Carbapenem resistance in Acinetobacter baumannii : the molecular epidemic features of an emerging problem in health care facilities

Acinetobacter baumannii is an opportunistic gram-negative pathogen with increasing relevance in a variety of nosocomial infections especially among intensive-care-unit (ICU) patients. Carbapenems have been widely used to treat serious multidrug-resistant A. baumannii infections; however, incidences of carbapenem-resistant A. baumannii are rising in several parts of the world and large and sustained outbreaks caused by such bacteria have been described. Carbapenem-resistant A. baumannii epidemics are sustained by clusters of highly similar strains that successfully spread among different cities and countries; their resistance phenotype is mainly due to the acquisition of carbapenem-hydrolyzing class D β-lactamase (CHDL) genes flanked by insertion sequence (IS) elements. Multi-facility outbreaks can be also sustained by inter-hospital transfer of colonized patients. Here, we review the global epidemiology of carbapenem-resistant A. baumannii, with the emphasis on the molecular epidemiology and genetic characterization of carbapenem resistance in epidemic strains.


Introduction
Acinetobacter spp.are glucose-non fermentative gram-negative coccobacilli that have emerged in recent years as a cause of healthcare-associated infections [1,2].Considered to be commensals of low-grade pathogenicity, i.e. opportunistic microorganisms, Acinetobacter were frequently ignored in the 1970s whenever isolated from clinical specimens [1].The genus Acinetobacter currently contains up to 32 described named and unnamed (genomic) species [1].Acinetobacter baumannii, genomic species 3 and 13TU, three of the most clinically relevant species, are genetically and phenotypically very similar to an environmental species, A. calcoaceticus, and are therefore grouped together into the so-called A. calcoaceticus-A.baumannii (Acb) complex [1].Because phenotypic identification of Acinetobacter isolates to the species level has proven to be insufficient, several genotypic methods have been developed for genomic species identification, that include amplified 16S rRNA gene restriction analysis (ARDRA), high-resolution fingerprint analysis by amplified fragment length polymorphism (AFLP), or sequence analysis of the 16S-23S rRNA gene spacer region [1,3,4].However, genotypic methods for species identification are often unavailable in developing countries, where Acinetobacter are frequently isolated but identified only at genus level.The species that is most commonly involved in hospital infections is A. baumannii, which causes a variety of health-care associated infections, comprising bacteremia, urinary tract infection, surgical-site infection, and nosocomial and ventilator-associated pneumonia, especially in intensive-care-unit (ICU) patients [1,2,[5][6][7].The rates of recovery of A. baumannii from natural environments and its incidence in the community are low, while its rate of carriage by hospitalized patients is high and its occurrence in the hospital setting is frequent [1]. A. baumannii has simple growth requirements and can survive in dry conditions.This might contribute to the fitness of A. baumannii in the hospital environment, which represents the main reservoir of the bacterium [1].

Carbapenem resistance mechanisms in A. baumannii
Resistance to antimicrobial agents may be the main advantage of A. baumannii in the nosocomial environment.Multidrug-resistant isolates of A. baumannii have been reported increasingly during the last decade, probably as a consequence of extensive use of antimicrobial agents in western countries [2,8].Also, as recently demonstrated by a retrospective, matched cohort study, patients with infection by multidrug-resistant Acinetobacter show higher mortality rate and length of hospitalization than patients with infection by susceptible Acinetobacter [5].
Mounting evidence indicates that A. baumannii possesses a broad range of mechanisms of resistance to all existing antibiotic classes as well as a prodigious capacity to acquire new determinants of resistance [1,2] Genome sequence analysis of six A. baumannii clinical strains has shown the presence of a resistance island with a variable composition of resistance genes interspersed with transposons, integrons, and other mobile genetic elements in three of them [9][10][11].Also, plasmids carrying resistance genes and/or resistance determinants involved in horizontal gene transfer have been described in several A. baumannii strains [12][13][14][15][16][17][18][19].
The broad-spectrum β-lactam antibiotics, carbapenems, were introduced by 1985 and have been for years the most important agents for the treatment of infections caused by multidrug-resistant A.
baumannii.Carbapenem resistance in Acinetobacter is now observed increasingly worldwide, and constitutes a sentinel event for emerging antimicrobial resistance [2,12].Carbapenem-resistant isolates of A. baumannii are usually resistant to all classes of antimicrobials, and show intermediate resistance to rifampin, while usually retaining susceptibility to tigecycline and colistin [2,12,20].Resistance against carbapenems is, in itself, considered sufficient to define an isolate of A. baumannii as highly resistant [12].The resistance of A. baumannii to carbapenems can be mediated by one of the resistance mechanisms that are known to occur in bacteria, including enzymatic inactivation, active efflux of drugs, and modification of target sites (Table 1).The production of carbapenem-hydrolizing beta-lactamases is the most common mechanism responsible for carbapenem resistance in A. baumannii.Several carbapenem-hydrolyzing βlactamases have been identified so far in A. baumannii.These include metallo-β-lactamases (VIM-, IMP-and SIM-types), which have been sporadically reported in some parts of the world and have been associated with class 1 integrons [2,7,12].Nevertheless, the most widespread carbapenemases in A. baumannii are class D β-lactamases.Three main acquired carbapenem-hydrolysing class D oxacillinase (CHDL) gene clusters have been identified either in the chromosome or in plasmids of A. baumannii strains, represented by the bla OXA-23 -, bla OXA-24/40 -, and bla OXA-58 -like genes [12].Different insertion sequence (IS) elements at the 5' and/or the 3' end of bla OXA-23 -, and bla OXA-58 -like genes, such as ISAba1, ISAba2, ISAba3, or IS18, have been demonstrated to regulate their expression [12,13,[15][16][17].Also, it has been recently demonstrated that the ISAba1 element is capable of transposition in E. coli and of mobilizing an antibiotic resistance gene [18].In addition to these CHDL genes, the chromosomal bla OXA-51-like gene, intrinsic to A. baumannii species, has been demonstrated to confer carbapenem resistance when an ISAba1 element is inserted upstream of the gene [19].Reduced susceptibility to carbapenems has also been associated with the modification of penicillin-binding proteins and porins or with upregulation of the AdeABC efflux system, and it has been suggested that the interplay of different mechanisms might result in high-level carbapenem resistance in A. baumannii (Table 1) [21][22][23].
Outbreaks caused by carbapenem-resistant A. baumannii have also been observed in developing countries such as Morocco, Thailand, India, and Indonesia [47,41].Furthermore, infections caused by Acinetobacter spp.without specifying whether they are caused by carbapenem-resistant strains have been reported in Africa (Lagos, Nigeria) and several Asian countries including Nepal [48][49][50].

Note References
-lactam hydrolysis IMP-1, -2, -4, -5, -6, -11 VIM-2, SIM-1 Class B metallo beta-lactamases.Class 1 integronassociated genes.baumannii genotypes belonging to ST group 2 in Greece and in Lebanon, and to two novel ST groups 4 and 5 in different Greek and Turkish cities, was also shown in the same study [33].The bla OXA-58 gene flanked by IS elements was present in all carbapenem-resistant genotypes analyzed from hospitals in Greece, Italy, Lebanon, and Turkey [7,16,33] (Figure 1).Of note, each of the IS elements flanking the 5' end of bla OXA-58 occurred in strains of distinct ST groups and PFGE profiles isolated in the same geographic region.Thus, ISAba2 element was detected in Greece and Italy, IS18 in Lebanon and Turkey, and ISAba1 in Turkey and Italy, suggesting that they might have been acquired through horizontal gene transfer [33].In further support of this hypothesis, plasmid-borne bla OXA-58 has been found in the majority of carbapenem-resistant A. baumannii strains isolated in Europe [6,7,10,13,14,16,33].The spread of carbapenemresistant A. baumannii carrying the bla OXA-58 gene might had also been contributed by international transfer of colonised patients, as recently demonstrated from Greece to Belgium [28], Greece to Australia [42], and Iraq to USA military services [44] (Figure 1).

Conclusions
Outbreaks of carbapenem-resistant A. baumannii are increasingly reported in several parts of the world that also include developing countries.They are sustained by clusters of highly similar strains that successfully spread among different cities and countries and are selected because of the acquisition of CHDLs genes flanked by IS elements.Multifacility A. baumannii outbreaks can be also sustained by inter-hospital transfer of colonized patients.This emphasizes the need to adopt surveillance and infection control programmes to prevent colonisation and infection by multidrug-resistant A. baumannii in the hospital setting.These programmes would include the study of global epidemiology of multidrug-resistant A. baumannii using molecular typing of bacterial isolates and characterization of antibiotic resistance in order to control the spread of A. baumannii infections over a wide geographic region.