the course of elaboration, with the aim of increasing the chances of decreasing bacterial resistivity to antibiotics.

teriologist Beijerinck [1], but it was not until 1960 that A. baumannii was declared in hospital. A. baumannii belongs to the large family of non-fermentable gram-negative bacteria capable of harming patients in surgical intensive care [2].
During the 20 past years it has developed a capital importance and its classification among the nosocomial infections makes it a priority to all the public health organizations considering its increase and recurrence [3]. A. baumannii is much more present in humans and is the origins of multiple diseases like septic fever, pneumonitis pachymeningitis and other disease [4]. Over time it has gained its resistance through diverse modifications and is presently resistant to approximately all the various groups of antibiotics even the most widely used drugs (fluoroquinolones, macrolides, trimethoprim, b-lactams, tetracyclines, aminoglycosides, and chloramphenicol) [5]. The bacterial efflux operation causes the formation of toxins and rejects antibiotics from the cells, which confers a specific invulnerability to antibiotics. Multidrug resistant (MDR) efflux pumps are now present in almost all microorganisms, in which bacteria is one of the main causes of obstruction to action of drugs [6]; several works have concluded that MDR is on origin of the decline progressive of drugs sensitization by bacterial mutation [7] that reduces largely the valid drug for cure. However using inhibitor components could restore bacterial susceptibility to antimicrobial agents. Efflux pumps inhibitors' (EPIs) synthetic or natural component is the potential drugs for treatment of MDR or PDR A. baumannii. After describing the general mechanisms of efflux pumps systems in bacterial resistance, we will explain regulation and physiology role of drug efflux pumps in the essential development of anti-resistivity drugs and report the evolution of the work done during the recent years especially in EPIs.

Mechanisms of Bacterial Resistance to Antimicrobial Agents
The bacterial resistance to antibiotics has emerged in the face of several pathogenic agents, besides A. baumannii, albeit efforts to treat these pathogenic germs still progressing [8]. Bacterial pathogens that have shown resistivity to a single drug or to several agents are considered MDR bacterial. With all the efforts united to resolve the problem via the outgrowth of new line of antibiotics, the bacteria does not cease also to mutate quickly to acquire new mechanism of resistivity or to improve their resistance to antibiotics [1] [4] [5] [9] [10] [11] [12] [13]. Many reports showed that bacterial resistance to antibiotics is believed that the pathogenic bacterium transfers certain genetic gene of resistance to drugs from one species to another and automatically acquires resistant phenotypes against the majority of the pre-existing antimicrobial agents [14]. This mechanism of opposition poses a critical problem to bacterial treatments. Another significant cause of this obstruction is the proximity of drugs to environment, agriculture and others, leading to the emergence and development of resistances. Clinically, a low or high or inappropriate use level of antibiotics will also imply in the increase in bacterial resistance [15]. Presently following many research

Alteration Drugs Target
Every drug has a specific target for destruction of bacteria, and as such when the target is changed bacteria can easily resist to antimicrobial agents, and these have been observed in quinolones (DNA gyrase variation or Qnr intercede purpose defense), aminoglycosides (16S rRNA methylation), and β-lactams (transformation in penicillin junction proteins). Reduced vulnerability to minocyclin and tigecyclin occurs via transformation in gene encoding S-adenosyl-L-methionine-dependent methyltransferase [16]. After undergone several transformation in gene lpx, pmrB and outer membrane induce the structure of lipopolysaccharide for causing polymyxin resistance [17] [18] [19].

Drug inactivation by A. baumannii Enzyme
A. baumannii synthesizes aminoglycoside-modifying enzymes well as AAD, APH, AAC3, and AAC6' that are frequently encrypted by a quota of aba obstruction island-homogeneous gene cassettes including class 1 integrons [20] [21]. A. baumannii also bring out a high quantity of antimicrobial -inactivating enzymes, which are encoded by plasmids and chromosome for the resistance of antibiotics developed from β-lactamine family [8]. These β-lactamases include,

Modification of Cell Wall Protein: Permeableness Barrier of OM
Target modification mechanisms, drug-specific inactivation, and efflux drugs crossways the cell membrane barriers play an essential role in influencing the sensitivity of Acinetobacter spp. to a wide range of antimicrobials. This trait is due to the non-appearance of conventional high permeability trimmers (porins of Enterobacteria spp.) [30] which has poor activity in Acinetobacter spp. and thus belong to the minor proteins [18]. First, matching who has correctly, study is A. baumannii and Pseudomonas aeruginosa (P. aeruginosa), OM also demon-

Drug Efflux Systems
During the recent years because of the poor OM absorbency drug efflux systems has become one of the most complicated mechanisms of bacterial resistance and has played an essential role on drug resistance specially to A. baumanii [39]. The

MDR Efflux Pumps: Structure and Regulation
The structure of the efflux system is comprised of 3 well-defined parts each playing a function in the drug efflux mechanism, including the outer membrane (OM) [40], the internal membrane (IM) and the fusion protein at the intermediate level (MFP); Each part of the structure EP has a certain factor causing resistance to approximately every groups of antibacterial [41] [42]. In reference to further research, it has been proven that there are five different families of efflux pumps present on A. baumannii: • major facilitation super family (MFS); • multidrug toxic composite extrusion (MATE) transporters; • resistance nodulation-division (RND) super family; • ATP binding cassette (ABC) transporters; • small multidrug resistance (SMR) family.
Recently other studies have reported a sixth efflux family named PACE (proteobacterial antimicrobial composite efflux) present in the A. baumannii [43].
However, because of inadequate data, we will concentrate much more on the first 5 families present in Figure 1 where a totally understanding of structure and regulation is not complete.

RND Efflux Pumps
The large family RND efflux is special of the rather complex compared to other family. it is very represented and has a special role in almost all major gram-negative bacteria and developing multiple resistance to antibiotic also call MDR such as A. baumannii, E. coli, and P. aeruginosa [46]. RND efflux on A. baumannii has  and [45]).

adeABC
The adeABC operon was newly discovered on the antibacterial agents of fluoroquinolones and aminoglycosides in the efflux system RND and divided into 3 part: adeB on inner membrane efflux transporters, adeA on membrane fusion proteins, and adeC on external membrane proteins [48]. AdeB has the largest representation on A. baumannii strains (80%), adeA and adeC has 42%, and 40% respectively [49]. The gene adeABC have almost the same structure that genes MexAB-OprM for P. aeruginosa and genes AcrAB-TolC for E. coli [48] [50]. Because of this high proportion of adeB gene compare to the others, its inactivation would dramatically cause sensitization to antimicrobial drugs in the hospital for A. baumannii [51]. The increasing concentration of MIC would be beneficial to important drug classes like aminoglycosides, tetracyclines-tigecycline, β-lactams, fluoroquinolones, macrolides, trimethoprim, and chloramphenicol [52]. Despite the advancement of research, rifampicin, flusidic acid and sometimes colistin remain resistant to isolate A. baumannii. Single last chances of fight against A. baumannii isolates are tigecycline but show a hard resistance to adeABC and also it presents a high resistance efflux. The MIC levels of tigecycline remain a clinical problem [42] [53]. Remarkably, about 20% of adeC was found to be involved in tigecycline resistance tests in A. baumannii demonstrating that in the adeABC gene, adeAB can keep walking without adeC except on [54]. The adeC plays a much more an almost negligible role in RND efflux system. The two components adeR and adeS are responsible for the regulation of the expression system of adeABC [55]. They are also called protein kinases and are found on both sides of adeABC in different trajectory. AdeRS, plays a determin-ing role in increasing resistance of adeABC. Some result shows that a dysfunction of adeR and or adeS will increase the resistance of tigecyclin, chloramphenicol, minocyline, erythromycine, cefotaxime, tetracycline, fluoroquinolones, and trimethoprim [19] [32]; as well increase the sensitization of amino-glycosides of A. baumannii isolate. Recently an intense sight of carbapenem resistance was discovered in A. baumannii isolate from the adeABC system such as class D carbapenemases, meropenem, and imipenem, and it remains a serious concern in clinical therapy [13] [56]. The ISAba1 insertion produced by the adeS mutation confers resistance over expression to tegecycline.

adeIJK
The second largest pump of the RND family's adeIJK also comprises of adeI, adeJ, adeK genes which occur on the three parts of the pump efflux structure respectively. AdeIJK was described initially in the years 2008 [5] [26] with the A. baumannii clinical strains fluctuating between 86% and 100% in a presence of the predominant gene adeJ. With various reported a MIC dimness of adeIJK mainly the resistance of A. baumannii to β-lactamines, lincosamides, fluoroquinolones, chloramphenicol, trimethoprime, and fusidic acid has been noticed [57]. The selection of the majority gene adeJ, will lead to an amplification in the sensitivity of chloramphenicol, macrolides, lincosamides, tetracyclines and quinolones and β-lactams [58] [59]. The regulation of adeIJK is less complex than that of adeABC, but at about 750 -850 kbp of adeIJK operon there is a regulator adeN belonging to the class of tetR [35]. The presence of this regulator adeN and mutation in different media led to an increase the resistance to antimicrobial drugs (ertapenem, aztreonam, tigecycline, meropenem, and minocycline) in A. baumannii [47]. Several studies have shown that the threshold of expression of adeIJK is lower than that of ABC, which indicated that the level of toxicity of adeIJK in the patient is well regulated [48] [52] [60]. It has been detected that adeIJK and adeABC have some similarity as the efflux of the same antibacterial drugs (fluoroquinolones, tetracyclines and chloramphenicol) [52] from A. baumannii and properties comparable to P. aeruginosa mexAB-OprM. Studies on production and regulation adeABC and adeIJK resulted in the formation of biofilms [36].

adeFGH
Outstanding variation of adeABC and adeIJK, has induced the discovery of adeFGH operon sometime after adeIJK identification. The presence of adeFGH in the genus A. baumannii through exposure to certain antibacterial agents (norfloxacin) [22] [61] and is also a true source of multidrug. The genes of the adeFGH operon, the adeG is the most representative of more than 80% of the others [37]. AdeFGH has also become popular in the species of A. baumannii due to its severe resistance to fluoro-quinolones, tetracyclines, tigecycline, chloramphenicol, trimethoprim, sulfamethoxazole and moderate resistance to eryt-F. T. D. Temgoua, L. Wu Journal of Biosciences and Medicines hromycin, rifampicin and aminoglycosides, [61] and also β-lactams. AdeFGH is regulated by LysR (LTTR), also called adeL. The adeL mutation will conduct to the adeFGH level rise. adeXYZ has also been found in A. baumannii genospecies 3 and has the same structure and positioning of MFP, OM, IM with propositions 80% (adeX), 89% (adeY) and 87% (adeZ) [39] [62] [63]. AdeXYZ and adeDE are also regularly present in A. baumannii GDG3 as opposed to GDG2 for ade IJK and adeABC. The engagement of adeXYZ in the resistance mechanism is not entirely described. Some research showed that the suppression or perturbation of adeFGH or of another in the RND system does not greatly modify the sensitivity to the antibiotics [2] [64].
It has been found that the A. baumannii (GDG3) gene and specific resistance to certain antibiotics (ceftazidime, tetracycline, amikacin, ciprofloxacin, erythromycin, rifampin, meropenem, chloramphenicol) [16] [26], is due to adeDE gene in A. baumannii chromosome, and have resistance to imipenem. Unlike to other efflux system (adeABC, adeIJK), the adeDE gene does not have an outer membrane [27] [32]. Previous studies have demonstrated inconsistency between adeDE and adeABC-adeIJK due to the presence of adeABC-adeIJK/inter 1-negative adeS in some isolates for the detection of adeE [65].

MFS Efflux Pumps
MFS is the subsequent most studied efflux mechanism in species A. baumannii have identified some genes cmlA, tet(A/B), craA, and floR as the most present and appertain to the superfamily MFS [66]. Plural research has explained a particularity of resistance caused by tetA and tetB [61]. These two genes are not involved in resistance to tigecycline, yet tetA leads to a tetracycline resistance while tetB induce the resistance to tetracyclin and minocyclin [67]. In A. baumannii isolate resistant to tetracycline, the overexpression rate of tetA is 30% -45% whereas tetB is 32% -72% [29] [67]. The cmlA gene of MFS is resistant to certain β-lactams, chloramphenicole, fluoroquinolones, tetracycline and rifampicin. craA is particularly resistant to chloranphenicol, imipenems, quinolones, aminoglycosides, and tetracyclines [68]. The MFS energy source is proton motive force (H + ) facilitating H + motive force inhibition to increase the sensitization of antimicrobial drugs [69]. Horizontal transmission was discovered by the association of two tetB-tetR genes in plasmid and the ISCR2 element of the MDR isolate. It has been reported that floR gene and cmlA gene were associate with abaR gene in A. baumannii chromosome [70] [71] [72].

MATE Efflux Pumps
The first and most frequent gene of the MATE family present in A. baumannii is the adeM gene. It represents between 63% -100% in MDR of A. baumannii [26] [71]. AdeM protein contains about 447 amino acids and multiform hydrophobic regions. The antimicrobial drugs resistant due to adeM gene are not related to adeABC and totally known. In certain studies it was noted that adeM is not as- have an implication of resistance in that of amino glycosides, trimethoprim, fluoroquinolones, erythrocin, and chloramphenicol. The MATE family is powered by double reservoir of energy PMF (motive force of the proton) and sodium ion gradient Na + [75]. This high energy source could be a particular reasons why adeM gene is seen as an important target for elaboration of efflux pumps inhibitory antibiotics that could help restores A. baumannii sensitization [76] [77].
The recapitulate of Efflux pumps family in A. baumannii and Antimicrobial drug target was listed in Table 1.

SMR Efflux Pumps
AdeS gene was characterized in 2009 initially in the efflux pump system by increasing the MIC (5 -6 times) on an E. coli strain for the resistance of novobiocin and erythromycin. AdeS gene is the main efflux pump of SMR family

ABC Efflux Pumps
ATP binding cassette (ABC) of super family efflux pumps are recognized to be censurable for multidrug-resistance due of P-glycoprotein (ABCB1) [80]. ABC

PACE Efflux Pumps
The proteo-bacterial antimicrobial compound efflux family (PACE) is uncommon of the newest families of efflux pumps identified in the latest 15 years [84].
PACE family described on plural gram-negative bacteria like E. coli, K. pneumoniae, Vibrio parahaemolyticus, Salmonella enterica, P. aeruginosa, Enterobactre cloacae, and serovar Typhi [36] [43] [85]. Its homologous aceI gene found to be resistant to chlorhexidine and its overexpression also lead to resistance to dequalinium, benzalkonium chloride, and acriflavine. In A. baumannii, aceI also induces the resistance of chlorhexidine and oxidants [85]. The aceI gene of PACE family could be the 6 th group of MDR efflux pumps [44].

Mechanisms of Transporters in the Efflux Pump Systems
According to structural and bioenergetics characteristics, carriers could be separated into two major groups [80] [86], 1) transporters that hydrolyze ATP as an energy source; they are also summons ABC transporters (ATP binding cassette) [81], and 2) transporters that use the proton H + (and/or Na + sodium MATE family) for energy source [81]. Transporter of proton is the main common conveyance present in gram-negative bacteria especially in multidrug resistant. The separate congregate and they are also responsible for transport of drugs, sugars, and intermediate metabolites [66]. Membrane proteins of the SMR family are engaged in the activity of lipophilic cationic drugs in A. baumannii [91]. These are the slightest known drug efflux proteins, with just 4 TMS predicted. They can function as either hetero-or homo-oligomeric complexes. Unlike pumps MFS families, RND and SMR, which act as anti-proton/anti-drug, the 12-TMS collapse pumps MATE family afresh recognize [108] are mainly anti-drugs Na + [90]. RND efflux systems presented a 3D structure of proteins of tripartite which is not totally understood by the configuration of these systems [26] [60]. It was also noted that RND pumps of A. baumannii and gram-negative a tripartite system. RND efflux protein are combined of 12 TMS including 2 large periplasmic which provide specify substrate [92] [93].

Mechanisms of Efflux Pumps Inhibitors (EPI)
In fighting bacterial resistance which has increased, it would be useful to employ inhibitors of resistance efflux pumps to restore the fundamental action of antibiotic. Efflux pumps are the newest bacterial resistance mechanism allowing resistance to almost all antibiotics [94]. Some molecules (chemical or natural) have the capacity to act specifically on the efflux system to restore the action of antibiotics and commonly called efflux pumps inhibitors (EPIs) [95]. In the A. baumannii species several chemical inhibitors have already been tested [96]. Only certain inhibitor has shown conclusive results but remains difficult to apply in clinical due to high levels of toxicity for the human organism [97] [98]. To discover adequate EPI, different strategies can be considered depending on the cause gene or the level of cellular resistance [99]. When MIC decrease eight-fold some antimicrobial drugs like levofloxacin, chloramphenicol, linezolid, ciprofloxacin, clarithromycin, tetracycline, and rifampicin, restores sensitivity to drugs with a density 100 μg/ml of the two EPIs, Figure 2. Various targets for inhibition of complex efflux pump (adapted from [6] and [102]).  [105].
In other studies, PAβN at 10 μg/ml decreased predominantly MIC concentrations of trimethoprim, clindamycin and chloramphenicol [97] [102]. Twice a time on clinical isolates, whereas PAβN at 20 μg/ml reduced nicidixic acid MIC to 16-fold but showed little effect on sensitiveness to ciprofloxacin [106]. At 100 μg/ml PAβN are also sensible minocycline activity by decreasing ≥ 04-fold MIC values [107]. Delightful, one study has propose a contradictory effect of NMP at 64 μg/ml on susceptibility to tetracyclines (i.e., increased susceptibility to minocycline, tetracycline, doxycycline) and tigecycline (reduced susceptibility) [107] [108] [109]. Presumably, the EPIs have powerful effect on resistivity reversal with molecules that have relatively acute MIC values such as clindamycin, chloramphenicol, linezolid rifampicin, trimethoprim clarithromycin [107] [108] [110]. Moreover, another study also examine the effect of phenothiazines, omeprazole (prochlorperazine, chlorpromazine, and promazine), verapamil and reserpine, on susceptibility cells with phenothiazines being the only emissary capable to re-establish sensibility to some antibiotics (≥8 time MIC decrease) [ [102]. Another lately kibdelomycin natural antibiotic was found, exhibits a broad-spectrum effect with the MIC 90 value of 0.125 facing A. baumannii [112], this agent appears to be a distressed substratum of efflux pumps. Finally, any agents that can traverse the OM of A. baumannii are expected to counter the activity of the efflux pumps in augmentation the drug ingress to their targets [114] [115]. In this regard, many plant extracts of EPI (in addition steroidal alkaloids conessine) are clever to break down the OM barrier to exert a synergistic efficacy on the amelioration of the activity of divers antimicrobials facing A. baumannii [116] [117] [118]. Natural efflux pump inhibitors (plant extracts): Biricodar and timcodar, Falvone, Berberis, Mahonia, Dalea versicolor, Lycopus europaeus, Rosmarinus officinalis, are the most common use against bacteria [116]. The analytical results of the natural inhibitor Rosmarinus officinalis and Lycopus europaeus have shown great efficacy on efflux pumps to restore the sensitivity of antibiotics against MDR strains of A. baumannii and P. aeruginosa [119]. The natural extract Geranium coespitosum, Punica granatum and Euphorbiaceae can inhibit the potentiating activity of strains MDR Staphylococcus aureus to restore the sensibility of erythromycin, fluoroquinolone, gentamicin, ampicillin, tetracycline, chloramphenicol [120] [121]. Extracts of Berberis aetnensis coming from volcano region can reduce the resistance of ciprofloxacin for P. aeruginosa, S. aureus, and E. coli [122] [123]. The natural inhibitors Mellisa officinalis, Daucus carota, Levisticum officinale, Glycyrrhiza glabra, has demonstrated a great activity facing S. tyhimuriun and K. pneumoniae by restore the sensibility of tetracycline, chloramphenicol and fluoroquinolones [94] [95]. these new type inhibitors could constitute a better and effective voice to resolve definitively the bacterial MDR problem (including A. baumannii). Therefore, control pharmacokinetic, pharmaco-dynamic complete and combined will give high efficacy and acceptable degree of toxicity.