Carbapenemases: Transforming Acinetobacter baumannii into a Yet More Dangerous Menace

Acinetobacter baumannii is a common cause of serious nosocomial infections. Although community-acquired infections are observed, the vast majority occur in people with preexisting comorbidities. A. baumannii emerged as a problematic pathogen in the 1980s when an increase in virulence, difficulty in treatment due to drug resistance, and opportunities for infection turned it into one of the most important threats to human health. Some of the clinical manifestations of A. baumannii nosocomial infection are pneumonia; bloodstream infections; lower respiratory tract, urinary tract, and wound infections; burn infections; skin and soft tissue infections (including necrotizing fasciitis); meningitis; osteomyelitis; and endocarditis. A. baumannii has an extraordinary genetic plasticity that results in a high capacity to acquire antimicrobial resistance traits. In particular, acquisition of resistance to carbapenems, which are among the antimicrobials of last resort for treatment of multidrug infections, is increasing among A. baumannii strains compounding the problem of nosocomial infections caused by this pathogen. It is not uncommon to find multidrug-resistant (MDR, resistance to at least three classes of antimicrobials), extensively drug-resistant (XDR, MDR plus resistance to carbapenems), and pan-drug-resistant (PDR, XDR plus resistance to polymyxins) nosocomial isolates that are hard to treat with the currently available drugs. In this article we review the acquired resistance to carbapenems by A. baumannii. We describe the enzymes within the OXA, NDM, VIM, IMP, and KPC groups of carbapenemases and the coding genes found in A. baumannii clinical isolates.


A Brief Summary of Acinetobacter baumannii as a Pathogen
Acinetobacter spp., a Gram-negative coccobacillus found in virtually all environments [1,2], used to be viewed as a "low-virulence" opportunistic pathogen of negligible significance. Despite signals about the potential this group of bacteria had as a nosocomial pathogen [3][4][5][6], its importance remained unappreciated until the mid-1990s [7]. Later, a better appreciation of the impact of Acinetobacter occurred after an increase in the understanding of its epidemiology identified it as the etiology of numerous hospital infections. In addition, an increase in virulence, difficulty of treatment due to drug resistance, and opportunities for infection, made this pathogen one of the most important threats

Mechanisms of Resistance to Carbapenems in A. baumannii
A. baumannii would not be the problematic pathogen it is without the multiple mechanisms of drug resistance the strains usually possess and its versatility to acquire new ones [9,51,[136][137][138][139][140]. Multiple intrinsic and acquired mechanisms of resistance have been detected in A. baumannii isolates [51]. These mechanisms include enzymatic modification of the antibiotic, changes in permeability, efflux pumps, or modifications in the target sites [51, 138,[141][142][143]. Multidrug-resistant (MDR; resistance to at least three classes of antimicrobials), extensively drug-resistant (XDR; MDR plus resistance to carbapenems), and pan-drug-resistant (PDR; XDR plus resistance to polymixins) [144] A. baumannii strains are currently being isolated in clinics making treatment increasingly harder [9,139,[145][146][147]. In particular, a "quantum leap" in the difficulty to treat A. baumannii infections occurred with the appearance of CRAB strains. Identified mechanisms of resistance to carbapenems include efflux pumps [51, [148][149][150][151][152], reduction or inactivation of expression of porins [51, [153][154][155], modification in expression or synthesis of new penicillin binding proteins [156,157], and presence of carbapenemases. Since the latter is the most frequently observed and worrisome mechanism present in CRAB strains, in this article, we review the carbapenemases detected in this bacterium.

Carbapenemases in A. baumannii: OXA β-Lactamases
One of the most common, if not the most common, mechanisms of resistance to β-lactam antibiotics is their enzymatic hydrolysis mediated by β-lactamases [158][159][160]. There is more than one classification scheme for β-lactamases, which number in the hundreds, but one of the most frequently used is that proposed by Ambler (amino acid sequence-based) that divides these enzymes into four molecular classes: A, B, C, and D [160][161][162]. Enzymes belonging to classes A, C, and D catalyze the hydrolysis of the β-lactam substrate forming an intermediate covalent acyl-enzyme complex with a serine residue within the active site. In the case of class B β-lactamases, the hydrolysis reaction is mediated by one or more zinc ions [160,161]. OXA β-lactamases are class D enzymes that were originally differentiated by TEM enzymes by their ability to hydrolyze oxacillin [163]. The first OXA enzymes showed relatively narrow spectrum and were plasmid-mediated [164][165][166][167]. Later, numerous cases of proteins, encoded by chromosome-or plasmid-located genes, with characteristics

Mechanisms of Resistance to Carbapenems in A. baumannii
A. baumannii would not be the problematic pathogen it is without the multiple mechanisms of drug resistance the strains usually possess and its versatility to acquire new ones [9,51,[136][137][138][139][140]. Multiple intrinsic and acquired mechanisms of resistance have been detected in A. baumannii isolates [51]. These mechanisms include enzymatic modification of the antibiotic, changes in permeability, efflux pumps, or modifications in the target sites [51, 138,[141][142][143]. Multidrug-resistant (MDR; resistance to at least three classes of antimicrobials), extensively drug-resistant (XDR; MDR plus resistance to carbapenems), and pan-drug-resistant (PDR; XDR plus resistance to polymixins) [144] A. baumannii strains are currently being isolated in clinics making treatment increasingly harder [9,139,[145][146][147]. In particular, a "quantum leap" in the difficulty to treat A. baumannii infections occurred with the appearance of CRAB strains. Identified mechanisms of resistance to carbapenems include efflux pumps [51, [148][149][150][151][152], reduction or inactivation of expression of porins [51, [153][154][155], modification in expression or synthesis of new penicillin binding proteins [156,157], and presence of carbapenemases. Since the latter is the most frequently observed and worrisome mechanism present in CRAB strains, in this article, we review the carbapenemases detected in this bacterium.

Carbapenemases in A. baumannii: OXA β-Lactamases
One of the most common, if not the most common, mechanisms of resistance to β-lactam antibiotics is their enzymatic hydrolysis mediated by β-lactamases [158][159][160]. There is more than one classification scheme for β-lactamases, which number in the hundreds, but one of the most frequently used is that proposed by Ambler (amino acid sequence-based) that divides these enzymes into four molecular classes: A, B, C, and D [160][161][162]. Enzymes belonging to classes A, C, and D catalyze the hydrolysis of the β-lactam substrate forming an intermediate covalent acyl-enzyme complex with a serine residue within the active site. In the case of class B β-lactamases, the hydrolysis reaction is mediated by one or more zinc ions [160,161]. OXA β-lactamases are class D enzymes that were originally differentiated by TEM enzymes by their ability to hydrolyze oxacillin [163]. The first OXA enzymes showed relatively narrow spectrum and were plasmid-mediated [164][165][166][167]. Later, numerous cases of proteins, encoded by chromosome-or plasmid-located genes, with characteristics of extended spectrum β-lactamase (ESBL) or carbapenemase OXA enzymes were described [160,163,168,169].

OXA-23-Like Group
The first OXA enzyme with carbapenemase activity to be identified in A. baumannii was the OXA-23 (first named ARI-1), found in an isolate from Scotland [170]. This enzyme gave the name to the first group of OXA enzymes with the capability to confer resistance to carbapenems (Table 1). Besides OXA-23, some enzymes within this group, such as OXA-27 or OXA-146, were subjected to more detailed analysis [171][172][173]. The bla OXA-23 gene is thought to have originated in A. radioresistens where close relatives are found located within the chromosome contiguous to a gene potentially coding for an ATPase [175,176]. After the first identification, the bla OXA-23 gene was detected in A. baumannii isolates from several countries, usually as part of Tn2006, Tn2007, Tn2009, Tn2008, and Tn2008B ( Figure 2). In all these elements, the bla OXA-23 is preceded by an IS element, ISAba1 or ISAba4, and contiguous to a truncated version of the ATPase [175,177]. Tn2006 has the typical structure of a composite transposon where the bla OXA-23 is part of a DNA stretch also containing the truncated ATPase gene and two genes, one of them coding for a helicase [177,178]. Tn2008 and Tn2008B consist of a copy of ISAba1 associated to the fragment including bla OXA-23 and the incomplete ATPase gene ( Figure 2). The difference between these two elements is the number of nucleotides between the ISAba1 and the OXA-23 initiation of translation codon, 27 and 34, respectively [177]. The ISAba1-bla OXA-23 -helicase region, i.e., the complete Tn2008 sequence, is contained within Tn2006. Furthermore, Tn2009 is a larger derivative of Tn2006 that acquired a DNA fragment between both ISAba1 copies [177]. Tn2006, Tn2008, and Tn2009 have been found within plasmids in addition to the chromosome [175,177,179,180]. Tn2007, detected in plasmids, consists of a DNA fragment including bla OXA-23 and a truncated version of the helicase gene preceded by a copy of ISAb4 ( Figure 2) [177]. Table 1 summarizes the number of OXA-23-like enzymes identified to date. The structure of Tn2007, which has not been proven to transpose, is shown on top. Tn2008 and Tn2008B differ in the number of nucleotides (27 and 34, respectively) that separate blaOXA-23 from ISAba1. Tn2006, the only structure experimentally shown to transpose [178], includes copies of ISAba1 in opposite orientations, while Tn2009 carries these insertion sequences in the same orientation. These two transposons are the only ones with the typical composite transposon structure. An extensive and detailed description of these elements was recently published [177]. ATPase*, gene coding for a putative ATPase truncated at the N-terminus. DADE, gene coding for a putative DEAD box family helicase [181]. The structure of Tn2007, which has not been proven to transpose, is shown on top. Tn2008 and Tn2008B differ in the number of nucleotides (27 and 34, respectively) that separate bla OXA-23 from ISAba1. Tn2006, the only structure experimentally shown to transpose [178], includes copies of ISAba1 in opposite orientations, while Tn2009 carries these insertion sequences in the same orientation. These two transposons are the only ones with the typical composite transposon structure. An extensive and detailed description of these elements was recently published [177]. ATPase*, gene coding for a putative ATPase truncated at the N-terminus. DADE, gene coding for a putative DEAD box family helicase [181]. Figure interpreted from Nigro and Hall (2016) [177].

OXA-24/40-Like Group
The number of beta-lactamases belonging to the OXA-24/40 group is less than that of the OXA-23 group and others to be discussed below. The first representative of this group to be isolated from A. baumannii was OXA-24, now known as OXA-40 after sequencing revision showed that they were identical enzymes [150,182]. The number of genes in this group, species that were found, and genetic location are shown in Table 1. Some plasmid-mediated genes belonging to the bla OXA24/40-like were found flanked by Xer site-specific recombination target sites, which led researchers to propose that this recombination mechanism plays a role in mobilization of these genes at the molecular level [183][184][185]. Xer site-specific recombination has been associated with plasmid evolution [186][187][188]. Structural studies showed that OXA-24/40 specifically utilizes carbapenems as substrate through a hydrophobic barrier for which the side chains of amino acids Y112 and M223 are essential [189]. This structure forms a tunnel-like entrance (hydrophobic bridge) to the active site. OXA-40 was also crystallized in complex to doripenem and inhibitors [190,191]. These studies were extended and details about the mechanism by which inhibitors inactivate OXA-24/40, such as decarboxylation of a K residue within, were clarified [189,191,192]. A clinical variant of OXA-24/40 identified in a clinical A. baumannii isolate has the mutation P227S, which results in an expanded substrate range that includes cephalosporins and aztreonam [193].

OXA-51-Like Group
OXA-51 was initially found as a chromosomally-mediated β-lactamase in a clinical A. baumannii isolate from Argentina [194]. Further studies showed the existence of numerous members of what is called the OXA-51-like group of enzymes [163]. The number of genes in this group, species that were found, and genetic location are shown in Table 1. Studies on the OXA-51 enzyme also showed that bla OXA-51-like genes are specific to the A. baumannii chromosome. As a consequence, bla OXA-51-like genes are used as a landmark in the identification of Acinetobacter isolates as A. baumannii [195,196]. A review describing available methodologies and their effectivity to identify A. baumannii has been recently published [197]. In contrast, identification of A. baumannii using bla OXA-51-like as a marker can present difficulties. Although these genes are mostly located within the chromosome, a few cases of plasmid location have been detected [198]. Plasmid-mediated bla OXA-51-like was the cause of misidentification of three non-baumannii Acinetobacter isolates that potentially acquired the gene from a plasmid originated in A. baumannii [199]. A. baumannii isolates from an outbreak in a hospital in Iran were hard to identify because the bla OXA-51-like was interrupted by an insertion of ISAba19 [200].
Early biophysical and biochemical studies on OXA-51 showed that the protein did not significantly denature when exposed to broad pH (4-10) and temperature (30-60 • C) ranges, and up to 75% of the enzymatic activity was retained in these conditions [201]. Enzymatic studies showed that OXA-51-like is a weak carbapenemase and the question of whether this enzyme or others within this group are responsible for carbapenem-resistance was the focus of numerous studies [152,163,198,[202][203][204]. Various bla OXA51-like genes were found associated to ISAbA1, which, when in the proper orientation, enhances expression and alone or in association with expression of other mechanisms can lead to phenotypic resistance to carbapenems [204][205][206]. However, levels of expression may not be the only way OXA-51-like enzyme confers diminished susceptibility to carbapenems. Variants with one or more amino acid substitutions with respect to OXA-51 exhibit enhanced hydrolytic activity [205,[207][208][209][210][211][212]. The molecular bases of several of these changes in activity associated to amino acids substitution were investigated by comparison of the three-dimensional structure of OXA-51 and the variant OXA-51 I129L in complex with doripenem [207,213]. This substitution increases the affinity of the protein for carbapenem molecules such as doripenem and imipenem. Variants including the substitution I129L show a significant increase in hydrolytic activity [208]. The results of the comparative analysis of these structures indicate that the presence of a Leu residue instead of Ile at position 129 causes the protein to better accommodate the substrate, increasing affinity and concomitantly enzymatic activity [207,213].
In conclusion, different OXA-51-like enzymes confer different degrees of or no clinical resistance to carbapenems, and they serve as a tool for molecular epidemiology of A. baumannii.

OXA-58-Like Group
This is a group of enzymes with a reduced number of variants ( Table 1). The first bla OXA-58 gene was present in a non-self-transmissible 30-kbp plasmid residing in a multiresistant A. baumannii isolated in the Rangueil University hospital, Toulouse, France [214]. The gene was flanked by ISAba3-like insertion element copies in opposite orientations. The same gene was detected in seven carbapenem-resistant isolates, six from patients, and one from the environment, in the burns unit of the same hospital [215], and rapidly spread to other countries [216]. A recent study showed that an A. seifertii plasmid is 99% homologous to one from A. baumannii, but the ISAba3-like insertion element located upstream of bla OXA-58 is interrupted by a copy of ISAba825 [217]. These studies argue in favor of horizontal transmission as the main mechanism of dissemination of bla OXA-58-like genes. Due to the difference in GC percent found between the gene and the A. baumannii chromosome, it is currently thought that bla OXA-58-like genes originated in a different bacterium [163].
The bla OXA-58 gene was carried by plasmids harbored by six clonally related A. baumannii strains isolated in 2005 from a hospital in Rome, Italy. The strains showed different levels of resistance to carbapenems, and the plasmids were nearly identical but differed at the regions carrying the bla OXA-58 gene. A structure consisting of IS26-ISAba2-bla OXA-58 -ISAba3 was identified that can induce amplification of the gene, producing an increase in MIC by gene dosage [218]. Gene amplification can occur through a wide variety of mechanisms [218], and its role in increasing levels of resistance has been shown multiple times [219][220][221][222][223].

OXA-143-Like Group
OXA-143 was first identified in a~30-kbp plasmid present in an A. baumannii blood culture isolate from a patient in an intensive care unit in Brazil in 2004 (Table 1) [224]. OXA-143 hydrolyzed penicillin and carbapenems but did not significantly hydrolyze expanded-spectrum cephalosporins. The bla OXA-143 gene was neither associated with insertion sequences nor located within an integron, but later, at least a bla OXA-143-like gene, bla OXA-253 , was located in a plasmid, downstream of ISAba47 [225]. On the other hand, bla OXA-143 and a downstream gene were flanked by copies of a gene coding for a replication protein, suggesting that its incorporation in the plasmid could have occurred by homologous recombination [224]. The OXA-143 amino acid sequence shows 88%, 63%, and 52% identity with OXA-40, OXA-23, and OXA-58, respectively. Later, other OXA-143-like enzymes were isolated in Brazil, Korea, Peru, and Honduras [225][226][227][228][229][230]. On the basis of the GC percent, these genes, which are located in plasmids, seem to have originated in a different Acinetobacter species [163].

OXA-235-Like Group
The OXA-235 and two variants, OXA-236 and OXA-237, which have one amino acid substitution (OXA-236 G173V and OXA-237 D208G), were identified in nine A. baumannii strains from the US and Mexico [231]. Location of the genes coding for these enzymes was chromosome or plasmid, with some strains showing positive signal within a plasmid and colocalization with bla OXA-51 . In all nine isolates having the bla OXA-235 , the gene was flanked by two copies of ISAba1. In a surveillance study performed between 2010 and 2016 in Canadian acute care hospitals, 94 carbapenem-resistant Acinetobacter sp. strains, of which 90 were A. baumannii, were selected for further analysis [232]. OXA-235-like proteins were detected in 48% of the strains. Furthermore, ISAba1 was associated to the bla OXA-235-like gene in all A. baumannii strains.
An outbreak that occurred across five healthcare facilities in Oregon, US, and lasted from June 2012 to October 2014, affected 16 patients and was caused by extensively drug-resistant A. baumannii [233]. The resistance to carbapenem was caused by the presence of the bla OXA-235-like gene bla OXA-237 , which was flanked by ISAba1 elements in opposite orientations in a 15.198-kbp plasmid present in all 16 isolates [233,234]. It was of special concern that bla OXA-237 is plasmid-mediated, a characteristic that enhances the potential for dissemination, and was found in a strain belonging to clonal group IC2, the most prominent worldwide [234].

Carbapenemases in A. baumannii: Metallo-β-Lactamases
The mechanism used by β-lactamases to catalyze hydrolysis of the antibiotic molecule can involve a two-step cycle consisting of acylation by formation of an acylenzyme intermediate by establishing a covalent bond between the β-lactam and a serine residue within the enzyme's active site followed by a deacylation step or a cation-facilitated hydrolytic reaction in which one or two essential zinc ions locate at the active site of the enzyme [235]. These enzymes, known as metallo-β-lactamases or Class B β-lactamases, are subdivided into three subclasses, B1-B3 [235].

NDM Group
NDM-1 is a metallo-β-lactamase (New Delhi metallo-β-lactamase) first isolated in India in 2008 from a patient with a urinary tract infection caused by a carbapenem-resistant Klebsiella pneumoniae [241]. The bla NDM-1 gene, located adjacent to a truncated IS26 element, was part of a 180-kbp plasmid that includes genetic determinants that confer resistance to all antibiotics except fluoroquinolones and colistin. It was also troublesome that an E. coli strain isolated form the same patient harbored a plasmid containing bla NDM-1 , suggesting that transfer by conjugation occurs at high frequency. This possibility was supported in mating assays and the authors predicted a worrisome scenario where the gene would spread to other bacteria [241]. Unfortunately, the predictions were correct, and bacteria harboring a plasmid containing the bla NDM-1 gene were soon isolated in infections across the world, [8,[242][243][244][245][246].
A search at the National Center for Biotechnology Information Pathogen Detection Browser (https://www.ncbi.nlm.nih.gov/pathogens) performed at the time this article was being written produced 2407 isolates carrying NDM-1. The number of NDM-1-possesing A. baumannii strains was 240, only surpassed by K. pneumoniae (1527) and Escherichia coli/Shigella (265 combined).
Except for monobactams, NDM-1 confers resistance to all other β-lactams [247]. Twenty-seven variants were described in addition to NDM-1 that present amino acid substitutions, and in one case there is a 5-amino acid repeat. A detailed description of the NDM-1 variants has been recently published [243]. According to this publication and newer data, NDM-1, NDM-2, NDM-3, NDM-5, and NDM-7 have been detected in A. baumannii [243,248,249]. Unfortunately, bla NDM-1 is usually associated to other genetic determinants that specify resistance to numerous antibiotics leaving only last-line antimicrobials, usually used in combination therapies, as options for treatment [250][251][252]. While the bla NDM-1 gene was found in more than one genetic context, in all cases described to date it is located downstream of ISAb125 sequences that provide the -35 region of the promoter [243]. In some instances, there is complete copy of the insertion sequence and in others only a fragment (identified as ISAba125*) [253]. This structure is associated to other genes, in many cases flanked by insertion sequences to form a composite transposon [243]. In most, but not all, cases, downstream of bla NDM-1 , there is a ble MBL gene (resistance to bleomycin) followed by trpF (phosphoribosylanthranilate isomerase), and dsbC (tat twin-arginine translocation pathway signal sequence domain protein). Other genes that usually follow these genes are cutA1 (periplasmic divalent cation tolerance protein) and groES-groEL (chaperonin) [243,254,255]. In many instances, this structure is followed by a copy of ISCR27 like in the A. baumannii transposon Tn125 and other elements ( Figure 3) [256][257][258][259]. These structures are found flanked by different elements that imply the existence of a wide variety of mechanisms of dissemination [243,253,255,[260][261][262][263]. In those cases where the structure is part of a composite transposon, this cluster of genes is flanked by ISAba125 (forming Tn125) [257], IS903, IS26, or IS3000, in direct or opposite orientations [243,252,253,256,264]. The structure ISAba125*-bla NDM-1 -ble MBL -trpF-dsbC-cutA1-groES-groEL has been also found flanked by short repeats with features characteristic of miniature inverted repeat elements [254].
An interesting feature of NDM-1 is related to its dependence on zinc ions, a property shared with other metallo-β-lactamases. It is well known that one of the mammals' innate immune responses to bacterial infections is the chelation of metal ions by high-affinity binding proteins [272,273]. Calprotectin, an important component of the cytosolic protein pool, was first identified by its ability to interfere with the growth of fungal and bacterial pathogens [274]. This property seems to be related to its ability to bind Zn +2 with high affinity. Calprotectin is released at the infection foci in the body, and one of its effects is to reduce the action of metallo-β-lactamases and induce degradation because the apo-enzyme is degraded in the periplasm [275]. Many metallo-β-lactamases elude this defense mechanism by being very efficient at binding the scarce zinc ions present in the periplasmic space [275]. NDM-1 differs from most metallo-β-lactamases in that limitation of zinc ions does not result in degradation because it is anchored to the outer membrane, a property that makes it refractory to destabilization [276]. Furthermore, this feature results in secretion of the protein with the release of outer membrane vesicles that shield infecting bacterial cells from high levels of β-lactams in their environment [276,277].

VIM Group
This type of metallo-β-lactamases were first identified in Pseudomonas aeruginosa isolates in Italy and France [278,279]. The variants were denominated VIM-1 and VIM-2, respectively, and were part of gene cassettes as part of class 1 integrons. However, while VIM-1-carrying integron was located within the chromosome and included a second aac(6′)-Ib-containing gene cassette in its variable region, the VIM-2-carrying integron was part of a ~45-kbp non conjugative plasmid and possessed a unique gene cassette. The geographical location of the first detection of these enzymes and their genetic environment are represented in the VIM name of the group (Verona integron-related metalloβ-lactamase). After the first identification, the number of VIM variants increased rapidly, mainly found in Enterobacteriaceae [160,280,281]. Forty-six VIM variants were identified in a search at the National Center for Biotechnology Information Pathogen Detection Browser (https://www.ncbi.nlm.nih.gov/pathogens) but 70 are presently uploaded in GenBank. Early studies to identify metallo-β-lactamases in a collection of isolates from Korean hospitals showed that in one study an A. baumannii isolate and in the other 27 out of 38 tested Acinetobacter spp. samples carried VIM-2 [282,283]. Five unrelated A. baumannii isolates obtained in two Greek hospitals in the years 2004 and 2005 included a class 1 integron that carried blaVIM-1 in the variable portion [284]. The nontransferable characteristic of blaVIM-1 in these isolates suggests chromosomal or a nonconjugative As it is the case with other resistance proteins like the pJHCMW1-coded AAC(6 )-Ib, which has a fusion at the N-terminus where the first six amino acids are identical to those of the TEM β-lactamase [141,142,166], analysis of the nucleotide sequence of bla NDM-1 and the upstream region showed that the first 6 amino acids of the protein are identical to those of the aminoglycoside phosphotransferase APH(3 )-Via [256], a protein associated to A. baumannii strains [265]. Furthermore, the GC content of the gene changes abruptly at the point of divergence. These facts, together with the known association of ISAba125 to A. baumannii, led to propose that bla NDM-1 originated through a fusion that took place in this bacterium [256]. With a few exceptions in which bla NDM-1 was found located within the chromosome, the gene is harbored in plasmids. Upwards of 350 different plasmids and 20 replicons harboring bla NDM genes were described [243]. The rich number of plasmid varieties and transposable elements that carry bla NDM genes explain their fast dissemination at the molecular and cellular levels [243]. The structure and biochemistry of NDM-1 has been thoroughly studied [252,[266][267][268][269][270][271].
An interesting feature of NDM-1 is related to its dependence on zinc ions, a property shared with other metallo-β-lactamases. It is well known that one of the mammals' innate immune responses to bacterial infections is the chelation of metal ions by high-affinity binding proteins [272,273]. Calprotectin, an important component of the cytosolic protein pool, was first identified by its ability to interfere with the growth of fungal and bacterial pathogens [274]. This property seems to be related to its ability to bind Zn 2+ with high affinity. Calprotectin is released at the infection foci in the body, and one of its effects is to reduce the action of metallo-β-lactamases and induce degradation because the apo-enzyme is degraded in the periplasm [275]. Many metallo-β-lactamases elude this defense mechanism by being very efficient at binding the scarce zinc ions present in the periplasmic space [275]. NDM-1 differs from most metallo-β-lactamases in that limitation of zinc ions does not result in degradation because it is anchored to the outer membrane, a property that makes it refractory to destabilization [276]. Furthermore, this feature results in secretion of the protein with the release of outer membrane vesicles that shield infecting bacterial cells from high levels of β-lactams in their environment [276,277].

VIM Group
This type of metallo-β-lactamases were first identified in Pseudomonas aeruginosa isolates in Italy and France [278,279]. The variants were denominated VIM-1 and VIM-2, respectively, and were part of gene cassettes as part of class 1 integrons. However, while VIM-1-carrying integron was located within the chromosome and included a second aac(6 )-Ib-containing gene cassette in its variable region, the VIM-2-carrying integron was part of a~45-kbp non conjugative plasmid and possessed a unique gene cassette. The geographical location of the first detection of these enzymes and their genetic environment are represented in the VIM name of the group (Verona integron-related metallo-β-lactamase). After the first identification, the number of VIM variants increased rapidly, mainly found in Enterobacteriaceae [160,280,281]. Forty-six VIM variants were identified in a search at the National Center for Biotechnology Information Pathogen Detection Browser (https://www.ncbi.nlm.nih.gov/pathogens) but 70 are presently uploaded in GenBank. Early studies to identify metallo-β-lactamases in a collection of isolates from Korean hospitals showed that in one study an A. baumannii isolate and in the other 27 out of 38 tested Acinetobacter spp. samples carried VIM-2 [282,283]. Five unrelated A. baumannii isolates obtained in two Greek hospitals in the years 2004 and 2005 included a class 1 integron that carried bla VIM-1 in the variable portion [284]. The nontransferable characteristic of bla VIM-1 in these isolates suggests chromosomal or a nonconjugative plasmid. The gene was also detected in eight out of 13 strains tested in a study in Saudi Arabia and Iran where bla VIM-2 was also present [285][286][287]. Both bla VIM-1 and bla VIM-2 were also detected in a study of 100 A. baumannii isolates from three teaching hospitals in Iran [288]. Other VIM-type enzymes identified in A. baumannii were VIM-3 and VIM-11 in strains from a university hospital in Taiwan [289]. Several studies identified unspecified VIM-type enzymes in A. baumannii clinical isolates [290][291][292][293][294][295][296]. Blast analysis, carried out at the time of writing of this article, demonstrated that the genes coding for VIM-6, VIM-11, and VIM-25 were also detected in A. baumannii.

IMP Group
The first metallo-β-lactamase in the IMP (active on imipenem) group was identified in an imipenem-resistant P. aeruginosa clinical strain collected in 1988 in Japan [297]. The bla IMP gene resided in a conjugative 47.7-kbp plasmid, pMS350, belonging to the P-9 incompatibility group that also conferred resistance to gentamicin and sulfonamide [297]. The blaI MP gene was then found in the chromosome and as part of an integron in a transferable plasmid of urinary tract infection Serratia marcescens isolates from Japan [298][299][300]. Rapid dissemination was soon described, an expected outcome due to the location in transferable plasmids. P. aeruginosa and K. pneumoniae clinical strains carrying bla IMP were identified, and a six-years study at the Nagasaki University Hospital, Japan, identified bla IMP in 80 clinical strains belonging to the species P. aeruginosa, P. putida, P. stutzeri, and Citrobacter freundii [301][302][303]. The first report of an IMP-type enzyme in Europe occurred in a multidrug-resistant A. baumannii strain isolated from the respiratory tract of an intensive care unit patient in Italy. Further analysis showed that the gene coded for IMP-2 and was carried in a gene cassette as part of a class I integron located in the chromosome (Figure 4) [304,305]. The bla IMP-2 gene cassette was located downstream of intI1, and the variable region also included aac(6 )-Ib and ant(3 )-Ia [304]. A Blast comparison between the aac(6 )-Ib-ant(3 )-Ia region from this transposon and that one from the Tn1331 [306][307][308] showed 99% homology with divergence at the point of action of IntI1 at the N-terminus coding sequences of aac(6 )-Ib, a well-known characteristic of this gene that exhibits heterogeneous N-termini [141,[309][310][311] (Figure 4). The second identification of an IMP-type enzyme in Europe was also on a nosocomial A. baumannii isolate in Portugal. Analysis of this gene concluded that it was a new member, named bla IMP-5 because bla IMP-3 and bla IMP-4 had been identified earlier in Asia [312][313][314][315]. IMP-4 was later found within a class 1 integron in A. baumannii isolates [316] and in similar integrons in A. pitti, K. pneumoniae, E. coli, and Enterobacter cloacae strains. In the cases of the E. coli and two E. cloacae strains, it was confirmed that the gene resided in a plasmid [317,318]. The genetic context of bla IMP-4 present in A. junii was not determined [319]. To date, A. baumannii strains carrying bla IMP-1 , bla IMP-2 , bla IMP-4 , bla IMP-5 , bla IMP-6 , bla IMP-8 , bla IMP-11 , bla IMP-14a , bla IMP-19 , and bla IMP-55 , have been reported. The bla IMP-55 was identified in a study of 65 multidrug-resistant A. baumannii strains isolated in two intensive care units in Iran which showed that 6 of them contained integrons including bla IMP-55 genes [320].

Figure 4.
Comparison of the In42 variable region and a Tn1331 resistance genes fragment. The genetic maps of the variable region of In42 and a resistance genes fragment of Tn1331 are aligned. All attC loci are shown equally in the figure, but they are not identical at the nucleotide sequence level. The attI1* structure and functionality have been described [306]. The aac(6′)-Ib genes code for proteins that show differences at the N-termini, a known characteristic of this gene [141,309,310].

Carbapenemases in A. baumannii: KPC β-Lactamases
Enzymes within the KPC (K. pneumoniae carbapenemase) group are class A serine carbapenemhydrolyzing β-lactamases that were initially reported in 2001, when KPC-1 was identified in a K. pneumoniae strain collected in 1996 from a hospital in North Carolina [321]. The gene quickly disseminated among Klebsiella strains and other bacteria and a high percentage of multidrug-resistant isolates were found to carry a blaKPC gene [322][323][324][325][326][327][328][329][330]. Unfortunately, bacteria carrying KPC enzymes are usually multidrug resistant, which seriously limits the antibiotic options for treatment; as a consequence, mortality among patients infected with KPC-harboring bacteria is high [328]. Interestingly, in some locations, there has been a decline in the isolates harboring these genes [325,331]. The quick and efficient dissemination of blaKPC genes is due to their usual location within transposable elements, mainly Tn4401 or close relatives [332] in conjugative plasmids [333][334][335][336]. Tn4401 is also commonly inserted within other transposon or insertion sequences generating multidrug resistance transposable elements [337,338]. It is worth noting that although unusual, blaKPC genes are found associated to non-Tn4401 genetic environments [339][340][341]. A search at the National Center for Biotechnology Information Pathogen Detection Browser (https://www.ncbi.nlm.nih.gov/pathogens) found 47 variants of KPC and 8716 records. There are numerous reports of A. baumannii carrying undetermined blaKPC genes [294,342,343]. Confirmed variants of blaKPC found in A. baumannii include blaKPC-2 and blaKPC-3, first identified in a study to characterize the resistance to carbapenem in A. baumannii (CRAB) isolates from patients with burn injury in Brazil [344]. Moreover, a study of Acinetobacter isolates from 17 hospitals in Puerto Rico identified blaKPC-4 and blaKPC-10 [345].

Final Remarks
A. baumannii emerged as a problematic nosocomial pathogen in the mid-1980s, when acquisition of resistance traits became significant, making treatment more challenging [11,353]. The genetic plasticity of Acinetobacter spp. permitted this bacterium to evolve rapidly, turning it into one the most serious threats to hospitalized patients [52,53, [354][355][356]. Currently, multi-and pan-drug-resistant A. baumannii are ubiquitous, and options for treatment are shrinking. The rise of carbapenem-resistant A. baumannii strains (CRABs) further compounds the problem, which requires urgent attention to avoid expanding the number of deaths due to nosocomial infection. Among the many mechanisms Figure 4. Comparison of the In42 variable region and a Tn1331 resistance genes fragment. The genetic maps of the variable region of In42 and a resistance genes fragment of Tn1331 are aligned. All attC loci are shown equally in the figure, but they are not identical at the nucleotide sequence level. The attI1* structure and functionality have been described [306]. The aac(6 )-Ib genes code for proteins that show differences at the N-termini, a known characteristic of this gene [141,309,310].

Carbapenemases in A. baumannii: KPC β-Lactamases
Enzymes within the KPC (K. pneumoniae carbapenemase) group are class A serine carbapenem-hydrolyzing β-lactamases that were initially reported in 2001, when KPC-1 was identified in a K. pneumoniae strain collected in 1996 from a hospital in North Carolina [321]. The gene quickly disseminated among Klebsiella strains and other bacteria and a high percentage of multidrug-resistant isolates were found to carry a bla KPC gene [322][323][324][325][326][327][328][329][330]. Unfortunately, bacteria carrying KPC enzymes are usually multidrug resistant, which seriously limits the antibiotic options for treatment; as a consequence, mortality among patients infected with KPC-harboring bacteria is high [328]. Interestingly, in some locations, there has been a decline in the isolates harboring these genes [325,331]. The quick and efficient dissemination of bla KPC genes is due to their usual location within transposable elements, mainly Tn4401 or close relatives [332] in conjugative plasmids [333][334][335][336]. Tn4401 is also commonly inserted within other transposon or insertion sequences generating multidrug resistance transposable elements [337,338]. It is worth noting that although unusual, bla KPC genes are found associated to non-Tn4401 genetic environments [339][340][341]. A search at the National Center for Biotechnology Information Pathogen Detection Browser (https://www.ncbi.nlm.nih.gov/pathogens) found 47 variants of KPC and 8716 records. There are numerous reports of A. baumannii carrying undetermined bla KPC genes [294,342,343]. Confirmed variants of bla KPC found in A. baumannii include bla KPC-2 and bla KPC-3 , first identified in a study to characterize the resistance to carbapenem in A. baumannii (CRAB) isolates from patients with burn injury in Brazil [344]. Moreover, a study of Acinetobacter isolates from 17 hospitals in Puerto Rico identified bla KPC-4 and bla KPC-10 [345].
Enzymes belonging to the KPC group are the only ones that recognize as substrates all FDA-approved β-lactams.

Final Remarks
A. baumannii emerged as a problematic nosocomial pathogen in the mid-1980s, when acquisition of resistance traits became significant, making treatment more challenging [11,353]. The genetic plasticity of Acinetobacter spp. permitted this bacterium to evolve rapidly, turning it into one the most serious threats to hospitalized patients [52,53,[354][355][356]. Currently, multi-and pan-drug-resistant A. baumannii are ubiquitous, and options for treatment are shrinking. The rise of carbapenem-resistant A. baumannii strains (CRABs) further compounds the problem, which requires urgent attention to avoid expanding the number of deaths due to nosocomial infection. Among the many mechanisms causing resistance, the acquisition of carbapenemase-coding genes are the most relevant. The majority of carbapenem-resistant A. baumannii isolates owes this property to the presence of OXA-23. The prevalence of bla OXA-23 is, at least in part, attributed to the spread of successful global clones such us GC1 and GC2. However, the number of isolates containing NDM-1 is rapidly growing. Research efforts geared to developing new antimicrobials must be complemented with strategies to overcome the presence of these enzymes such as the introduction of new inhibitors to be used in combination with antibacterial drugs. Despite the grim perspective of witnessing the rise of strains resilient to all available treatments, investigative attempts to introduce new inhibitors and drugs give hope that options to control these infections will continue to be available.