Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options

Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of β-lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized.

Among Acinetobacter species, Acinetobacter baumannii is the most important member associated with hospital-acquired infections worldwide . This aerobic Gramnegative coccobacillus had been regarded as a low-grade pathogen, but it is a successful pathogen responsible for opportunistic infections of the skin, bloodstream, urinary tract, and other soft tissues (Peleg et al., 2008). Because many A. baumannii infections have suddenly been reported among veterans and soldiers who served in Iraq and Afghanistan (Centers for Disease and Prevention, 2004), A. baumannii is referred to as "Iraqibacter." Multidrug-resistant (MDR) A. baumannii has spread to civilian hospitals in part by crossinfection of injured military patients repatriated from war zones (Peleg et al., 2008). Most A. baumannii infections occur in critically ill patients in the intensive care unit (ICU) setting (Fournier and Richet, 2006) and account for up to 20% of infections in ICUs worldwide (Vincent et al., 2009). Furthermore, FIGURE 1 | Biology of Acinetobacter baumannii. Studies of virulence factors, pathogenesis, antimicrobial resistance, treatment options of A. baumannii will provide an important aid for discovering new antibiotics and determining efficient combination therapy, which are essential strategies for combating multidrug-resistant A. baumannii infections. the frequency of community-acquired A. baumannii infections has been increasing gradually . Several virulence factors have been identified by genomic and phenotypic analyses, including outer membrane porins, phospholipases, proteases, lipopolysaccharides (LPS), capsular polysaccharides, protein secretion systems, and iron-chelating systems (Antunes et al., 2011;McConnell et al., 2013;. Many reports have shown that A. baumannii rapidly develops resistance to antimicrobials, and multidrug-resistant strains have been isolated (McConnell et al., 2013). The WHO declared that A. baumannii is one of the most serious ESKAPE organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter species) that effectively escape the effects of antibacterial drugs (Boucher et al., 2009). A number of A. baumannii resistance mechanisms are known, including enzymatic degradation of drugs, target modifications, multidrug efflux pumps, and permeability defects Kim et al., 2012;. In this review, we summarize the virulence factors of A. baumannii, antibiotic resistance mechanisms, and the therapeutic options available for treating A. baumannii infections. Figure 1 depicts all the features described in this review.

ACINETOBACTER BAUMANNII VIRULENCE FACTORS AND PATHOGENESIS
Although recent genomic and phenotypic analyses of A. baumannii have identified several virulence factors responsible for its pathogenicity, relatively few virulence factors have been identified in A. baumannii, compared to those in other Gram-negative pathogens (McConnell et al., 2013). The proposed A. baumannii virulence factors are summarized in Table 1.

Porins
Porins are outer membrane proteins associated with modulating cellular permeability. OmpA is a β-barrel porin and one of the most abundant porins in the outer membrane. In A. baumannii, OmpA is the very well-characterized virulence factor with a variety of interesting biological properties identified in in vitro model systems (Smith et al., 2007;McConnell et al., 2013). A random mutagenesis screen showed that the A. baumannii ompA mutant is defective in inducing apoptosis in human epithelial cells (Choi et al., 2005). Purified OmpA binds host epithelial cells, targets mitochondria, and induces apoptosis by releasing proapoptotic molecules, such as cytochrome c and apoptosisinducing factor (Choi et al., 2005;Lee et al., 2010). Another study showed that OmpA translocates to the nucleus by a novel monopartite nuclear localization signal and induces cell death (Choi et al., 2008a). OmpA also plays a major role in adherence and invasion of epithelial cells by interacting with fibronectin (Choi et al., 2008b;Gaddy et al., 2009;Smani et al., 2012), and binds to factor H in human serum , which may allow A. baumannii to avoid complement-mediated killing. The Virulence factor ompA gene is necessary for persistence of A. baumannii in the mouse lung (Wang et al., 2014).
Furthermore, OmpA is also involved in antimicrobial resistance of A. baumannii (Sugawara and Nikaido, 2012;Smani et al., 2014). The major A. baumannii porin is OmpA, which has 70-fold lower pore-forming activity than that of OmpF (Sugawara and Nikaido, 2012). Furthermore, disrupting the ompA gene significantly decreases the minimal inhibitory concentrations (MICs) of several antibiotics (chloramphenicol, aztreonam, and nalidixic acid), suggesting that OmpA participates in the extrusion of antibiotics from the periplasmic space through the outer membrane and couples with inner membrane efflux systems (Smani et al., 2014). OmpA enhances survival and persistence of A. baumannii by facilitating surface motility and biofilm formation (Gaddy et al., 2009;Clemmer et al., 2011;McConnell et al., 2013). OmpA also regulates biogenesis of outer membrane vesicles (Moon et al., 2012). These results suggest that the OmpA protein is an attractive target for developing novel antibiotics and prevention strategies. Two recent reports based on immuno-proteomics and reverse vaccinology suggested that OmpA is a potential vaccine candidate against A. baumannii (Fajardo Bonin et al., 2014;Hassan et al., 2016). Actually, the OmpA protein is immunogenic in healthy individuals and patients with A. baumannii invasive infections . In a mouse model of A. baumannii infection, mice immunized with OmpA had a significantly higher survival rate than that of control mice (Luo et al., 2012;Zhang et al., 2016).
The 33-to 36-kDa Omp protein (Omp33-36), which acts as a water passage channel, is another outer membrane porin associated with A. baumannii cytotoxicity (Smani et al., 2013;Rumbo et al., 2014). The omp33-36 deletion strain significantly reduces adherence and invasion of human lung epithelial cells and cytotoxicity to these cells (Smani et al., 2013). Deletion of the omp33-36 gene in a murine sepsis model attenuates lethality and reduces bacterial concentrations in the spleen and lungs (Smani et al., 2013). One study showed that purified Omp33-36 induces apoptosis in several different cell types, including immune and connective tissue cells, by activating caspases and modulating autophagy (Rumbo et al., 2014). Omp33-36 is also involved in antibiotic resistance. A. baumannii strain JC10/01 resistant to carbapenem antibiotics (imipenem and meropenem) exhibits loss of Omp33-36 and episomal expression of Omp33-36 in this strain clearly reduces the MICs of imipenem and meropenem (del Mar Tomas et al., 2005).
Omp22 has also been identified as a novel, conserved, and safe antigen for developing effective vaccines to control A. baumannii infections , although the contribution of Omp22 to A. baumannii pathogenicity has not been determined. Both active and passive immunizations with Omp22 increase the survival rates of mice, suppress bacterial burdens in the organs and peripheral blood, and reduce serum levels of inflammatory cytokines and chemokines . Other porins, such as carbapenemassociated outer membrane protein (CarO) and OprD-like, are also virulence-related factors associated with attenuated virulence in a mouse model (Fernandez-Cuenca et al., 2011).

Capsular Polysaccharides and Lipopolysaccharides (LPS)
Beyond OmpA, the A. baumannii envelope is associated with many factors that contribute to pathogenicity. Among these, capsular exopolysaccharides and LPS are A. baumannii pathogenicity factors. Notably, many isolates from patients with A. baumannii infections express surface capsular polysaccharides and contain a conserved gene cluster, called the K locus, which may determine production of capsular polysaccharides (Koeleman et al., 2001;Hu et al., 2013;Kenyon and Hall, 2013;Geisinger and Isberg, 2015). A random transposon screening to identify genes essential for growth in an inflammatory exudative fluid lead to the identification of the ptk and epsA genes, which are predicted to be required for capsule polymerization and assembly . The ptk and epsA mutants are deficient in capsule production and have a growth defect in human serum, resulting in a highly significant decrease in survival in soft tissue infection sites . Mutation in the pglC or pglL gene, which is responsible for synthesis of the O-pentasaccharide found on glycoproteins and capsular polysaccharides, also attenuate lethality in a mouse septicemia model and form abnormal biofilm structures (Iwashkiw et al., 2012;Lees-Miller et al., 2013). Therefore, capsular polysaccharides have been proposed to be a target for protective antibody-based interventions (passive immunization; Russo et al., 2013).
One study showed that capsular polysaccharides are involved in antimicrobial resistance of A. baumannii (Geisinger and Isberg, 2015). Mutants deficient in capsular polysaccharides have lower intrinsic resistance to peptide antibiotics. In addition, the presence of antibiotics induces hyperproduction of capsular polysaccharides (Geisinger and Isberg, 2015). Antibiotic-induced production of capsular polysaccharides increases resistance to killing by host complement and increases virulence in a mouse model of systemic infection (Geisinger and Isberg, 2015). That study also demonstrated that increased capsule production after exposure to an antibiotic depends on transcriptional increases in K locus gene expression, and that expression of K locus genes is regulated by the bfmRS two-component regulatory system (Geisinger and Isberg, 2015). bfmR is a gene essential for growth in human ascites, which is an ex vivo medium that reflects the infection environment (Umland et al., 2012), and is important for persistence in the lung in a murine pneumonia model (Wang et al., 2014). BfmS is also a virulence factor that plays an important role in biofilm formation, adherence to eukaryotic cells, and resistance to human serum . On report showed BfmR-mediated resistance to complementmediated bactericidal activity and resistance to the clinically important antimicrobials (meropenem and colistin; Russo et al., 2016). However, that study suggested that BfmR effects are independent of capsular polysaccharide production. Therefore, the relationship between BfmRS and capsular polysaccharides must be described in more detail.
LPS is the major component of the outer leaflet of the outer membrane in most Gram-negative bacteria and is an immunoreactive molecule that induces release of tumor necrosis factor and interleukin 8 from macrophages in a Toll-like receptor 4 (TLR4)-dependent manner (Erridge et al., 2007). LPS is composed of an endotoxic lipid A moiety, an oligosaccharide core, and a repetitive O-antigen (Lee et al., 2013b). In A. baumannii, LPS plays a major role in virulence and survival of A. baumannii Lin et al., 2012;McQueary et al., 2012). Mutant cell lacking LpsB glycotransferase have a highly truncated LPS glycoform containing only two carbohydrate residues bound to lipid A, resulting in decreased resistance to human serum and decreased survival in a rat model of soft tissue infection McConnell et al., 2013). Inhibiting LpxC, an enzyme involved in the lipid A biosynthesis, dose not inhibit growth of the bacterium, but suppresses A. baumannii LPS-mediated activation of TLR4 . Inhibition of LpxC in mouse model enhances clearance of A. baumannii by enhancing opsonophagocytic killing and reduces serum LPS concentration and inflammation, which completely protects mice from lethal infection Lee et al., 2013b). These results indicate that blocking LPS synthesis is a powerful strategy for discovering novel antibiotics. Modification of LPS contributes to resistance to antimicrobials. Many studies have shown that modifications in LPS decrease the susceptibility of A. baumannii to many clinical important antibiotics, such as colistin (Moffatt et al., 2010;Arroyo et al., 2011;Beceiro et al., 2011;Pelletier et al., 2013;Boll et al., 2015;Chin et al., 2015).

Phospholipase
Phospholipase is a lipolytic enzyme essential for phospholipid metabolism and is a virulence factor in many bacteria, such as P. aeruginosa, Legionella monocytogenes, and Clostridium perfringens (Camarena et al., 2010;Flores-Diaz et al., 2016). Three classes of phospholipases, such as phospholipase A (PLA), phospholipase C (PLC), and phospholipase D (PLD) have been defined based on the cleavage site. PLA hydrolyzes fatty acids from the glycerol backbone, whereas PLC cleaves the phosphorylated head group from the phospholipid. PLD is a transphosphatidylase that only cleaves off the head group. Degradation of phospholipids affects the stability of host cell membranes, and the cleaved head group can interfere with cellular signaling, resulting in changes in the host immune response (Songer, 1997;Flores-Diaz et al., 2016). PLC and PLD have been identified as virulence factors in A. baumannii (Camarena et al., 2010;Jacobs et al., 2010;Stahl et al., 2015). Acinetobacter baumannii ATCC17978 has two PLCs (A1S_0043 and A1S_2055) and inactivation of the A1S_0043 gene leads to a modest reduction in the cytotoxic effect of A. baumannii on epithelial cells compared to that of the parental strain (Camarena et al., 2010;Fiester et al., 2016). Disrupting one (A1S_2989) of the two PLD genes present in A. baumannii strain 98-37-09 results in reduced resistance to human serum, decreased capacity for invading epithelial cells, and decreased virulence in a murine model of pneumonia . Another report showed that A. baumannii ATCC 19606 has three PLD genes and all three play important roles in virulence and host cell invasion in a concerted manner (Stahl et al., 2015). These results suggest that phospholipase enzymes are important virulence factors in A. baumannii pathogenesis.

Outer Membrane Vesicles (OMVs)
OMVs are spherical, 20-200 nm diameter vesicles secreted by the outer membranes of various Gram-negative pathogenic bacteria (Kulp and Kuehn, 2010). They are composed of LPS, outer membrane and periplasmic proteins, phospholipids, and DNA or RNA, and are recognized as delivery vehicles for bacterial effectors to host cells (Ellis and Kuehn, 2010). OMVs deliver diverse virulence factors to the interior of host cells simultaneously and allow the pathogens to interact with the host without close contact between bacteria and host cells (Jun et al., 2013). Many A. baumannii strains secrete OMVs containing various virulence factors, including OmpA (Kwon et al., 2009;Jin et al., 2011;Moon et al., 2012), proteases (Kwon et al., 2009), and phospholipases (Kwon et al., 2009). OMVs derived from A. baumannii interact with host cells and deliver bacterial effectors to host cells via lipid rafts, resulting in cytotoxicity (Jin et al., 2011). Purified OMVs of A. baumannii ATCC 19606 induce expression of pro-inflammatory cytokine genes in epithelial cells in a dose-dependent manner (Jun et al., 2013). Notably, OMVs treated with proteinase do not induce a significant increase in the expression of pro-inflammatory cytokine genes, suggesting that the membrane proteins in OMVs are responsible for eliciting a potent innate immune response (Jun et al., 2013). One study supports the role of OMVs in A. baumannii pathogenesis. An A. baumannii strain that produces abundant OMVs with more virulence factors induces a stronger innate immune response and is more cytotoxic compared with those of a strain producing fewer OMVs (Li Z. T. et al., 2015).
Due to the importance of OMVs in A. baumannii virulence, several reports have shown that A. baumannii OMVs could be used as an acellular vaccine to elevate protective immunity (McConnell et al., 2011;Huang et al., 2014). In a mouse model of disseminated sepsis, vaccination with A. baumannii ATCC 19606 strain OMVs protects mice from challenge with homologous bacteria and provides protection against other clinical isolates (McConnell et al., 2011). Similar results were obtained in a pneumonia mouse model. Bacterial burden, inflammatory cell infiltration, and inflammatory cytokine accumulation in the pneumonia model were significantly suppressed by both active and passive immunization with OMVs (Huang et al., 2014). These results indicate that A. baumannii OMVs can be used as an acellular vaccine to effectively control A. baumannii infections. Interestingly, A. baumannii OMVs are also related with the spread of antibiotic resistance and induce the horizontal transfer of the OXA-24 carbapenemase gene .

Metal Acquisition System
Although iron is one of the most abundant elements in environmental and biological systems, ferric iron is relatively unavailable to bacteria in the preferred state, because of its poor solubility (10 −17 M solubility limit for ferric iron) under aerobic and neutral pH conditions as well as due to chelation by lowmolecular-weight compounds, such as heme, or high-affinity iron-binding compounds, such as lactoferrin and transferrin (Rakin et al., 2012;Saha et al., 2013). To overcome this iron limitation, most aerobic bacteria produce a high-affinity iron chelator known as a siderophore (Saha et al., 2013). Siderophores are low molecular weight compounds (400-1,000 kDa) with high affinity for iron. The range of Fe 3+ -siderophore association constants is 10 12 -10 52 (Saha et al., 2013). Siderophores have been classified into catecholates, hydroxymates, and a mixed type based on the moiety that donates oxygen ligands to coordinate Fe 3+ (Saha et al., 2013). Acinetobacter baumannii also has iron siderophores and acinetobactin, the best-characterized A. baumannii siderophore, is a mixed type siderophore with an oxazoline ring derived from threonine (McConnell et al., 2013). Acinetobactin is an A. baumannii virulence factor (Gaddy et al., 2012;Penwell et al., 2012;Megeed et al., 2016). Impaired acinetobactin biosynthesis and transport functions significantly reduce the ability of A. baumannii ATCC 19606 cells to persist within epithelial cells and cause cell damage and animal death (Gaddy et al., 2012). Mutation in the entA gene, which is essential for biosynthesis of the acinetobactin precursor 2,3dihydroxybenzoic acid, also significantly reduces the capacity of A. baumannii ATCC 19606 cells to persist within human alveolar epithelial cells and diminishes the ability to infect and kill Galleria mellonella larvae . One study showed that acinetobactin production occurs significantly more frequently in MDR A. baumannii isolates than that in avirulent isolates (Megeed et al., 2016).
The A. baumannii NfuA Fe-S scaffold protein, that participates in the formation of Fe-S clusters and plays a role in cell responses to iron chelation and oxidative stress, has also been identified as a virulence factor (Zimbler et al., 2012). The nfuA mutant is more sensitive to reactive oxygen species (ROS), such as hydrogen peroxide and cumene hydroperoxide, and shows significantly reduced growth in human epithelial cells. In addition, a G. mellonella infection model showed that more than 50% of injected G. mellonella larvae die 6 days after infection with the parental strain, whereas less than 30% of the larvae die when infected with the nfuA mutant (Zimbler et al., 2012). One report showed that iron starvation increases production of PLCs, which increase hemolytic activity of A. baumannii (Fiester et al., 2016). These reports indicate that iron acquisition functions play a critical role in A. baumannii virulence.
The innate immune metal-chelating protein calprotectin inhibits bacterial growth by host-mediated chelation of metals, such as zinc (Zn 2+ and Zn) and manganese (Mn 2+ and Mn) (Corbin et al., 2008). However, A. baumannii can cause disease in the presence of this nutritional immune protein in vivo (Juttukonda et al., 2016). To combat the zinc limitation, A. baumannii uses a zinc acquisition system (ZnuABC), which is up-regulated under Zn-limiting conditions, and the znuB mutant strain experiences Zn starvation at higher Zn concentrations than that of the wild-type (Hood et al., 2012). ZnuB contributes to the pathogenesis of A. baumannii pulmonary infections. Notably, a zinc limitation reduces the imipenem MIC of MDR A. baumannii to below the clinical breakpoint for imipenem resistance in A. baumannii (Hood et al., 2012), possibly because many carbapenemases are metalloenzymes that require Zn for their hydrolyzing activity. Besides the ZnuABC system, the novel Zn metallochaperone ZigA has been characterized in A. baumannii (Nairn et al., 2016). ZigA tightly interacts with Zn, which is required for bacterial growth under Zn starvation conditions and for disseminated infection in mice (Nairn et al., 2016).
The mechanism employed by A. baumannii to overcome a Mn limitation has been identified. Calprotectin induces Mn starvation in A. baumannii, which increases transcription of an NRAMP (Natural Resistance-Associated Macrophage Proteins) family Mn transporter and a urea carboxylase to resist the antimicrobial activities of calprotectin (Juttukonda et al., 2016). A urea carboxylase enzyme (MumC) is important for growth of A. baumannii in the presence of calprotectin and an NRAMP family transporter (MumT) contributes to the fitness of A. baumannii in a murine pneumonia model (Juttukonda et al., 2016), suggesting that the two proteins are virulence factors. Acinetobacter baumannii can utilize urea as a sole nitrogen source, and this urea utilization is required for MumC (Juttukonda et al., 2016). Based on the contribution of MumC to A. baumannii resistance to calprotectin, the authors suggest a connection between metal starvation and metabolic stress, such as nitrogen starvation.

Protein Secretion Systems
Several protein secretion systems have been identified in A. baumannii (Weber et al., 2015a). The most recently described A. baumannii secretion system is a type II secretion system (T2SS) (Johnson et al., 2015). The T2SS is a multi-protein complex that is structurally very similar to type IV pili systems, which is an appendage that is commonly found in Gram-negative bacteria (Korotkov et al., 2012). T2SS translocates a wide range of proteins from the periplasmic space to the extracellular milieu out of the cell or the outer membrane surface. The T2SS is composed of 12-15 proteins comprised of four subassemblies: a pseudopilus, a cytoplasmic secretion ATPase, an inner-membrane platform assembly, and a dodecameric outermembrane complex (Korotkov et al., 2012;Harding et al., 2016). Secretion by T2SS is a two-step process. The target proteins are first translocated to the periplasm by the general secretory (Sec) system or the twin arginine transport (Tat) system, where the target proteins are then secreted out of the cell through the T2SS (Korotkov et al., 2012). Deleting A. baumannii genes for the T2SS components, gspD or gspE, results in loss of LipA secretion, indicating that LipA is a T2SS substrate (Johnson et al., 2015). Because LipA is a lipase that breaks down long-chain fatty acids, lipA, gspD, and gspE mutant strains are incapable of growing on long-chain fatty acids as a sole carbon source and are defective in in vivo growth in a neutropenic murine model of bacteremia (Johnson et al., 2015). The role of a functional T2SS for full virulence of A. baumannii has been shown in G. mellonella and murine pulmonary infection models (Harding et al., 2016). Lipases (LipA, LipH, and LipAN) and the metallopeptidase CpaA have been identified as T2SS substrates (Elhosseiny et al., 2016;Harding et al., 2016). Notably, two proteins (LipA and CpaA) among these secreted proteins require specific chaperones for secretion. These chaperones are encoded adjacently to their cognate effector, and their inactivation abolishes secretion of LipA and CpaA (Harding et al., 2016).
Acinetobacter baumannii also has a type VI secretion system (T6SS). The T6SS was first identified in Vibrio cholera and P. aeruginosa (Mougous et al., 2006;Pukatzki et al., 2006). Many bacteria use the T6SS to inject effector proteins, providing a colonization advantage during infection of eukaryotic hosts (Mougous et al., 2006) or to kill competing bacteria . The T6SS leads to DNA release and horizontal gene transfer in V. cholera, which may contribute to spread of antibiotic resistance (Borgeaud et al., 2015). The T6SS is composed of many conserved structural proteins and accessory factors, and bears a contractile bacteriophage sheath-like structure forming a needle or spike structure used to penetrate the target cell (Shneider et al., 2013). Hcp is a structural protein forming a polymerized tubular structure that is secreted out of the cell, and VgrGs are involved in attaching effector domains to the spike, and a proline-alanine-alanine-arginine (PAAR) repeat protein forms the sharp tip of the distinctive needle-like structure (Shneider et al., 2013;Zoued et al., 2014).
The presence of T6SS in A. baumannii was initially predicted by bioinformatic analysis (Weber et al., 2013). Although the role of T6SS in A. baumannii ATCC 17978 has not been determined (Weber et al., 2013), research on A. baumannii strain M2 showed that this strain produces a functional T6SS and that the T6SS mediates killing of competing bacteria (Carruthers et al., 2013). Another study showed that the T6SS is active in six pathogenic strains of A. baumannii . However, the T6SS seems to play an important role in A. baumannii virulence in a strain-specific manner (Repizo et al., 2015). They compared T6SS functionality of several A. baumannii strains, including ATCC17978 (a type strain), various MDR strains implicated in hospital outbreaks (Ab242, Ab244, and Ab825), and DSM30011 (a non-clinical isolate). Although the T6SS genomic locus is present in all of these strains, only DSM30011 has a fully active T6SS that mediates E. coli killing (Repizo et al., 2015). In addition, the T6SS of DSM30011 is required for host colonization of the G. mellonella model organism (Repizo et al., 2015). Similar results were obtained from a comparative analysis of the genomes of MDR A. baumannii clinical strains (Wright et al., 2014;Jones et al., 2015). A. baumannii isolates of a particular clade exhibit complete loss of the T6SS genomic locus. Therefore, these results suggest that more extensive investigations are required to analyze the role of T6SS in A. baumannii virulence, even though this system seems to play an important role in A. baumannii virulence in some strains. Notably, one study showed that several MDR A. baumannii strains have a large, selftransmissible plasmid that carries negative regulators for T6SS (Weber et al., 2015b). The T6SS is silenced in plasmid-containing, antibiotic-resistant cells, whereas plasmid-losing cells have an active T6SS. Although plasmid-losing cells are capable of T6SSmediated killing of competing bacteria, they become susceptible to antibiotics (Weber et al., 2015b). This result suggests a molecular switch between T6SS and antibiotic resistance. The type V system autotransporter Ata has also been characterized in A. baumannii (Bentancor et al., 2012a). This is a trimeric membrane protein that mediates biofilm formation, adherence to extracellular matrix components such as collagen I, III, and IV, and virulence in a murine systemic model of Acinetobacter infection (Bentancor et al., 2012a). Another experiment using a pneumonia model of infection in immunocompetent and immunocompromised mice showed that Ata is a vaccine candidate against A. baumannii infections (Bentancor et al., 2012b). A type IV secretion system present in the plasmid was bioinformatically identified in A. baumannii , but no experimental evidence describing its function has been presented.
Penicillin-Binding Protein 7/8 (PBP7/8) and β-Lactamase PER-1 Although PBPs are commonly involved in resistance to βlactam antibiotics, PBP7/8 encoded by the pbpG gene is a virulence factor in A. baumannii. The pbpG mutant strain grows similar to its wild-type strain in Luria-Bertani medium, but the mutant shows reduced growth in human serum and its survival significantly decreases in rat soft-tissue infection and pneumonia models (Russo et al., 2009). An investigation of bacterial morphology using electron microscopy suggested that loss of PBP7/8 may have affected peptidoglycan structure, which may affect susceptibility to host defense factors (Russo et al., 2009).

Others
Acinetobacter baumannii CipA is a novel plasminogen binding and complement inhibitory protein that mediates serum resistance (Koenigs et al., 2016). CipA-binding plasminogen is converted to active plasmin that degrades fibrinogen and complement C3b, which contributes to serum resistance of A. baumannii. Therefore, the cipA mutant strain is efficiently killed by human serum and also shows a defect in the penetration of endothelial monolayers (Koenigs et al., 2016). Similar to CipA, the A. baumannii translation elongation factor Tuf is also a plasminogen-binding protein. Tuf-binding plasminogen can be converted to active plasmin, which proteolytically degrades fibrinogen as well as component C3b (Koenigs et al., 2015). RecA, which is involved in homologous recombination and the SOS response, has been identified as an A. baumannii virulence factor. The recA mutant shows significantly reduced survival within macrophages and decreases lethality in a mouse model of systemic infection . The surface antigen protein 1 (SurA1) plays an important role in fitness and virulence of A. baumannii (Liu D. et al., 2016). Serum resistance of the surA1 mutant significantly decreases compared with that of the wild-type strain CCGGD201101. In the G. mellonella insect model, a surA1 mutant strain exhibits a lower survival rate and decreased dissemination (Liu D. et al., 2016).
A growth analysis of 250,000 A. baumannii transposon mutants within G. mellonella larvae identified 300 genes required for survival or growth of A. baumannii inside G. mellonella larvae (Gebhardt et al., 2015). The 300 genes were classified into six categories of micronutrient acquisition, cysteine metabolism/sulfur assimilation, aromatic hydrocarbon metabolism, cell envelope/membrane/wall, stress response genes, antibiotic resistance, and transcriptional regulation. Among them, four transcriptional regulators required for growth in G. mellonella larvae were called the gig (growth in Galleria) genes. Loss of these genes (gigA-D) led to a significant defect in both growth within and killing of G. mellonella larvae (Gebhardt et al., 2015). This study identified stress proteins, such as UspA, as factors required for growth in G. mellonella. Another study showed that UspA is essential for pneumonia and sepsis pathogenesis of A. baumannii (Elhosseiny et al., 2015). Among the 300 genes, several genes are involved in aromatic hydrocarbon metabolism (Gebhardt et al., 2015). Another study showed that GacS, which is a transcriptional factor that regulates expression of genes, such as paaE, and is responsible for the phenylacetic acid catabolic pathway, affects A. baumannii virulence . Experiments using a paaE deletion mutant confirmed the role of aromatic hydrocarbon metabolism in A. baumannii virulence , but its molecular mechanism remains unknown. Interestingly, a recent report showed that accumulation of phenylacetate in A. baumannii induces rapid neutrophil influx to a localized site of infection and increases bacterial clearance (Bhuiyan et al., 2016). They suggested that phenylacetate is a neutrophil chemoattractant inducing bacterialguided neutrophil chemotaxis. This report may reveal a novel molecular mechanism about the role of the phenylacetic acid catabolic pathway in A. baumannii virulence.
Biofilm formation plays an important role in immune evasion by A. baumannii (de Breij et al., 2010), and pili are essential for A. baumannii adherence to and biofilm formation on abiotic surfaces as well as virulence (Tomaras et al., 2003(Tomaras et al., , 2008. Notably, imipenem treatment of the imipenem-resistant A. baumannii isolate induces expression of important genes responsible for synthesis of type IV pili (Dhabaan et al., 2015), suggesting that the ability to overproduce pili confers a biological advantage to A. baumannii.

ANTIMICROBIAL RESISTANCE OF A. BAUMANNII
Acinetobacter baumannii has become one of the most successful pathogens in modern healthcare because of its amazing ability to acquire antimicrobial resistance. Several strains of A. baumannii are highly resistant to most clinically available antibiotics . A. baumannii has a number of resistance mechanisms, including β-lactamases, aminoglycoside-modifying enzymes, efflux pumps, permeability defects, and modifications of target sites. The accumulation of several resistance mechanisms in A. baumannii has gradually decreased the number of antibiotic classes available to treat A. baumannii infections in clinical practice. Table 2 shows the antibiotic resistance mechanisms found in A. baumannii. We will discuss the details below.

β-Lactamases
Inactivation of β-lactams by β-lactamases is a major antibiotic resistance mechanism in A. baumannii. Based on sequence homology, β-lactamases are grouped into molecular classes, A, B, C, and D (Jeon et al., 2015). All four classes of βlactamases were identified in A. baumannii. Recent studies have shown that A. baumannii has natural competence to incorporate exogenous DNA and its genome has foreign DNA at high frequencies, implying frequent horizontal gene transfer in this pathogen (Ramirez et al., 2010a;Touchon et al., 2014;Traglia et al., 2014). Additionally, albumin, a main protein in blood, enhances natural competence of A. baumannii (Traglia et al., 2016). Therefore, natural competence of A. baumannii may contribute to identification of a large number of β-lactamases in this threatening human pathogen.
Unlike the serine-dependent β-lactamases (classes A, C, and D), class B β-lactamases are metallo-β-lactamases (MBLs) that require zinc or another heavy metal for catalysis (Jeon et al., 2015). Due to a broad substrate spectrum, MBLs catalyze the hydrolysis of virtually all β-lactam antibiotics including carbapenems, but not monobactams (Jeon et al., 2015). A variety of class B β-lactamases have been identified in A. baumannii ( Table 2).

Efflux Pumps
Efflux pumps are associated with resistance against many different classes of antibiotics, such as imipenem (Hu et al., 2007) and tigecycline (Peleg et al., 2007;Ruzin et al., 2007), in A. baumannii. Reversal of antibiotic resistance by efflux pump inhibitors, such as 1-(1-naphthylmethyl)-piperazine and carbonyl cyanide 3-chlorophenyl-hydrazone, supports the importance of efflux pumps in A. baumannii antibiotic resistance (Pannek et al., 2006;Deng et al., 2014). Four categories of efflux pumps, such as the resistance-nodulation-division superfamily, the multidrug and toxic compound extrusion family, the major facilitator superfamily, and the small multidrug resistance family transporters, are related to antimicrobial resistance in A. baumannii ( Table 2; . AdeABC in the resistance-nodulation-division superfamily is associated with aminoglycoside resistance (Magnet et al., 2001) and with decreasing susceptibility to tigecycline (Ruzin et al., 2007) and non-fluoroquinolone antibiotics (Higgins et al., 2004). AdeABC seems to be cryptic in wild-type A. baumannii because of stringent control by the AdeRS twocomponent system (Marchand et al., 2004), but point mutations or insertion of the ISAba1 sequence in the adeS gene leads to overexpression of AdeABC (Marchand et al., 2004;Sun et al., 2012Sun et al., , 2016Hammerstrom et al., 2015). Cell density (Fernando and Kumar, 2012) and the BaeSR two-component system (Lin et al., , 2015, which is involved in an envelope stress response, also seem to regulate transcription of the adeA gene and thus affect tigecycline susceptibility. Other resistancenodulation-division type efflux pumps, such as AdeFGH and AdeIJK, are synergistically associated with tigecycline resistance (Damier-Piolle et al., 2008). AdeFGH and AdeIJK expression is regulated by the LysR-type transcriptional regulator AdeL and the TetR-type transcriptional regulator AdeN (Coyne et al., 2010;Rosenfeld et al., 2012).
Acinetobacter baumannii clinical isolates possess a strong ability to form biofilms (Rodriguez-Bano et al., 2008). Notably, the subinhibitory concentrations of antibiotics encountered by low-dose therapy seem to strongly induce biofilm formation (Kaplan, 2011). A recent result revealed the mechanism. Overexpression of the AdeFGH efflux pump by low-dose antimicrobial therapy increases the synthesis and transport of autoinducer molecules, which induce biofilm formation (He X. et al., 2015). These results suggest a link between low-dose antimicrobial therapy and a high risk for biofilm infections caused by A. baumannii.
CmlA and CraA are major facilitator superfamily efflux pumps related with chloramphenicol Roca et al., 2009), and TetA is associated with tetracycline resistance (Ribera et al., 2003a). The novel efflux pump AmvA mediates resistance to different classes of antibiotics, disinfectants, detergents, and dyes, such as erythromycin, acriflavine, benzalkonium chloride, and methyl viologen (Rajamohan et al., 2010). AbaF was recently identified as a novel efflux pump associated with fosfomycin resistance (Sharma et al., 2016).
AbeM is in the multidrug and toxic compound extrusion family and confers resistance to imipenem and fluoroquinolones (Su et al., 2005). AbeS is the small multidrug resistance family transporter and affects resistance to various antimicrobial compounds. Deletion of the abeS gene results in increased susceptibility to various antimicrobial compounds, such as chloramphenicol, nalidixic acid, and erythromycin (Srinivasan et al., 2009). Some other efflux pumps, such as MacAB-TolC (Kobayashi et al., 2001) and EmrAB-TolC (Lomovskaya and Lewis, 1992), have been well described in E. coli, but their role in A. baumannii has been recently explored. The EmrAB-TolC efflux pump is also present in A. baumannii where it conferred resistance to netilmicin, tobramycin, and imipenem (Nowak-Zaleska et al., 2016). Another report identified three novel efflux pumps (A1S_1535, A1S_2795, and ABAYE_0913) in A. baumannii using multiplexed phenotypic screening . A1S_1535 confers resistance to various antibiotics, including gentamicin, kanamycin, chloroxylenol, oxytetracycline, 1,10-phenanthroline, and chloramphenicol . A1S_2795 is the first major facilitator superfamily efflux pump found to confer resistance to the sulphonamide sulfathiazole, and ABAYE_0913 is associated with resistance to chloramphenicol and fusidic acid .

Permeability Defects
A change in envelope permeability can influence antibiotic resistance. For example, porins form channels that allow transport of molecules across the outer membrane and play a significant role in A. baumannii virulence ( Table 1). Because porins affect membrane permeability, they also play a significant role in the mechanism of resistance. Reduced expression of some porins, including CarO (Mussi et al., 2005(Mussi et al., , 2007 (Quale et al., 2003), Omp43 (Dupont et al., 2005), Omp44 (Quale et al., 2003), and Omp47 (Quale et al., 2003), is associated with carbapenem resistance in A. baumannii. Loss of Omp29 in A. baumannii producing OXA-51-like or OXA-23-like carbapenemases results in increased imipenem resistance (Jeong et al., 2009;Fonseca et al., 2013). OmpA is also related with resistance to aztreonam, chloramphenicol, and nalidixic acid (Smani et al., 2014). One study showed that OmpA and CarO physically interact with OXA-23 carbapenemase, and these interactions are associated with antibiotic resistance (Wu et al., 2016). These results provide a novel view to increase understanding of bacterial antibiotic resistance mechanisms.
Besides outer membrane proteins, envelope components, such as LPS and peptidoglycans, also affects antibiotic resistance of A. baumannii. Loss or modification of LPS decreases membrane integrity and increases colistin resistance in A. baumannii (Adams et al., 2009;Moffatt et al., 2010).

Aminoglycoside-Modifying Enzymes
Aminoglycoside-modifying enzymes are the major mechanism by which A. baumannii confers resistance to aminoglycosides. Aminoglycoside-modifying enzymes can be classified into acetyltransferases, adenyltransferases, and phosphotransferases. These enzymes are typically present on transposable elements and are transferred among pathogenic bacteria . Several reports show that many MDR A. baumannii isolates produce a combination of aminoglycoside-modifying enzymes (Gallego and Towner, 2001;Nemec et al., 2004). A study from China identified a MDR A. baumannii strain carrying four aminoglycoside-modifying enzymes (Zhu et al., 2009). Another study from Greece reported that all A. baumannii strains contain aminoglycoside-modifying enzymes (Ploy et al., 1994), indicating the high prevalence of these enzymes in A. baumannii.

Alteration of Target Sites
Modifications in antibiotic target sites for antibiotics can induce antibiotic resistance in A. baumannii. In the absence of other known resistance mechanisms, only overexpression of altered PBPs with a low affinity for imipenem induce imipenem resistance (Gehrlein et al., 1991). Quinolone resistance is associated with modifications in GyrA (one subunit of DNA gyrase) and ParC (one subunit of topoisomerase IV) in epidemiologically unrelated A. baumannii isolates (Vila et al., 1995). Acinetobacter baumannii TetM, which has 100% homology with S. aureus TetM, has been proposed to be associated with tetracycline resistance through ribosomal protection (Ribera et al., 2003b). Similar to other pathogenic bacteria, dihydrofolate reductases (DHFR and FolA) responsible for trimethoprim resistance have been found in nosocomial MDR A. baumannii isolates (Mak et al., 2009;Lin M. F. et al., 2013;Taitt et al., 2014). The 16S rRNA methylase ArmA responsible for aminoglycoside resistance is also found in many A. baumannii strains and always coexists with OXA type carbapenemases such as OXA-23 (Yu et al., 2007;Cho et al., 2009;Karthikeyan et al., 2010;Brigante et al., 2012;Hong et al., 2013;Bakour et al., 2014;Tada et al., 2014;Hasani et al., 2016). As described above, many studies have shown that modifications or/and loss of LPS decrease the susceptibility of A. baumannii to many clinical important antibiotics, such as colistin.

Others
AdeABC is associated with decreased susceptibility to tigecycline (Ruzin et al., 2007). However, some clinical isolates without overexpressed AdeABC, AdeFGH, and AdeIJK have decreased susceptibility to tigecycline. Several reports have suggested the mechanism. One study analyzed eight A. baumannii clinical isolates and revealed that the deletion mutation in the trm gene, which encodes S-adenosyl-L-methionine-dependent methyltransferase, decreases susceptibility to tigecycline (Chen et al., 2014). The same result was reported using a highly efficient and versatile genome-editing platform enabling markerless modification of the A. baumannii genome. Deletion of AdeR, a transcription factor that regulates AdeABC efflux pump expression in tigecycline-resistant A. baumannii, reduces the MIC of tigecycline. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after the adeR deletion according to a highly efficient and versatile genome-editing platform (Trebosc et al., 2016). Whole-genome sequencing in two tigecycline-resistant adeR deletion strains revealed that a mutation in the trm gene makes the adeR mutant resistant to tigecycline. In addition, a trm disruption was identified in most tigecycline-resistant clinical isolates (Trebosc et al., 2016). However, its exact mechanism was not determined. Another study revealed that a frameshift mutation in plsC, encoding 1acyl-sn-glycerol-3-phosphate acyltransferase, is associated with decreased susceptibility to tigecycline .
The abrp gene, which encodes the peptidase C13 family, is associated with decreased susceptibility to tetracycline, minocycline, doxycycline, tigecycline, chloramphenicol, and fosfomycin . Deletion of abrp increases cell membrane permeability, displays slower cell growth rate, and confers reduced susceptibility to these antibiotics . However, its exact mechanism was not determined. Some genes involved in cell division, including blhA, zipA, zapA, and ftsK, are associated with intrinsic β-lactam resistance in A. baumannii (Knight et al., 2016).
Increased expression of mutagenesis-related genes, such as the SOS response genes, is a well-understood mechanism of E. coli and other bacteria to obtain antibiotic resistance (Cirz and Romesberg, 2007). Acinetobacter baumannii also seems to have an inducible DNA damage response in which RecA plays a major regulatory role and seems to acquire antibiotic resistances under clinically relevant DNA-damaging conditions (Aranda et al., , 2014Norton et al., 2013). Furthermore, RecA is involved in the A. baumannii pathogenicity .

PROSPECTIVE TREATMENT OPTIONS
Although carbapenems are effective antibiotics to treat A. baumannii infections (Cisneros and Rodríguez-Baño, 2002;Turner et al., 2003), the rate of carbapenem-resistant A. baumannii isolates has been increasing gradually (Mendes et al., 2010;Kuo et al., 2012;. Only a few effective antibiotic options are available to treat MDR A. baumannii infections (Gordon and Wareham, 2009;. To combat MDR or pandrugresistant (PDR) A. baumannii, which are resistant to all available antibiotics, combination therapies, including colistin/imipenem, colistin/meropenem, colistin/rifampicin, colistin/tigecycline, colistin/sulbactam, colistin/teicoplanin, and imipenem/sulbactam, have been extensively studied. Prospective treatment options of Acinetobacter baumannii infections are summarized in Table 3. We will discuss the most recent published reports.

Carbapenems and β-Lactamase Inhibitors
Carbapenems, including imipenem, meropenem, and doripenem, have generally been considered the agents to treat A. baumannii infections, due to their effective activity against this organism and their favorable safety (Doi et al., 2015). However, the decreased susceptibility of A. baumannii to carbapenems has forced clinicians and researchers to explore alternative therapeutic approaches (Doi et al., 2015). Because carbapenem-resistant A. baumannii strains are often resistant to all other commonly used antibiotics as well, these strains remain susceptible to only limited antibiotics, such as minocycline/tigecycline and polymyxins (colistin and polymyxin B; Doi et al., 2015). Carbapenem therapies combined with a few effective antibiotics was extensively tested and many cases showed a synergistic effect against A. baumannii infections (Table 3). However, recent increase of tigecyclineor colistin-resistant A. baumannii increasingly poses a serious Carbapenem +ampicillin+sulbactam+

In vivo
Carbapenem-resistant Combination therapy with ampicillin-sulbactam and meropenem is effective against skin and soft tissue infection Hiraki et al., 2013 In vivo

Multidrug-resistant
The combination of a carbapenem and ampicillin/sulbactam was associated with a better outcome than the combination of a carbapenem and amikacin, or a carbapenem alone Kuo et al., 2007 Carbapenem +minocycline

In vitro
Multidrug-resistant Minocycline in combination with rifampicin, imipenem, and colistin showed bactericidal synergy in most of the isolates which did not harbor the tetB gene, but the combinations were not synergistic in  In vitro Carbapenem-resistant, colistin-susceptible Effective for strains highly resistant to imipenem and moderately resistant to rifampicin Montero et al., 2004 In vitro Multidrug-resistant, colistin-susceptible Synergistic effect against 11 of 13 isolates Hogg et al., 1998 In vitro Extensively drug-resistant Combinations of colistin/rifampicin, colistin/meropenem, colistin/minocycline and minocycline/meropenem are synergistic Liang et al., 2011 In vitro Multidrug-resistant, colistin-susceptible Colistin/rifampicin was fully synergistic against 4 of 5 isolates; colistin/meropenem and colistin/azithromycin were synergistic against 3 of 5 isolates; colistin/doxycycline was partially synergistic or additive against 5 isolates Timurkaynak et al., 2006 Case report Carbapenem-resistant, colistin-susceptible Rifampicin/colistin and ampicillin/sulbactam resulted in microbiological clearance in 9 of 14 critically ill patients Petrosillo et al., 2005 In vitro Carbapenem-resistant, colistin-heteroresistant Rifampicin/colistin and imipenem/colistin were synergistic against heteroresistant isolates and prevented the development of colistin-resistant strains Rodriguez et al., 2010 Case report Carbapenem-resistant, colistin-susceptible Synergistic effect in patients with ventilator-associated pneumonia Aydemir et al., 2013 (Continued) Frontiers in Cellular and Infection Microbiology | www.frontiersin.org

In vitro
Extensively drug-resistant The daptomycin-colistin combination was the most effective; the colistin/imipenem combination was also effective Cordoba et al., 2015 Colistin+vancomycin

In vitro/in vivo
Multidrug-resistant, colistin-susceptible Highly active both in vitro and in an animal model of Galleria mellonella  Colistin+fosfomycin

colistin-resistant
In vitro synergy between colistin and fusidic acid that is comparable to the synergy between colistin and vancomycin; the synergy with fusidic acid is strain-dependent and applicable to strains for which the colistin MICs are relatively low Bowler et al., 2016 In vitro Novobiocin

In vitro
Carbapenem-susceptible Inhibition of frequency of the occurrence of rifampin resistance mutants Jara et al., 2015 Bacteriophages

In vitro
Colistin-susceptible, colistin-resistant Synergistic effect targeting to strains with specific colistin resistance mechanisms; synergy against both colistin-susceptible strains and colistin-resistant strains with mutations in pmrB and phosphoethanolamine modification of lipid A, but not against colistin-resistant strains with loss of lipopolysaccharide Martinez-Guitian et al., 2016 Bulgecin A+ carbapenem

In vitro
Colistin-resistant Farnesol increased sensitivity to colistin for colistin-resistant strains Kostoulias et al., 2015 Oleanolic acid+gentamicin or kanamycin  threat to public health worldwide (Peleg et al., 2007;Hornsey et al., 2010;Cai et al., 2012). Sulbactam is a β-lactamase inhibitor and also has affinity for penicillin-binding proteins of A. baumannii (Rafailidis et al., 2007;Doi et al., 2015). Combined therapy of ampicillin with sulbactam is effective for treating bloodstream infections due to MDR A. baumannii (Smolyakov et al., 2003). Ampicillin/sulbactam/carbapenem combination therapy is also effective for treating MDR A. baumannii bacteremia (Kuo et al., 2007) and skin and soft tissue infection of carbapenem-resistant A. baumannii (Hiraki et al., 2013), but not in ventilatorassociated pneumonia (Kalin et al., 2014). The population pharmacokinetics and pharmacodynamics of sulbactam were determined in critically ill patients with severe sepsis caused by A. baumannii (Jaruratanasirikul et al., 2016) and in patients with impaired renal function (Yokoyama et al., 2015). Another βlactamase inhibitor tazobactam increases the activity of peptide antibiotics, such as colistin and daptomycin, in a murine model of A. baumannii pneumonia (Sakoulas et al., 2016). The authors suggested that β-lactamase inhibitors may exert similar effects in potentiating peptide antibiotics, because of structural similarities between β-lactamase inhibitors and peptide antibiotics (Sakoulas et al., 2016).

Minocycline/Tigecycline
Minocycline, is a broad-spectrum tetracycline antibiotic that has been proposed for treating drug-resistant A. baumannii based on its high degree of susceptibility to this drug and its favorable pharmacokinetic profile (Ritchie and Garavaglia-Wilson, 2014). The mean susceptibility rate of A. baumannii to minocycline is approximately 80% worldwide (Castanheira et al., 2014). Therefore, minocycline therapy has high treatment success rates and good tolerability (Ritchie and Garavaglia-Wilson, 2014). However, since the introduction of minocycline, approximately 20% of A. baumannii isolates are not susceptible to minocycline. The TetB efflux pump is the main determinants of minocycline resistance (Vilacoba et al., 2013). Minocycline therapy combined with colistin is effective for treating minocycline-resistant A. baumannii infections , and minocycline therapy combined with rifampicin, colistin, or imipenem has a synergistic effect in most of isolates without the tetB gene, but combined therapies are not synergistic in isolates with the tetB gene .
Tigecycline is the first glycylcycline class antibiotic that exhibits bacteriostatic activity by binding to the 30S ribosomal subunit, and is active against A. baumannii infections (Pachon-Ibanez et al., 2004;Anthony et al., 2008;Koomanachai et al., 2009). Tigecycline shows a synergistic effect with some classes of antibiotics, such as amikacin (Moland et al., 2008) and colistin (Mutlu Yilmaz et al., 2012). However, limitations of tigecycline use have emerged with its increasing use. Tigecycline is less effective than imipenem to treat pneumonia in a murine pneumonia model (Pichardo et al., 2010). β-Lactam or carbapenem instead of tigecycline was recommended for A. baumannii infections with tigecycline MIC of more than 2 mg/L, due to high mortality from the tigecycline treatment (Curcio and Fernandez, 2008). In a study of 266 patients with MDR A. baumannii infections, tigecycline-based therapy was not more effective than non-tigecycline-based therapies (Lee Y. T. et al., 2013). Tigecycline resistance associated with overexpression of efflux pumps, such as AdeABC, has been reported in clinical isolates of A. baumannii (Peleg et al., 2007;Ruzin et al., 2007;Hornsey et al., 2010. Multiple MDR A baumannii clones resistant to tigecycline have been reported in many medical centers (Navon-Venezia et al., 2007). Therefore, tigecycline can only be used in limited cases for treating A. baumannii infections.
Unfortunately, the emergence of colistin-resistant A. baumannii strains has increased worldwide (Cai et al., 2012). The mechanisms of colistin resistance include loss of LPS (Moffatt et al., 2010) and the addition of phosphoethanolamine to LPS by the PmrAB two-component system (Adams et al., 2009). Mutations in pmrA and pmrB activate pmrC, which adds phosphoethanolamine to the hepta-acylated form of lipid A . Interestingly, an investigation of the in vitro activities of various antimicrobial combinations against colistin-resistant A. baumannii showed that the most effective combinations against colistin-resistant A. baumannii are colistinrifampin and colistin-teicoplanin, indicating that colistin is the most common constituent of antimicrobial combinations even against colistin-resistant A. baumannii (Bae et al., 2016). Similarly, minocycline therapy in combination with colistin is effective to treat infections caused by minocycline-resistant A. baumannii. Minocycline/colistin therapy significantly improves survival of mice infected with minocycline-resistant A. baumannii and reduces the number of bacteria present in the lungs of mice .
A urinary tract Enterococcus faecalis isolate that apparently requires vancomycin to grow was reported in 1994, and this phenomenon is called "antimicrobial agent dependence." Colistin dependence was reported in an A. baumannii-A. calcoaceticus complex (Hawley et al., 2007). Partial colistin dependence has been detected in several LPS-deficient strains with mutations in lpxA, lpxC, and lpxD (Garcia-Quintanilla et al., 2015). Many colistin-susceptible A. baumannii isolates develop colistin dependence in vitro after exposure to colistin (Hong et al., 2016). Although the clinical implication of colistin dependence and its molecular mechanism remain unclear, it is interesting that patients with colistin-dependent A. baumannii isolates show a high rate of treatment failure (Hong et al., 2016).
Unlike colistin, polymyxin B is not converted from a prodrug form into an active form; thus, plasma concentrations of polymyxin B more quickly reach target levels (Sandri et al., 2013). In addition, polymyxin B is available for direct parenteral administration (Zavascki et al., 2007). Despite the favorable pharmacokinetics of polymyxin B, dose-related nephrotoxicity limits the concentration of polymyxin B used in combination therapy (Dubrovskaya et al., 2015). Therefore, almost all studies on polymyxins are carried out for colistin. However, because some carbapenems have comparatively safer dose modulation to optimize killing during combination therapy (Cannon et al., 2014), several studies have analyzing the pharmacodynamics of carbapenems in combination with polymyxin B (Lenhard et al., 2016a,b;Rao et al., 2016a). One study showed that intensified meropenem dosing combined with polymyxin B is a good strategy to treat carbapenem-resistant A. baumannii, regardless of the meropenem MIC (Lenhard et al., 2016a). Combination therapy with doripenem and polymyxin B also showed similar results. Early aggressive dosing of doripenem combined with polymyxin B is effective for treating heteroresistant A. baumannii infections (Rao et al., 2016a). A combined pharmacodynamics analysis of four different carbapenems with polymyxin B showed that doripenem, meropenem, or imipenem display similar pharmacodynamics in combination, and the decision to use carbapenem in combination with polymyxin B is usually based on toxicodynamic profiles (Lenhard et al., 2016b). Polymyxin B also shows good bactericidal activity in combination with high tigecycline concentrations (Hagihara et al., 2014;Rao et al., 2016b). Therefore, polymyxin B combination therapies seem to be one of the most promising options for minimizing the emergence of polymyxin resistance. Increasing the dose intensity of polymyxin B amplifies polymyxin B resistance in A. baumannii Tsuji et al., 2016). In conclusion, although polymyxin B displays dose-related nephrotoxicity, it is a potential therapeutic alternative to colistin when use together with intensified doses of other antibiotics. Large-scale screening of Streptomyces secondary metabolites was performed to develop a novel combination therapy using minimal concentrations of polymyxin B, and the reliable polymyxin synergist netropsin was identified . Survival of G. mellonella infected with colistin-resistant clinical A. baumannii isolates is significantly higher when treated with polymyxin B combined with netropsin than when treated with polymyxin B or netropsin alone .

Other Antibiotics
Trimethoprim-sulfamethoxazole is a two antibiotics combination that exerts a synergistic effect by inhibiting successive steps in the folate synthesis pathway against a number of bacteria (Wormser et al., 1982). The in vitro killing activity of trimethoprim-sulfamethoxazole against carbapenem-resistant A. baumannii was recently studied. Trimethoprim-sulfamethoxazole alone effectively kills all carbapenem-resistant A. baumannii strains and trimethoprimsulfamethoxazole combined with colistin also rapidly kills all strains for up to 24 h (Nepka et al., 2016). These results suggest that trimethoprim-sulfamethoxazole might be an effective therapy for severe carbapenem-resistant A. baumannii infections. Plazomicin is a next-generation aminoglycoside synthetically derived from sisomicin that enhances activity against many MDR Gram-negative bacteria (Garcia-Salguero et al., 2015). A synergistic effect was observed with carbapenems along with plazomicin during treatment of A. baumannii infections (Garcia-Salguero et al., 2015), indicating the potential utility of plazomicin combined with carbapenems.
The inducible DNA damage response in A. baumannii plays an important role in acquiring antibiotic resistance under clinically relevant DNA-damaging conditions (Aranda et al., , 2014Norton et al., 2013). The aminocoumarin novobiocin is a well-established antimicrobial agent that inhibits the DNA damage response in Gram-positive bacteria by interfering with ATPase activity of DNA gyrase (Schroder et al., 2013). One study showed that novobiocin also inhibits acquisition of antimicrobial resistance in MDR A. baumannii through DNA damage-induced mutagenesis (Jara et al., 2015).

Non-antibiotic Therapies: Phage and Others
The worldwide spread of MDR pathogens has renewed interest in the therapy using bacteriophage, which is a virus that infects and lyses bacteria. Various lytic A. baumannii bacteriophages, such as vB_Ab-M-G7 (Kusradze et al., 2016) and Bφ-C62 (Jeon et al., 2016), have been used to treat infections caused by MDR A. baumannii. Bacteriophage-encoded endolysin has also received attention. Endolysin is a lytic enzyme that degrades the cell wall of bacterial hosts and shows promise as a novel class of antibacterials with a unique mode of action (Defraine et al., 2016). For example, endolysin from A. baumannii bacteriophage ØABP-01 degrades the crude cell wall of A. baumannii strains and elevates antibacterial activity when combined with colistin (Thummeepak et al., 2016). However, most Gram-negative pathogens are generally not susceptible to endolysins, due to their protective outer membrane (Lee et al., 2013a). To overcome this problem, endolysins have recently been engineered with specific outer membrane-destabilizing peptides to obtain the ability to penetrate outer membrane and these engineered endolysins are called "artilysins" (Rodriguez-Rubio et al., 2016). Several engineered artilysins have been developed to combat MDR A. baumannii and show highly effective antimicrobial activity against A. baumannii (Briers et al., 2014;Yang et al., 2015;Defraine et al., 2016;Thandar et al., 2016). These results suggest that artilysins can be a treatment option for MDR A. baumannii. The diversity of the phage population was determined by analysis of viromes, endolysins, and CRISPR spacers (Davison et al., 2016). These results can be used to assist in finding an effective endolysin for combating MDR A. baumannii. Various peptides, such as American alligator plasma peptide (Barksdale et al., 2016) and antimicrobial peptide dendrimer G3KL (Pires et al., 2015), have in vitro antimicrobial activity against MDR A. baumannii. However, the use of antimicrobial enzymes or peptides also has some important problems, such as their short half-life in serum and high production costs compared with those of smaller molecules.
An in silico analysis predicted that OXA-58, OXA-23, and OXA-83 are translocated to the periplasm via the Sec system (Liao et al., 2015;Chiu et al., 2016). A SecA inhibitor (rose bengal) inhibits periplasmic translocation of these carbapenemhydrolyzing class D β-lactamases, indicating that these βlactamases are selectively released via a Sec-dependent system (Liao et al., 2015;Chiu et al., 2016). Imipenem or meropenem combined with rose bengal shows synergistic effects for carbapenem-resistant A. baumannii clinical isolates (Chiu et al., 2016). Similarly, β-aminoketone (MD3), an inhibitor of bacterial type I signal peptidases that cleaves the amino-terminal signal peptides of translocated proteins, shows a synergistic effect when combined with colistin against colistin-resistant A. baumannii strains .
Bulgecin A is a natural product of P. mesoacidophila and a lytic transglycosylase inhibitor that works synergistically with β-lactams (Skalweit and Li, 2016). Bulgecin A restores the efficacy of meropenem in suppressing growth of carbapenemresistant A. baumannii strains, suggesting that Bulgecin A may be an adjunctive compound to extend the life of carbapenems against A. baumannii infections (Skalweit and Li, 2016). Similarly, farnesol, a natural product of Candida albicans for quorum-sensing, disrupts A. baumannii cell membrane integrity, alters cell morphology, and increases sensitivity of MDR A. baumannii strains to colistin (Kostoulias et al., 2015). Many herbal active compounds have potent antibacterial activities against many bacteria including carbapenem-resistant A. baumannii (Lin et al., 2015). For example, oleanolic acid is a triterpenoid compound that widely exists in food, medicinal herbs, and many plants and can potently inhibit various pathogenic bacteria. One study showed that oleanolic acid increases aminoglycoside uptake by changing membrane permeability and energy metabolism in A. baumannii (Shin and Park, 2015).
Cyanide 3-chlorophenylhydrazone (CCCP) is an efflux pump inhibitor that decreases the MIC of colistin in colistin-susceptible and colistin-resistant A. baumannii strains (Park and Ko, 2015;Ni et al., 2016). Other efflux pump inhibitors, such as ABEPI1 and ABEPI2, inhibit efflux-mediated minocycline tolerance of A. baumannii. Adding these compounds during growth in human serum leads to the accumulation of minocycline within A. baumannii and inhibits efflux potential of the bacterium (Blanchard et al., 2014).
Gallium is a semi-metallic element in group 13 of the periodic table that binds to biological complexes containing Fe 3+ and disrupts essential redox-driven biological processes (Bernstein, 1998). Gallium has been used as a simple inorganic or organic salt or complexed with organic compounds. Several studies have shown that gallium nitrate or gallium protoporphyrin IX could be a viable therapeutic option for treating MDR A. baumannii (Antunes et al., 2012;Arivett et al., 2015). Some D-amino acids, such as D-His and D-Cys, inhibit bacterial growth, biofilm formation, and adherence to eukaryotic cells in A. baumannii .
Probiotics are "live microorganisms that confer a health benefit on the host when administered in adequate amounts" (Reid et al., 2005) and assist in protecting against MDR A. baumannii infections. For example, the ability of the probiotic Bifidobacterium breve to protect against MDR A. baumannii infections has been investigated (Asahara et al., 2016). This probiotic markedly potentiates protection against fatal intestinal infections caused by MDR A. baumannii (Asahara et al., 2016). With probiotics, immunomodulators, such as lysophosphatidylcholine (Parra Millan et al., 2016) and macrolide antibiotics such as clarithromycin (Konstantinidis et al., 2016), can reduce A. baumannii infection severity by stimulating the immune response, when combined with antibiotics such as colistin, tigecycline, or imipenem.

CONCLUSION
The number of studies about A. baumannii is increasing dramatically because of its increasing clinical importance. Use of animal models has produced important data regarding virulence factors that contribute to A. baumannii pathogenesis. Notably, some studies on metal acquisition and protein secretion systems are interesting. Besides iron acquisition systems such as acinetobactin, the discovery of zinc and manganese acquisition systems in A. baumannii broadens our understanding of A. baumannii pathogenesis. More extensive studies on various protein secretion systems present in A. baumannii are required. About 300 genes required for in vivo survival of A. baumannii were identified using transposon screening in G. mellonella larvae (Gebhardt et al., 2015). Because many of these genes were not known to be associated with A. baumannii pathogenesis, more detailed studies are required to determine whether these genes are related to the pathogenesis of A. baumannii. In addition, transposon screening in other model animals will provide novel insight into A. baumannii pathogenesis. Knowledge of virulence factors responsible for A. baumannii pathogenicity will be the cornerstone for developing novel antibiotics. For example, LPS is an important virulence factor and LpxC inhibitor, which inhibits LPS synthesis, completely protects mice from lethal infection . These results indicate that blocking LPS synthesis is a powerful strategy for discovering novel antibiotics. However, despite recent extensive studies about A. baumannii pathogenesis, the toxicity and pathogenicity of A. baumannii remain unclear.
Recent interest about A. baumannii is mostly due to its seemingly endless capacity to acquire antibiotic resistance. A. baumannii has almost all bacterial resistance mechanisms. All class β-lactamases have been detected in A. baumannii and the frequency of carbapenem-resistant A. baumannii isolates is very high. Furthermore, almost all A. baumannii contain aminoglycoside-modifying enzymes and many efflux pumps responsible for resistance to various clinically important antibiotics have been identified in A. baumannii. Due to these abilities, available antibiotics to treat A. baumannii infections are significantly limited. Colistin is used as the antibiotic treatment of last resort, due to its relatively low resistance rate. However, emergence of colistin-resistant A. baumannii strains has increased worldwide with increasing use of colistin. Notably, some more recent studies have proposed that another polymyxin antibiotic, polymyxin B, is a potential therapeutic alternative to colistin (Lenhard et al., 2016a,b;Rao et al., 2016a;Repizo et al., 2015). Polymyxin B has not been a good antibiotic owing to dose-dependent nephrotoxicity, but recent reports show that a novel combination therapy with carbapenems or tigecycline using minimal concentrations of polymyxin B can be a good strategy to treat carbapenem-resistant A. baumannii infections. These results indicate the requirement for extensive studies that analyze the pharmacodynamics of polymyxin B in combination therapy.
Various trials to identify a novel alternative to carbapenem or colistin have been performed. Among them, engineered endolysins (artilysins) are particularly interesting, despite evident defects. A lytic enzyme degrading peptidoglycan of bacteria is a promising novel class of antimicrobial agents due to its unique mode of action. Similar to β-lactam antibiotics that are one of the most successful antibiotics, inhibition of peptidoglycan synthesis is a promising target of antimicrobial agents. Because lytic enzymes directly degrade peptidoglycans, but not proteins, the possibility of the emergence of a resistance mechanism is relatively low. In addition, enzymes with relatively high molecular weight are not inhibited by efflux pumps. If the short stability of artilysin in serum and high cost in its production compared with small molecules can be resolved, the improved artilysin can be a good treatment option for carbapenem-or colistin-resistant A. baumannii infections. In conclusion, novel, rationally designed strategies and screening-based approaches are required to discover new classes of antibiotics. If we continue to take all efforts at maintaining the effectiveness of antibiotics and developing novel antibiotics, effective control of A. baumannii infections can be successful.