Acinetobacter spp. are glucose-non-fermentative, non-motile, non-fastidious, catalase-positive, oxidase-negative, aerobic Gram-negative coccobacilli[1]. Since 1986, the taxonomy of the genus Acinetobacter has been modified several times. Currently, the original single species classification of Acinetobacter calcoaceticus (A. calcoaceticus) has been abandoned, and at least 34 genomic species can be distinguished within the genus Acinetobacter, 23 of which have been assigned species names[2]. The challenge in the taxonomy of Acinetobacter is due to the clusters of closely related species that are difficult to distinguish using phenotypic traits and chemotaxonomic methods. The A. calcoaceticus-Acinetobacter baumannii (A. baumannii) complex, comprising A. calcoaceticus, A. baumannii, and the genomic species 3 and 13TU, is the most well-known example[3]. Because the antibiotic susceptibilities and clinical relevance of the different genomic species are significantly different[4-7], genomic methods for Acinetobacter species identification are necessary. A number of genomic fingerprinting methods have been proposed, including pulsed-field gel electrophoresis (PFGE); ribotyping; polymerase chain reaction (PCR)-based fingerprinting techniques, such as random amplified polymorphic DNA analysis; repetitive extragenic palindromic sequence-based PCR (rep-PCR); amplified ribosomal DNA restriction analysis; RNA spacer fingerprinting; and amplified fragment length polymorphism analysis[8]. In addition, new methods, such as 16S-23S ribosomal intergenic spacer, 16S rRNA gene, rpoB gene and gyrB gene sequence analyses, have been developed for Acinetobacter species identification[9,10].

Of all of the Acinetobacter species, A. baumannii is the most important member associated with infections in clinical practice and causes most of the reported outbreaks. In addition to chart review and statistical epidemiology, some DNA fingerprinting methods are valuable in outbreak investigations and strain discrimination. Rep-PCR, PFGE, and multilocus sequence typing (MLST) have all been used in previous studies. Rep-PCR has been proven to be a useful and expedient method for the epidemiological characterization of A. baumannii nosocomial outbreaks[11]. Rep-PCR has also been used as a tool for determining species lineages of A. baumannii in a hospital[12] and for differentiating pan-European, multi-resistant A. baumannii clone III from clones I and II[13]. Despite the inter-laboratory variability of rep-PCR, this method has the advantage of being faster to perform than PFGE and MLST. The intra-laboratory clustering of A. baumannii has been shown to be well conserved[14] and to correlate well with PFGE[15] or MLST results[16], demonstrating the robustness of rep-PCR. We have found one rep-PCR major cluster (84%) of A. baumannii carrying a class I integron that spread among four regional hospitals in northern Taiwan[17]. However, PFGE is still considered the gold standard for typing outbreak-related isolates of A. baumannii[18-21], whereas MLST provides a high level of resolution and is an excellent tool for studying the population structure and long-term epidemiology of A. baumannii[22]. Recently, the A. baumannii MLST database (http://pubmlst.org/abaumannii/) was developed for the BIGSdb genomics platform[23] to assist the broader community in elucidating the structure and function of this microorganism.

A. baumannii, named after Paul Baumann, is ubiquitous in soil and water[24]. Previously, A. baumannii was regarded as a low-virulence commensal bacterium. However, it has become a successful pathogen[25] and has emerged as a major cause of healthcare-associated infections, most of which have occurred in critically ill patients in the intensive care unit (ICU) setting[26]. In recent decades, infections caused by A. baumannii have also occurred outside the ICU or in trauma patients after natural disasters, and they have even affected patients with co-morbidities in the community[27]. Reports of community-acquired Acinetobacter infections have increased over the past decade[28]. Several different types of infections, including pneumonia, urinary tract infections, bacteremia, wound infections and even meningitis, are caused by this organism[29]. These infections often occur in older patients, many of whom have chronic underlying diseases and have previously received antimicrobial treatment[30,31]. The mortality of patients with A. baumannii infections in hospitals and in the ICU has ranged from 7.8% to 23% and from 10% to 43%, respectively[32].

In recent years, many studies have reported the risk factors for acquiring A. baumannii infections and have particularly focused on those caused by multidrug-resistant strains. The acquisition of MDRAB is related to multiple factors, including environmental contamination and contact with transiently colonized healthcare providers[33]. The independent risk factors for the acquisition of imipenem-resistant A. baumannii (IRAB) include a hospital size of > 500 beds, previous antimicrobial treatment, a urinary catheter, surgery[34], previous ICU stay, and prior exposure to imipenem or third-generation cephalosporins[35]. The only significant independent risk factor for the appearance of imipenem-resistant multidrug-resistant A. baumannii (MDRAB) in patients formerly infected with imipenem-susceptible MDRAB is imipenem or meropenem exposure[36]. For extensively drug-resistant A. baumannii (XDRAB) infections, the prior use of imipenem, meropenem, piperacillin/tazobactam or fourth-generation cephalosporins and > 30 d of being bed-ridden have been found to be independent risk factors[37]. A systematic review concluded that the acquisition and spread of A. baumannii appeared to be related to a large number of variables, the most important of which were deficiencies in the implementation of infection control guidelines and the use of broad-spectrum antibiotics[38].

The risk factors that are associated with A. baumannii bacteremia are immunosuppression, unscheduled hospital admission, respiratory failure at ICU admission, previous antimicrobial therapy, previous sepsis in the ICU, and the invasive procedures index[39]. Resistance to carbapenems, mechanical ventilation, and the presence of malignancy have also been found to be associated with high mortality rates in patients with A. baumannii bacteremia[40]. Regarding ventilator-associated pneumonia caused by A. baumannii, the risk factors include neurosurgery, adult respiratory distress syndrome, head trauma, and large-volume pulmonary aspiration[41]. Because various studies showed certain differences in the risk factors of acquiring drug-resistant A. baumannii bacteremia or pneumonia[42-46], a separate investigation should be performed in each hospital setting to limit the spread of this pathogen[38].

Previously, A. baumannii was regarded as a low-grade pathogen; however, it contains virulence factors that enhance its bacterial toxicity and pathogenicity. A combined approach of genomic and phenotypic analyses led to the identification of several virulence factors, including extracellular components with hemolytic, phospholipase, protease and iron-chelating activities, biofilm formation, surface motility, and stress resistance[47]. The biofilm formation of A. baumannii facilitates its attachment to abiotic and biotic surfaces[48], including those of medical devices and host tissues. The initiation and maturation of biofilms are related to pilus assembly and the production of the biofilm-associated protein (Bap), which is regulated by the two-component system BfmRS[49]. The Bap protein plays a role in adhesion to human epithelial cells[50], and the inhibition of this protein can prevent A. baumannii infection[51]. In fact, in a multicenter cohort study, all catheter-related urinary or blood stream infections due to A. baumannii were caused by biofilm-forming strains[52]. A 2D proteomic analysis of pellicle-forming A. baumannii isolates showed that overexpression of CarO, which is an OprD-homolog, siderophore iron uptake, and pili systems are involved in the development of biofilms[53].

Iron uptake systems are essential to the survival and pathogenicity of bacteria, especially in the low-iron environment of the human host. A. baumannii grown under iron-limited conditions undergo major transcriptional changes of not only many iron acquisition-related genes but also of genes involved in motility[54]. A. baumannii is well-equipped with metal homeostatic systems that are required for the colonization of a diverse array of tissues[55]. Genome investigations have revealed wide distributions of endogenous siderophores in clinical A. baumannii isolates, arguing for their role in pathogenic capabilities[47]. The zinc acquisition system has also been found in A. baumannii, which is required for efficient zinc uptake in vitro and full pathogenesis in vivo[56].

A. baumannii adheres to human bronchial epithelial cells in vitro, and its prevalent European clone II has a relatively high capacity for adhering to these cells[57]. Additionally, the K1 capsular polysaccharide has been shown to prevent A. baumannii from being phagocytized by macrophages, to optimize its growth in human ascites fluid and serum, and to enhance its survival in a rat soft tissue infection model[58]. Moreover, several proteins have been implicated as possible virulence factors in A. baumannii. Omp38 induces the apoptosis of host cells[59], the absence of the RecA protein decreases survival in response to both heat shock and desiccation[60], and the inactivation of phospholipase D diminishes A. baumannii pathogenesis[61]. Importantly, the outer membrane protein A of A. baumannii (AbOmpA) is the most abundant surface protein that has been associated with the apoptosis of epithelial cells through mitochondrial targeting[62]. AbOmpA is also the major nonspecific channel in A. baumannii and appears to be essential for this organism’s high levels of intrinsic resistance to a number of antibiotics[63]. A. baumannii can rapidly develop resistance to polymyxin antibiotics through the loss of the lipid A component of lipopolysaccharide[64], which subsequently alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface[65].

Currently, centain strains of A. baumannii is highly resistant to most antibiotics available in clinical practice. A number of resistance mechanisms to many classes of antibiotics are known to exist in A. baumannii, including β-lactamases, multidrug efflux pumps, aminoglycoside-modifying enzymes, permeability defects, and the alteration of target sites[109-111]. Most of these resistance mechanisms can target different classes of antibiotics. However, several different mechanisms can work together to contribute to the resistance to a single class of antibiotics. For example, the resistance mechanisms in CRAB are diverse[112]. In addition to β-lactamases with carbapenem-hydrolyzing activity as a major carbapenem resistance mechanism, which include carbapenem-hydrolyzing class D β-lactamases (CHDLs) and metallo-β-lactamases (MBLs), porins such as CarO[66] and penicillin-binding protein modifications might also be involved in carbapenem resistance[113]. The spread of multidrug-resistance determinants in A. baumannii is mostly through plasmid conjugation, transposon acquisition or integron mobilization to gain clusters of genes encoding resistance to several antibiotic families[110]. Furthermore, the functional insertion sequences are important in amplifying antimicrobial resistance and gene plasticity[114-118]. Table 1 shows the various antimicrobial resistance mechanisms of A. baumannii. The details are further discussed below.

β-lactamase

Inactivation of β-lactams constitutes an important part of multidrug resistance in A. baumannii, especially for β-lactam antibiotic resistance. All four Ambler classes of β-lactamases (i.e., classes A, B, C and D) can be identified in this organism[66]. Although a wide range of class A β-lactamases, including those of temoneira (TEM)[119-121], sulfhydryl variable (SHV)[122], cefotaxime hydrolyzing capabilities (CTX-M)[123,124], guiana extended-spectrum (GES)[115,125], self-transferable plasmid from E. coli (SCO)[126], Pseudomonas extended resistant (PER)[127-130], vietnam extended-spectrum β-lactamase (VEB)[96,131-133], carbenicillin hydrolyzing β-lactamase (CARB)[134,135] and K. pneumoniae carbapenemase (KPC)[136], have been reported in A. baumannii (Table 1), they are generally regarded to play a minor role in its resistance phenotype, especially in carbapenem resistance. Some of these enzymes are narrow-spectrum β-lactamases, e.g., TEM-1[119-121], SCO-1[126] and CARB-4[135]; however, a number of these enzymes are still responsible for the hydrolysis of extended-spectrum β-lactams (ESBL). PER-1 was the first ESBL enzyme identified in A. baumannii[137], whereas TEM-92 and CARB-10 were the first reported TEM-type[120] and CARB-type[134] ESBLs, respectively. Later, the chromosomally encoded ESBLs SHV-5[122], PER-2[132] and PER-7[129,130] were also described. A. baumannii strains carrying the extended spectrum VEB-1 enzyme were first reported in an outbreak in France[131]. GES-11, an integron-associated GES variant, can even confer reduced susceptibility to carbapenems[115,125]. In addition, CTX-M enzymes are transmitted by integrons or plasmids, indicating the potential dissemination in outbreaks between different strains[123,124]. Finally, KPC-10 was the first KPC β-lactamase to be identified[136].

Class B β-lactamases can confer resistance to the majority of β-lactams because of their broad range, potent carbapenemase activity and resistance to inhibitors[138]. Although MBLs are not the predominant carbapenemases in A. baumannii, verona integron-encoded metallo-β-lactamase (VIM), imipenemase (IMP) and seoul imipenemase (SIM) MBLs have been found contribute to the high-level resistance to carbapenems. The first VIM enzyme was described by Yum in 2002[139]. Thereafter, several other VIM variants, including VIM-1[140-142], VIM-3[143], VIM-4[141,142], and VIM-11[143], were identified in A. baumannii. IMP enzymes have also been reported in several Gram-negative bacteria worldwide, including A. baumannii. At least nine variants of IMP enzymes have been identified in A. baumannii: IMP-1[144], IMP-2[145], IMP-4[146,147], IMP-5[148], IMP-6[149], IMP-8[143], IMP-11[150], IMP-19[150] and IMP-24[143]. SIM-1 is the only SIM enzyme that has been reported in A. baumannii[151]. More recently, NDM (new Deli metallo-β-lactamase)-1[152-154] and NDM-2[155] were observed in A. baumannii. bla_NDM-1 is integrated in the chromosome within a new transposon structure with two copies of the insertion sequence ISAba125 in one clinical strain of A. baumannii. Such variability of the genetic environment of bla_NDM-1 likely facilitates its rapid dissemination[153].

The nucleotide sequence of the chromosomal cephalosporinase gene, which encodes an AmpC β-lactamase, in A. baumannii was first characterized in a clinical isolate from Spain in 2000[156]. Different isolates of A. baumannii have been shown to have almost identical AmpC sequences (no more than two amino-acid substitutions)[157]. A phylogenetic analysis showed that Acinetobacter ampC genes are descended from a common ancestor and are more closely related to each other than the ampC genes found in other species of bacteria[158]. The class C chromosomal β-lactamase AmpC in A. baumannii has a typical cephalosporinase substrate profile[156]. The presence of AmpC β-lactamase plays an important role in β-lactam resistance in A. baumannii, and in fact, a high percentage of drug-resistant A. baumannii possess bla_ampc[119]. In a study of 23 MDRAB clinical isolates from five proximal hospitals in Taiwan, all isolates had AmpC-type bla[77]. The presence of an insertion sequence with a strong promoter upstream of ampC in A. baumannii clinical isolates has the potential to overexpress AmpC, resulting in high-level ceftazidime resistance[159,160]. ISAba1-like sequences have been identified immediately upstream of the bla_ampC gene in ceftazidime-resistant A. baumannii isolates but have been shown to be absent in ceftazidime-susceptible A. baumannii isolates[157].

Class D β-lactamases were designated OXAs in reference to their preferred substrate oxacillin[161]. Some OXAs are also able to hydrolyze extended-spectrum cephalosporins, and some can even inactivate carbapenems by acting as carbapenemases[66]. At least 121 different variants of class D β-lactamases have been identified at the protein level, and in contrast to other class D β-lactamases, 45 of these variants exhibit carbapenem-hydrolyzing activities[162]. The bla_oxa genes can be located either on a chromosome or a plasmid and can sometimes be found in integrons[163,164]. Among the four classes of β-lactamases, MBLs and CHDLs are the two main groups of carbapenemases in A. baumannii, the latter of which is responsible for the most common type of carbapenem resistance via enzymatic degradation[165]. Currently, nine major subgroups of OXA carbapenemases have been identified based on amino acid homologies[166]. Four subgroups of OXA with carbapenemase activity, including the OXA-23, OXA-40/24, OXA-51 and OXA-58 clusters, are prevalent in A. baumannii[162,166].

New OXA-type carbapenemases have been frequently discovered since the first clinical isolate of A. baumannii with OXA-23 was characterized[66]. The bla_OXA-23 carbapenemase gene has also been disseminated worldwide[167]. The countries that have reported A. baumannii with OXA-23 carbapenemase include France[168-170], Germany[171], Bulgaria[172], Romania[173], the United States[105], Colombia[174], Brazil[175], Australia[176], Taiwan[177,178], China[92,179], Korea[180], Singapore[147,181], Italy[182] and Spain[183]. A. radioresistens has been proposed as a silent source of bla_OXA-23 for A. baumannii[184], and a novel variant, named bla_OXA-133, has been reported by the Asia-Pacific SENTRY surveillance program[185].

OXA-51/69-like β-lactamases are intrinsic chromosomal enzymes in A. baumannii[166,186] that emerged as a new subgroup of carbapenemases in MDRAB in 2004[187] and that show increased carbapenemase activity when ISAba1 is upstream of the promoter region[188,189]. However, drug export by an efflux pump might be more important in some clinical isolates[190]. A comparative genomics study by Adams et al[105] showed that the studied A. baumannii strains, including wild-type strains and clinical isolates of MDRAB, all possessed genes belonging to the OXA-51 group. The recently identified OXA-51 group of β-lactamases comprises a novel cluster among the OXA-type carbapenemases, and the cluster includes many variant oxacillinases that have been reported in several studies, including those by Heriter in 2005[186], Brown in 2005[191], Turton in 2006[192], Vahaboglu in 2006[193], Koh in 2007[147], Evans in 2007[194], Naas in 2007[122], Tsakris in 2007[195] and Higgins in 2009[196]. The CHDLs that have been found are listed in Table 1.

The OXA-40/OXA-24 CHDL group is made up of OXA-25, OXA-26, OXA-40, and OXA-72 (an original sequencing error occurred in sequencing OXA-24; it is now known as OXA-40)[166]. These enzymes only differ by a few amino acid substitutions. OXA-40/OXA-24 was originally identified as chromosomally encoded in a carbapenem-resistant A. baumannii isolate recovered from Spain[197]. OXA-25, OXA-26, and OXA-27 were later characterized to be associated with carbapenem resistance in clinical isolates of A. baumannii from Spain, Belgium and Singapore[198]. Thereafter, the OXA-40/OXA-24 gene in A. baumannii was reported in several areas[199], including Spain[188,200,201], Portugal[202] and the United States[203]. The plasmid-mediated bla_OXA-24 gene was noted in the isolates from an outbreak in Spain[204]. Additionally, OXA-72 has been identified in A. baumannii isolates from Taiwan[205], China[92] and Croatia[206].

OXA-58 was first identified from an isolate of MDRAB in France[207]. The bla_OXA-58 gene was found to be plasmid borne. Many OXA-58-producing A. baumannii isolates were reported worldwide in subsequent years, including isolates in Europe[208-211], Argentina[208], Kuwait[208], the United Kingdom[208], Australia[212], Taiwan[116], the United States[213,214] and China[215]. A number of outbreaks have also been reported in many countries, including Italy[216], Belgium[79], France[217], Turkey[193], Greece[218,219], and the United States[214]. OXA58 can lead to high-level carbapenem resistance in A. baumannii via the upstream IS1008 insertion[116] or the presence of the ISAba825-ISAba3-like hybrid promoter[118]. OXA-97 is a point mutation variant of OXA-58 that shares the same hydrolytic properties and has been recently identified in A. baumannii isolates from Tunisia[220]. Another point mutation derivative is OXA-96, which was identified in A. baumannii from Singapore[147].

In 2009, a novel CHDL, OXA-143, was identified that shares 88% amino acid identity with OXA-40, 63% identity with OXA-23, and 52% identity with OXA-58[196]. Another novel oxacillinase, OXA-182, was identified in imipenem-nonsusceptible Acinetobacter isolates in Korea[221] and showed 93% identity with OXA-143 and 89% identity with OXA-40 based on amino acid sequence alignment. OXA-235, and the amino acid variants OXA-236 and OXA-237, were identified from A. baumannii isolates from the United States and Mexico[222]. The deduced amino acid sequences shared an 85% identity with OXA-134, 54 to 57% identities with the acquired OXA-23, OXA-24, OXA-58, and OXA-143, and a 56% identity with the intrinsic OXA-51. Thus, OXA-235, OXA-236 and OXA-237 represent a novel subclass of OXAs. The expression of OXA-235 in A. baumannii leads to reduced carbapenem susceptibility, while the cephalosporin minimal inhibition concentrations (MICs) are unaffected.

While multidrug-resistant efflux pumps have been shown to have roles in bacterial pathogenicity[223], the contribution of efflux pumps to bacterial multidrug resistance is often reported[224,225]. Efflux-based mechanisms are responsible for resistance against many different classes of antibiotics, including tigecycline resistance[226,227] or imipenem resistance[190] in A. baumannii. Furthermore, the linear relationship between the log-transformed expression values of the AdeABC efflux pump genes and the log-transformed MIC values is statistically significant, indicating that overexpression of the AdeABC efflux pump is a prevalent mechanism for decreased susceptibility to tigecycline[228]. The importance of efflux pumps in multidrug resistance in A. baumannii is supported by the fact that the presence of efflux pump inhibitors, such as 1-(1-naphthylmethyl)-piperazine[229,230], phenyl-arginine-β-naphthylamide[231,232], or carbonyl cyanide 3-chlorophenylhydrazone[232], can reverse the resistance pattern.

Four categories of efflux pumps, including the resistance-nodulation-division (RND) superfamily, the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion (MATE) family and the small multidrug resistance (SMR) family transporters, have been reported to be related to antimicrobial resistance in A. baumannii[233,234]. Of these different pumps, the RND and MFS transporters are mentioned most often. AdeABC, a RND-type efflux pump with a three-component structure, is not only associated with aminoglycoside resistance[235] but is also associated with decreasing susceptibility to several antimicrobials, including tigecycline[226]. Differences in the expression of adeABC were shown to contribute to both inter- and intra-clone variation in tigecycline MICs in a study of A. baumannii epidemic clones[236]. Both the increase in tigecycline resistance during therapy[236] and the decrease in susceptibility to non-fluoroquinolone antibiotics during an outbreak[237] are mediated by the up-regulation of AdeABC in A. baumannii. The AdeABC pump in wild A. baumannii is cryptic due to stringent control by the AdeRS two-component system[238]. Point mutations in AdeS and AdeR or a truncation of AdeS due to an ISAba1 insertion may be related to the overexpression of AdeABC, which leads to multidrug resistance[238,239]. However, the existence of tigecycline-nonsusceptible and adeB-overexpressing A. baumannii clinical isolates without known adeRS mutations[240] and the low expression of adeABC in a clinical strain of A. baumannii with the ISAba1 insertion in the adeRS operon[239] suggest that the regulation of adeABC gene expression is complex. Additionally, the cell density-dependent expression of adeB suggests the presence of global regulatory mechanisms for the expression of this gene in A. baumannii[241]. BaeSR, which functions as an envelope stress response system to external stimuli, is proposed to influence the transcription of adeAB and thus tigecycline susceptibility in A. baumannii by functioning as a regulator of global transcription[242].

In addition to the AdeABC efflux pump, the inactivation of other RND-type efflux pumps, including AdeFGH[243] and AdeIJK[232,244,245], demonstrates their contribution to multidrug resistance in A. baumannii. The AdeABC and AdeIJK efflux systems can contribute synergistically to tigecycline resistance[244]. An open reading frame encoding a LysR-type transcriptional regulator, named adeL, is located upstream of the adeFGH operon and is responsible for the overexpression of AdeFGH[243], whereas the expression of AdeIJK in Acinetobacter baumannii is regulated by AdeN, a TetR-Type regulator[246]. Although the RND efflux pump AdeDE was initially identified in Acinetobacter genomic group 3[247], adeE was later found to coexist with adeB in some clinical isolates of A. baumannii[245].

A number of MFS efflux pumps, including TetA[248], CmlA[225], MdfA[233], CraA[249] and AmvA[250], that mediate resistance to different types of antibiotics in A. baumannii have been characterized. AbeM, a H-coupled pump that belongs to the MATE family[251], was reported to be present in the clinical isolates of A. baumannii in several studies[77,232,245] and to confer resistance to fluoroquinolones or imipenem in A. baumannii. A. baumannii with a mutant AbeS SMR pump exhibits erythromycin and chloramphenicol resistance[252].

Aminoglycoside-modifying enzymes (AMEs) are the principal mode of resistance to aminoglycosides. This resistance is primarily mediated by three classes of enzymes, including acetyltransferases, adenyltransferases and phosphotransferases, that typically reside on transposable elements; these enzymes chemically modify aminoglycosides[253]. The coding genes for these enzymes can be transferred among different bacterial types through plasmids, transposons, integrons, and natural transformation or transduction[254]. A phenotypic analysis of aminoglycoside resistance profiles indicated that many isolates could produce a combination of aminoglycoside-modifying enzymes[255,256]. The co-carrying of four AME genes, including a novel AME gene aac(6’)-Ib, was reported in a PDRAB strain from China[257]. The identification of MDRAB isolates harboring genes for the bla_OXA-23-like genes, AME (aac(6′)-Ib) and the 16S rRNA methylase (armA) implicates AMEs in multidrug resistance[258].

Different types of AMEs have been reported in A. baumannii. Amikacin resistance has been reported to be associated with a gene encoding APH(3’)-VI phosphotransferase[255]. Furthermore, AME aac(6’)-Iad plays an important role in amikacin resistance in Acinetobacter spp. in Japan[259]. Of the 106 MDRAB isolates identified in one study, 95% possessed at least one type of AME, including aacA4, aacC1, aacC2, aadB, aadA1, aphA1 and aphA6[256]. In another study in Greece, all of the collected A. baumannii strains contained AMEs, which were either aac(6’)-Ib or aac(6’)-Ih[260]. Class I integrons containing the gene cassettes aacA4-catB8-aadA1, dhfrXII-orfF-aadA2, or aacC1-orfP-orfP-orfQ-aadA1 have been proposed to be associated with the horizontal transfer of diversified aminoglycoside-resistant genes among clinical isolates of A. baumannii[17,256,261].

Porins, which perform multiple functions in membranes, are proteins that can form channels to allow the transport of molecules across lipid bilayer membranes[233]. These outer membrane proteins not only influence the virulence of A. baumannii, e.g., through Omp38-induced epithelial cell apoptosis[59], biofilm formation related to OmpA[262], OmpA-dependent host cell death[263], and attenuated virulence by the decreased expression of genes encoding CarO- and OprD-like proteins[263], but also play a significant role in the mechanisms of resistance. For example, the loss of a 29 kDa outer-membrane protein, which was later shown to be CarO, contributes to imipenem resistance[263-267]. Several other studies have also identified a number of OMPs involved in the carbapenem resistance of A. baumannii. A reduction in the expression of two porins of 22 and 33 kDa was involved in the carbapenem resistance of A. baumannii strains in an outbreak in Spain[268]. In one study, CRAB isolates found in New York had reduced expression of the 47-, 44-, and 37-kDa outer-membrane proteins[91], while in other studies, a 33- to 36-kDa OMP was also shown to be associated with carbapenem resistance in A. baumannii[269,270]. A 43-kDa OMP, belonging to the OprD family, has been identified as a basic amino acid and imipenem porin through electrophoresis and MALDI-MS analyses[271].

In the presence of OXA carbapenemases, including OXA-51-like or OXA-23-like enzymes, the loss of the 29-kDa outer-membrane protein is associated with a higher imipenem MIC in A. baumannii[272,273]. Moreover, a novel insertion sequence, ISAba10, inserted into ISAba1 adjacent to the bla_OXA-23 gene, can disrupt the outer-membrane protein gene carO in A. baumannii[180]. The loss of lipopolysaccharide (LPS) from the outer membrane, resulting in a decrease in membrane integrity, occurred in a colistin-resistant clinical isolate of A. baumannii in Australia[64]. Disruption of the ompA gene can lead to decreases in the MICs of chloramphenicol, aztreonam, and nalidixic acid[274].

Changes in penicillin-binding proteins (PBPs), mutations of DNA gyrase, ribosomal protection by the TetM protein and the involvement of dihydrofolate reductase in trimethoprim resistance all occur via mechanisms that alter the target sites for antibiotics[275]. Imipenem resistance has been associated with the overexpression of certain PBPs with a low affinity for imipenem in the absence of other known resistance mechanisms[276]. While an insertion sequence disrupting the gene encoding PBP6b has been identified in an endemic carbapenem-resistant clone, its role must be further evaluated[277]. Furthermore, mutations in DNA gyrase gene gyrA and parC, which encode topoisomerase IV, have been reported in an A. baumannii outbreak investigation[237]. The gyrA mutation at Ser-83 was shown to be associated with quinolone resistance in epidemiologically unrelated isolates of A. baumannii[278]. While tetA and tetB genes are well recognized for their role in tetracycline resistance in A. baumannii through efflux pumps[225,279], tetM is proposed to be another resistance mechanism that acts through ribosomal protection[280]. Trimethoprim resistance through dihydrofolate reductase in A. baumannii is similar to that of other bacteria. Plasmids containing folA genes and integrons harboring dfr or dhfr genes in A. baumannii have been found[17,279,281]. Recently, the coexistence of the 16S rRNA methylase armA gene and genes encoding OXA carbapenemases were reported in many countries, including China[282], South Korea[253,283], India[284], Italy[285], Japan[286], and Yemen[258], indicating the contribution of the armA gene to the multidrug resistance of MDRAB.

The horizontal transfer of resistance genes is a successful mechanism for the transmission and dissemination of multiple drug resistance determinants among bacterial pathogens[287]. Integrons, which are located on either bacterial chromosomes or plasmids, are assembly platforms that incorporate exogenous ORFs by site-specific recombination and convert them to functional genes by ensuring their correct expression[288]. Integrons share common features: a gene encoding an integrase, a specific recombination site that is recognized by the integrase and into which the cassettes are inserted, and a promoter that directs the transcription of the cassette-encoded genes. Currently, there are four classes of integrons, and class 1 integrons are the most common in bacteria[289].

The role of integrons in the development of multidrug resistance relies on their unique capacity to cluster and express drug resistance genes[287]. Many studies regarding integrons harboring different types of resistance genes have been reported worldwide in recent decades. Class I integrons were detected in 52.8% of A. baumannii strains in the Nanjing area of China in 2007[290], whereas an epidemic, class 1 integron-carrying MDRAB clone was found to be widespread in Taiwan in the same year[291]. Four different integron structures were detected in 84% of all collected isolates of A. baumannii in a Spanish study[255]. However, while no clear antibiogram differences could be associated with the presence or absence of integron structures in the Spanish study, other reports have suggested that integrons play a major role in multidrug resistance in A. baumannii[261,291,292]. Additionally, epidemic strains of A. baumannii have been found to contain significantly more integrons than non-epidemic strains[293]. Therefore, integrons are regarded as useful markers for epidemic strains of A. baumannii, and their typing can provide valuable information for epidemiological studies[294,295].

A study performed in Italy found that 44% of the epidemiologically unrelated A. baumannii isolates collected over an 11-year period were integron-positive[296]. Most integron-positive strains carried the same array of cassettes, despite their notable genetic diversity that was identified through a ribotyping analysis, implying that horizontal transfer of the entire integron structure or an ancient acquisition occurred. Additionally, while the same integron can be present in unrelated strains[17], related strains can also have different integrons[297].

Although different relationships exist among different classes of antibiotics and integrons[298,299], most studies have emphasized the association of integrons with cassette genes and aminoglycoside resistance[261]. The diversity of the genes encoding AMEs and their association with class 1 integrons was observed in a study involving three pan-European clones of A. baumannii[256]. Six different class 1 integron variable regions were detected in 74% of the collected strains. Furthermore, Huang et al[291] collected 283 MDRAB isolates from three medical centers in Taiwan from 1996 to 2004 and found seven types of gene cassettes, most of which contained AMEs, including aacA4, aacC1, aac(6^’)-II, aadA1, aadA2, aadA4 and aadDA1.

Variable CHDL genes, including bla_OXA-3[292], bla_OXA-10[96,290], bla_OXA-20[19,292,296], bla_OXA-21[297], and bla_OXA-37, have been reported in integrons[164,297]. Integron-associated imipenem resistance in A. baumannii has also been documented[300]. Genes encoding carbapenemases, such as MBLs bla_VIM, bla_IMP and bla_SIM, have been found in integrons. bla_VIM-1-carrying integrons[140] and bla_VIM-2-carrying integrons[139] have been noted in Greece and Korea, respectively. In Taiwan, integron-mediated gene spreading has been demonstrated hospitals[301], especially in a unit with high antibiotic selective pressure[302]. bla_VIM-11-carrying integrons have also been identified in MDRAB isolates, and this MBL gene has been postulated to spread among Pseudomonas aeruginosa and A. baumannii strains[143,291]. Other reported MBLs include bla_IMP-1[303], bla_IMP-2[145], bla_IMP-4[146,147], bla_IMP-5[148], bla_IMP-8[291] and bla_SIM-1[151]. The genes for chloramphenicol resistance in the integrons of A. baumannii are catB2[135], catB3[146,147,151] and catB8[294,304].

While ampicillin/sulbactam has been shown to be effective in treating blood stream infections caused by MDRAB[337], a later meta-analysis revealed that sulbactam-based therapy is not superior to other therapeutic approaches, including colistin, cephalosporins, anti-pseudomonas penicillins, fluoroquinolones, minocycline/doxycycline, aminoglycosides, tigecycline, polymyxin, imipenem/cilastatin, and combination therapies[338]. Although sulbactam-based therapy failed to prove its superiority to other regimens for the treatment of A. baumannii infections, a case of skin and soft tissue infection caused by CRAB that was treated successfully with ampicillin/sulbactam and meropenem combination[339] raises the possibility of ampicillin/sulbactam as a component of combination therapy against CRAB. The combination of ampicillin/sulbactam with a carbapenem for treating MDRAB bacteremia has been shown to be associated with a better outcome[340], but such beneficial effects were not observed for MDRAB VAP[341].

In 2004, tigecycline was reported to have a good in vitro bacteriostatic effect against A. baumannii, including strains resistant to imipenem[342]. Another in vitro study using a time-kill assay demonstrated the potential role of tigecycline in the treatment of A. baumannii and proposed that doxycycline could be a suitable and cost-effective option in some instances[343]. Tigecycline efficacy was shown to correlate well with the free concentration-time curve of MIC in a murine Acinetobacter spp. model[344]. Additionally, several cases affiliated with severe infections by MDRAB were successfully treated with tigecycline in terms of their clinical and microbiological outcomes[345].

With its increasing use, the limitations and adverse aspects of tigecycline in treating MDRAB infections have begun to be realized. Tigecycline was less effective than imipenem in the treatment of pneumonia caused by non-IRAB strains in a murine pneumonia model[346]. In a study consisting of 34 patients with MDRAB infections, the mortality rate reached up to 41%. The authors found that the correlation of clinical and microbiological outcomes was poor and concluded that tigecycline had excellent in vitro activity against MDRAB, but its clinical efficacy was still uncertain[336]. One of the possible causes for the discrepancy of treatment outcomes may be variable tigecycline MICs. MIC determination for tigecycline before treatment, with the broth dilution method being favored[347], might increase clinical success[345]. A. baumannii isolates with tigecycline MICs of ≥ 2 mg/L were associated with higher mortality rates; thus, treatment with β-lactams or carbapenems instead of with tigecycline is preferred[348]. This notion was further supported in a matched cohort study in Taiwan that dealt with the effectiveness of tigecycline-based versus colistin-based therapy for the treatment of pneumonia caused by MDRAB[349]. The excess mortality rate of 16.7% in the tigecycline-based group compared with the colistin-based group was mostly attributed to subjects with MIC > 2 μg/mL.

In a meta-analysis of the efficacy and safety of tigecycline, clinical failure, superinfection and adverse events were more frequent with the use of tigecycline[350]. The authors suggested that physicians should avoid tigecycline monotherapy for the treatment of severe infections caused by MDRAB and that they should use it as a last-resort antibiotic. There was no antagonism found when tigecycline was used with other antimicrobials possessing activities against Gram-negative bacteria[351]. However, tigecycline-based therapy for MDRAB infections is not satisfactory. In a study of 266 patients with healthcare-associated MDRAB infections, the mortality rate was not significantly different between those receiving tigecycline-based therapy and those receiving non-tigecycline therapy[352].

While tigecycline has an expanded spectrum of antibacterial activity and a synergic effect with some classes of antibiotics, such as amikacin[353], earlier studies have shown that tigecycline resistance in A. baumannii has emerged[354] and is associated with multidrug efflux systems, especially overexpression of the adeABC pump[226,227]. The increased expression of the adeABC operon can be found in clinical isolates of A. baumannii post-tigecycline therapy[236,355]. High resistance rates and high MICs of tigecycline in multiple clones of MDRAB were noted in a medical center in Israel[356]. This phenomenon led to concern regarding the use of tigecycline as one of the few treatment choices for infections caused by MDRAB.

Colistin has been described as a last resort for the treatment of MDRAB[357], and this drug is often used in combination therapy. In a report on the clonal spread of MDRAB in eastern Taiwan, antibiotic susceptibility testing showed that 10.4%, 47.8% and 45.5% of MDRAB isolates were resistant to colistin, rifampicin, and tigecycline, respectively, implying that colistin was the only effective antimicrobial agent in that area for treating MDRAB[358]. In addition to its intravenous injection for MDRAB infections, colistin can be given via intraventricular and intrathecal routes for meningitis[359] and via nebulization for pneumonia[360,361].

Unfortunately, colistin-resistant A. baumannii strains have been reported all over the world[357] and are attributed to the loss of lipopolysaccharide[64] or/and phosphoethanolamine modification of lipid A mediated by the PmrAB two-component system[362,363]. Because colistin monotherapy is unable to curb the appearance of resistance, colistin-based combination therapy might be the optimal antimicrobial strategy. Colistin combined with different classes of antibiotics, including tigecycline, cefoperazone/sulbactam or piperacillin/tazobactam, revealed synergistic effects in some CRAB strains[364]. Time-kill assays have also shown that colistin/meropenem, colistin/rifampicin, and colistin/minocycline are synergistic in vitro against XDRAB strains[365]. The beneficial effects of colistin and rifampicin combination for patients with VAP caused by CRAB have been documented in terms of clinical and microbiological outcomes[366]. However, another multi-center, randomized clinical trial concluded that 30-d mortality was not reduced by the addition of rifampicin to colistin in serious XDRAB infections[367]. Additionally, such a regimen might be hindered by a high level of rifampicin resistance in A. baumannii[368]. Treatment with combination therapy, including colistin/carbapenem and colistin/sulbactam, for XDRAB blood stream infections led to higher microbiological eradication and lower mortality rates in comparison with the colistin monotherapy group[369]. The combination therapy of colistin and tigecycline has also been proposed as a reasonable treatment of choice for XDRAB pneumonia, especially in the first 48 h, in a rat lung model[370]. Interestingly, a significant synergy has been observed for the combination of colistin and teicoplanin against MDRAB in vitro[371]. Telavancin, a similar lipoglycopeptide of teicoplanin, has been shown to be efficacious in vivo when used in colistin combination therapy in a Galleria mellonella model of A. baumannii infection[372].

Doripenem, a novel broad-spectrum carbapenem, has displayed in vitro synergistic activity with tigecycline, colistin and amikacin against MDRAB strains with doripenem resistance[373]. One recent prospective, observational Spanish study did not support an association of combination therapy with reduced mortality in MDRAB infections[374]. Overall, the choice of combination therapy should take several key factors into consideration, including the antimicrobial resistance phenotype, resistance mechanisms, and MIC[375].