Genetic basis of antibiotic resistance in pathogenic Acinetobacter species
Antibiotic resistance in Acinetobacter spp., particularly Acinetobacter baumannii, is increasing rapidly. A. baumannii possesses two intrinsic β-lactamase genes, in addition to weak permeability and efflux systems, that together confer a natural reduced susceptibility to antibiotics. In addition, numerous acquired mechanisms of resistance have been identified in A. baumannii. The very high genetic plasticity of A. baumannii allows an accumulation of resistance determinants that give rise to multidrug resistance at an alarming rate. The role of novel genetic elements, such as resistance islands, in concentrating antibiotic resistance genes in A. baumannii requires detailed investigation in the near future. © 2011 IUBMB IUBMB Life, 63(12): 1061–1067, 2011
Members of the genus Acinetobacter seem to have the ability to develop resistance to new antibiotics extremely rapidly. Most multidrug-resistant clinical isolates of Acinetobacter belong to Acinetobacter baumannii or its close relatives. Resistance of A. baumannii isolates to broad-spectrum antibiotics such as amikacin, expanded-spectrum cephalosporins, carbapenems, and tigecycline is on the rise, with the result that physicians are increasingly left with very few effective therapeutic options. A.baumannii possesses a range of intrinsic resistance determinants, but recent data indicate that A. baumannii can also acquireresistance determinants identified in other clinically-significant Gram-negative species such as Enterobacteriaceae and Pseudomonas aeruginosa. This review considers the nature and expression of the intrinsic and acquired resistance determinants found in A. baumannii, with particular emphasis on resistance toβ-lactams and carbapenems, as well as the role played by insertion sequences (ISs) and novel genetic elements, such as resistance islands, in contributing to the genome plasticity found in this species and the consequent rapid accumulation of determinants contributing to multidrug resistance.
GENETIC BASIS OF RESISTANCE TO β-LACTAMS
A. baumannii produces an intrinsic AmpC-type cephalosporinase that is normally expressed at a low level and does not modify the efficacy of expanded-spectrum cephalosporins or carbapenems (1). Insertion of ISAba1 (belonging to the IS4 family) upstream of the blaampC gene enhances expression by providing promoter sequences, resulting in resistance to expanded-spectrum cephalosporins (2, 3). Extended-spectrum ampC-type β-lactamases (ESAC) have also been identified in A. baumannii, possessing activity toward expanded-spectrum cephalosporins and monobactams due to a Pro210Arg substitution and a duplication of an Ala residue at position 215 (inside the Ω-loop). These enzymes hydrolyze all cephalosporins, but do not compromise the efficacy of carbapenems (4).
A. baumannii also expresses another chromosomally-encoded intrinsic β-lactamase, OXA-51, for which many point-mutant variants have been described (5). These enzymes normally have low levels of carbapenemase activity, but may be overproduced when their genes are supplied with efficient promoters by ISAba1 or ISAba9, and may subsequently impact susceptibility to carbapenems in A. baumannii (6, 7).
Acquired Narrow-spectrum β-lactamases
Narrow-spectrum clavulanic acid inhibited penicillinases (TEM-1 and TEM-2, CARB-5) and clavulanic acid-resistant oxacillinases (OXA-20, OXA-21) have been reported in A. baumannii (8). The clavulanate-inhibited penicillinase SCO-1 has also been identified in various Acinetobacter spp., including A. baumannii, Acinetobacter junii, Acinetobacter baylyi, and Acinetobacter johnsonii (9).
Acquired clavulanic acid Inhibited Extended-spectrum β-lactamases (ESBLs)
The first ESBL identified in A. baumannii was PER-1 (10). Sequence analysis revealed that a novel IS element, ISPa12, was upstream of blaPER-1, while blaPER-1 was bracketed downstream by another IS element, ISPa13, which was structurally unrelated to ISPa12. Together with ISPa12, ISPa13 forms a composite transposon named Tn1213 (11). ISPa12 also plays a role in the expression of the blaPER-1 gene by providing promoter sequences.
The blaPER-7 gene, encoding greater resistance to third-generation cephalosporins and monobactams than PER-1, has also been identified in A. baumannii, with an ISCR1 element immediately upstream blaPER-7. ISCR1 differs from other ISs since it lacks terminal inverted repeats and might transpose by a mechanism termed rolling-circle transposition. ISCR1 possesses a promoter sequence named PCR1-1 at its right extremity that is responsible for the expression of blaPER-7 (12). PER-2, which is distantly related to PER-1, has been found exclusively in South American isolates of A. baumannii, associated with a single copy of ISPa12 (13).
Another important ESBL found in A. baumannii is VEB-1. Found initially in France, where it disseminated nationwide in hospitals, the blaVEB-1 gene was identified as a gene cassette in class 1 integrons (see below) that varied in size and structure (14, 15).
A single A. baumannii isolate producing CTX-M-2 has been identified in Japan (16), and CTX-M-43 and CTX-M-2 producers have been detected in A. baumannii isolates from Bolivia and Pennsylvania, USA, respectively (17, 18). There are also rare reports of blaSHV and blaTEM type ESBL genes in A. baumannii, located either on the chromosome (blaSHV-5) or on plasmids (blaTEM-92, blaSHV-12, blaTEM-116) (19–21).
RTG-4 was the first reported carbenicillinase with ESBL properties and was identified in an A. baumannii isolate from France (22). RTG-4 is an atypical ESBL in that it hydrolyzes cefepime and cefpirome significantly, but not ceftazidime or cefotaxime (22). ISAba9 was identified upstream of the blaRTG-4 gene and might be involved in transposition of the blaRTG-4 gene by a single-ended transposition mechanism (22).
Overall, expression of ESBLs in A. baumannii may contribute significantly to resistance to broad-spectrum cephalosporins and to the increasingly observed multidrug resistance patterns. The ESBL genes are often associated with aminoglycoside resistance genes (see below). Detection of ESBLs in A. baumannii can be difficult since they may be associated with over-expression of naturally-occurring cephalosporinases.
Acquired Carbapenem-hydrolyzing β-lactamases
Carbapenems have been the mainstay of treatment of A. baumannii infections for the past two decades. However, a number of acquired β-lactamases have been identified as a source of carbapenem resistance in A. baumannii. They belong to either the class D (oxacillinases), class B (metallo-β-lactamases), or class A families (Table 1) (8).
Table 1. Acquired carbapenem-hydrolyzing ß-lactamases identified in A. baumannii
|IMP-1||B||Plasmid||Integron||Italy, Japan, South Korea|
|OXA-23||D||Plasmid or chromosomal||Tn2006, Tn2007, Tn2008||UK, French Polynesia, Korea, Brazil, Iraq, China|
|OXA-40||D||Plasmid or chromosomal||a||France, Spain, Portugal, USA|
|OXA-58||D||Plasmid or chromosomal||RE element||France, Spain, Italy, Greece, Austria, UK, Romania, Iraq, Argentina, Kuwait|
|OXA-97||D||Plasmid or chromosomal||RE element||Tunisia|
Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are often involved in resistance to carbapenems in A. baumannii (11). Although they only confer reduced susceptibility to carbapenems (and do not possess significant activity against expanded-spectrum cephalosporins), their association with additional resistance mechanisms such as efflux or impermeability gives rise to a high level of carbapenem resistance.
The first CHDL identified was OXA-23 (ARI-1) which, together with its point mutant derivative OXA-27, constitutes a major subgroup of CHDLs. OXA-23 is the most widespread CHDL in A. baumannii and has been identified worldwide (23). The blaOXA-23 gene has been identified as part of various transposon structures, namely Tn2006, Tn2007, and Tn2008 (23, 24). Interestingly, the natural reservoir of the blaOXA-23 gene has been identified as Acinetobacter radioresistens, which is normally a non-pathogenic environmental species (25).
A second group of CHDLs identified on either the chromosome or plasmids in A. baumannii comprises OXA-40 (also named OXA-24), OXA-25, and OXA-26. OXA-40 producers have also been reported worldwide, but are found particularly in the USA, Spain, and Portugal (5).
A third group of CHDLs comprises OXA-58 and its variant OXA-97. Again, epidemiological surveys have identified the blaOXA-58 gene in worldwide isolates of A. baumannii (5). The blaOXA-58-like genes have been identified as being plasmid-encoded and associated with specific ISs (but are not part of transposons). These ISs play a role in enhancing expression of the genes encoding these enzymes, but not in their acquisition, which may occur by homologous recombination processes rather than by transposition (26). OXA-58 has also been identified in other Acinetobacter species, such as A. junii, and Acinetobacter genomic species 3 and 13TU (5).
Metallo-β-lactamases (MBLs) are much powerful carbapenemases (27). Although reported mainly in Pseudomonas aeruginosa, four groups of MBLs have also been described in A. baumannii, namely IMP-like, VIM-like, SIM-1, and NDM-type enzymes (5, 8). Analysis of the genetic support of the MBL-encoding genes identified in A. baumannii showed that the blaIMP, blaVIM, and blaSIM genes each occur as gene cassettes embedded in class 1 integron structures (see below). The most recent MBLs identified in A. baumannii are the emerging NDM enzymes. The blaNDM genes have mostly been reported in Enterobacteriaceae, but there have been reports of NDM-1 in A. baumannii from India (28) and Germany (29). These isolates were resistant to high levels of all β-lactams, including carbapenems. Additionally, the first known variant of the blaNDM-1 gene, namely blaNDM-2, encoding NDM-2, was identified in an A. baumannii isolate from Egypt (30) and was associated with ISAba125, which provided promoter sequences involved in blaNDM-2 expression. Overall, MBLs confer high levels of carbapenem resistance in A. baumannii, and also confer resistance to other β-lactams with the exception of aztreonam.
Finally, an emerging group of carbapenem-hydrolyzing β-lactamases in A. baumannii comprises the class A carbapenemases. Ten KPC-positive Acinetobacter isolates belonging to the Acinetobacter calcoaceticus–A. baumannii complex were identified during PCR-based surveillance of β-lactam resistance in Puerto Rico. DNA sequencing of the blaKPC genes identified KPC-2, −3, and −4, and a novel variant, KPC-10 (31). In addition, a carbapenem-hydrolyzing GES-type enzyme was identified in A. baumannii from a hospital patient in Paris, France. This ESBL, named GES-14, differs from GES-1 by two amino acid substitutions, and can significantly compromise the efficacy of all β-lactams, including cephalosporins, aztreonam, and carbapenems. The blaGES-14 gene was located in a peculiar class 1 integron structure located on a ca. 95-kb self-transferable plasmid (32).
Non-enzymatic Resistance to β-lactams and in particular carbapenems
Resistance to carbapenems in A. baumannii may be enhanced by interactions between broad-spectrum β-lactamases and other resistance mechanisms, including porin(s) loss, active drug efflux, and (rarely) modification of penicillin-binding proteins (PBPs) (8). Several reports have associated decreased expression of certain porins with antimicrobial resistance in A. baumannii, including several outer membrane proteins (OMPs) that have some homology with the monomeric OmpA porin found in Enterobacteriaceae. Porins of this family have been characterized in several species of Acinetobacter, including A. radioresistens, A. junii and A. baumannii, and are known as slow porins that allow the penetration of β-lactams (33). Three OMPs have been associated with resistance or decreased susceptibility to carbapenems, namely a 33–36 kDa protein, a 29 kDa protein also known as CarO, and a 43 kDa protein, that show homologies with OprD from P. aeruginosa (34). An additional OMP (OmpW) has been identified in A. baumannii that shows significantly decreased expression in ceftriaxone-resistant clinical isolates (34). However, additional studies are required in order to determine their genetic basis of these proteins and to better evaluate their respective contributions in term of antibiotic resistance.
Efflux-mediated resistance is a common factor affecting antibiotic susceptibility in Gram-negative bacteria, and several efflux pumps have been described in A. baumannii (35). The AdeABC (Acinetobacter drug efflux) pump belongs to the Resistance-Nodulation-cell Division (RND) family (35). The adeABC operon encodes the AdeA Major Fusion Protein (MFP), the inner membrane protein AdeB and the outer membrane factor (OMF) AdeC. Inactivaction experiments in clinical isolates over-expressing AdeABC showed that cefepime, cefpirome, and cefotaxime were the most affected β-lactams (35). Over-expression of the naturally-occurring AdeABC efflux pump in association with carbapenem-hydrolyzing oxacillinases may confer a high-level of resistance to carbapenems (36). A second RND efflux pump that contributes to β-lactam resistance in A. baumannii is AdeIJK, but no effect of this pump on carbapenem resistance has been observed (35).
GENETIC BASIS OF RESISTANCE TO QUINOLONES AND FLUOROQUINOLONES
Quinolones and fluroquinolones act by binding to bacterial gyrase (encoded by gyrA andgyrB genes) and topoisomerase IV (encoded by parA and parC genes) enzymes (37). In Enterobacteriaceae, specific mutations in the Quinolone-Resistance-Determining-Region (QRDR) are known to impact on susceptibility to quinolones and fluoroquinolones. These changes result in a lower affinity for the binding of the quinolone to the enzyme-DNA complex. Mutations in the target sites of quinolones and fluoroquinolones have also been extensively reported in A. baumannii. In particular, mutations resulting in a Ser-86-Leu substitution in GyrA, and a Ser-80-Leu substitution in ParC increase the MICs of ciprofloxacin in clinical isolates (34).
The other quinolone resistance mechanism found in A. baumannii involves active efflux by one of the five efflux pumps, namely three RND efflux pumps, AdeABC, AdeIJK, and the recently described AdeFGH, the small multidrug-resistant efflux pump (SMR) AbeS, and the multi-drug and toxic compound extrusion efflux pump (MATE) AbeM. These systems can pump-out fluoroquinolones and may contribute by themselves to low-level resistance to these antibiotics (35).
All of these resistance mechanisms are chromosomally-encoded and, to date, no plasmid-mediated quinolone resistance determinants have been identified in A. baumannii.
GENETIC BASIS OF RESISTANCE TO AMINOGLYCOSIDES
Multiple aminoglycoside-modifying enzymes, including phosphotransferases, acetyltransferases, and adenyltransferases, have been reported in the last 20 years. Most aminoglycoside resistance in Acinetobacter spp. involves production of aminoglycoside-modifying enzymes, and all three classes have been found in A. baumannii (38). The genes encoding these enzymes are often embedded in class 1 integrons (see below). Among the latest aminoglycoside acetyltransferases, AAC(6′)I-ad has been identified in Acinetobacter spp. and may play a significant role in resistance to amikacin (39). In addition, plasmid-mediated RNA methylases such as ArmA have been identified in Acinetobacter isolates from Japan and the USA (39, 40); these mediate resistance to most aminoglycosides by modifying 16S rRNA. ArmA was identified inside transposon Tn1548, a large composite transposon formed by two copies of IS26 (41). A complex class 1 integron structure (associated with insertion sequence ISCR1) has been identified downstream of the armA gene.
GENETIC BASIS OF RESISTANCE TO RIFAMPICIN
Rifampicin binds to conserved amino acids in the active site of the bacterial RNA polymerase and blocks transcription initiation. A large proportion of rifampicin resistance results from chromosomal mutations leading to amino acid changes in the active site (38). Transferable resistance to rifampicin associated with the arr-2 gene has also been described in A. baumannii (42). This gene encodes a rifampicin ADP-ribosylating transferase that inactivates rifampicin by ribosylation. The arr-2 gene was identified as a gene cassette within a class 1 integron structure, in association with the ESBL gene blaVEB-1. The arr-2 gene has also been identified in association with the blaPER-7 ESBL gene in a complex class 1 integron (15).
GENETIC BASIS OF RESISTANCE TO CYCLINES
Cyclines exhibit bacteriostatic action by reversibly binding to the 30S ribosomal subunit and inhibiting protein translation (43). Resistance to tetracyclines has been well-documented in A. baumannii (38). The TetA and TetB resistance determinants are specific efflux pump proteins that remove tetracyclines from the cell. Ribosome protection mediated by the widely distributed TetM determinant may also occur (39, 40). The glycylcyclines are derivatives of the tetracycline antibiotics, with structural modifications that confer antimicrobial activity against several multidrug-resistant species. Tigecycline is the first commercially available member of the glycylcyclines, and multidrug-resistant strains of A. baumannii often retain susceptibility to tigecycline. Known mechanisms of resistance to tigecycline mainly involve active efflux by AdeABC, AdeIJK, and AdeFGH (35, 44). Several worldwide reports indicate an increasing trend of resistance to tigecycline in isolates of A. baumannii (40).
GENETIC BASIS OF RESISTANCE TO COLISTIN AND POLYMYXINS
These compounds bind to and disrupt the negatively charged outer-membrane of Gram-negative bacteria (45). The main mechanisms of resistance these antibiotics involve: (i) reduction of the net negative charge of the outer-membrane protein by modification of lipid A, an essential component of the bacterial lipopolysaccharide (LPS); and (ii) proteolytic cleavage of the antimicrobial compound and exclusion of peptides by a broad-spectrum efflux-pump. In A. baumannii, point mutations and frameshifts in the pmrA and pmrB genes have been shown to cause an increase in colistin MICs (46). PmrAB is a two-component system involved in environmental sensing of pH and ion concentration.
GENETIC BASIS OF RESISTANCE TO OTHER CLASSES OF ANTIBIOTICS, DISINFECTANTS AND HEAVY METALS
Resistance to sulfonamides is highly prevalent in A. baumannii and other Acinetobacter spp. since the sul1 gene, encoding an efflux-based mechanism of resistance to sulfonamides, is always associated with class 1 integrons, that are themselves highly prevalent in Acinetobacter spp. (39). Resistance to trimethoprim, another antibiotic acting on folate synthesis, is often mediated by dfr genes that are distributed widely in Gram-negative bacteria in general. Resistance to trimethoprim in A. baumannii may also be associated with over-expression of intrinsic efflux pumps such as AdeABC, AdeFGH, and AbeM (35).
Chloramphenicol inhibits bacterial protein synthesis and possesses broad-spectrum activity that includes Acinetobacter spp. Resistance to chloramphenicol is often encoded by genes located on integrons, such as the cmlA or cat genes that encode an efflux pump and a chloramphenicol-modifying enzyme, respectively. The role of intrinsic efflux pumps has also been demonstrated, particularly for the CraA (‘chloramphenicol resistance Acinetobacter’) pump, which pumps-out chloramphenicol specifically, as well as AbeM belonging to the MATE family, and AbeS belonging to the SMR family (35).
Resistance to macrolides is intrinsic to Acinetobacter spp., but plasmid-mediated resistance to macrolides associated with Tn1548 (harboring a 16S RNA methylase armA gene) has also been reported. The mel and mph genes, encoding an efflux pump and a macrolide-modifying enzyme, respectively, have also been identified in A. baumannii (41).
Resistance to heavy metals, dyes, and disinfectants is mainly mediated by intrinsic efflux pumps. Genome analysis has revealed that A. baumannii contains several operons with genes encoding efflux pumps that confer resistance to different heavy metals and disinfectants (42). The operon encoding resistance to mercury is encoded by transposon Tn21 (42).
GENETIC VEHICLES FOR RESISTANCE DETERMINANTS IN ACINETOBACTER SPP.
The main genetic structures associated with acquired antibiotic resistance genes in A. baumannii are integrons and transposons. Class 1 integrons are genetic elements that contain the determinants of a site-specific recombination systems through which they can capture resistance genes (38), while transposons can be classified into two main classes: (i) Tn3-type transposons (also named class II transposons); and (ii) composite transposons (also named class I transposons), made of two copies of same or closely-related ISs. These structures are usually found as part of larger DNA structures, including the main chromosome and numerous plasmids.
Whole-genome sequencing of the epidemic A. baumannii strain AYE, responsible for a large epidemic outbreak of infection in France during 2004, revealed a large cluster of resistance determinants on the chromosome of this clinical strain (42). This 86-kb resistance island, designated AbaR1, contained >40 antibiotic or heavy metal resistance genes, including the blaVEB-1 ESBL gene (42). Other AbaR elements have been identified in different multidrug-resistant A. baumannii isolates, and one has been identified in the multidrug-susceptible A. baumannii strain SDF (47–49). To date, all AbaR elements have been chromosomally located, being inserted into the comM gene that encodes a putative ATPase. A 5-bp site duplication (ACCGC) was identified on both extremities of AbaR1 suggesting that its acquisition could be result of a transposition event (42). It has been suggested that comM could be an integration hotspot for AbaR elements, and it has been demonstrated that the junctions observed between the AbaR elements and the comM gene were largely conserved in a collection of A. baumannii clinical isolates (49).
The findings reviewed above underline the genetic adaptability of A. baumannii. Many of the acquired resistance determinants found in A. baumannii are similar to those found in other Gram-negative bacteria, but some of these resistance determinants may be difficult to detect in a routine laboratory since A. baumannii already has a significant degree of intrinsic resistance to many antibiotics. Lack of detection of these resistance mechanisms may further enhance their spread. Multidrug resistance seems to result from both the accumulation of multiple mutations and the acquisition of resistance genes from other bacterial genera, with the latter occurring by a variety of mechanisms, including the transfer of plasmids, transposons, and integrons, sometimes leading to the formation of clusters of resistance genes termed resistance islands. Overall, it seems that A. baumannii is becoming well-adapted to the development of modern medicine and the growing number of immunocompromised patients that necessitates a large usage of broad-spectrum antibiotics. A. baumannii seems to be a bacterial species with very high genetic plasticity. The role of novel genetic elements such as resistance islands in concentrating antibiotic resistance genes in A. baumannii will require detailed study in the near future.