Oxacillinase (OXA)-producing Acinetobacter baumannii in Brazil: clinical and environmental impact and therapeutic options

Following a worldwide trend, infections caused by MDR OXA-type (Ambler class D) carbapenemase-producing Acinetobacter baumannii are currently regarded as a clinical and epidemiological emergency in Brazil. OXA-producing A. baumannii strains have been identified in the states of Alagoas, Amazonas, Bahia, Distrito Federal, Espírito Santo, Goiás, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Norte, Rio Grande do Sul, Santa Catarina and São Paulo. In some settings, the presence of OXA-23-and/or OXA-143 -producing A. baumannii (so far restricted to Brazil) has been endemic and A. baumannii strains carrying blaOXA-23 genes have been detected in hospital wastewater effluents, hence a potential risk to the community and the environment. Although molecular typing by multilocus sequence typing (MLST - Bartual scheme, University of Oxford, http://pubmlst.org/abaumannii/) has revealed the international spread of a clonal complex (CC) denominated CC92, in Brazil most OXA-23-producing A. baumannii belong to CC113, CC109 or CC104 clonal complexes. Finally, from a clinical point of view, the main problem of A. baumannii infections is the limited use of antibacterial agents with in vitro activity, often restricted to ampicillin/sulbactam, polymyxin B and/or colistin (polymyxin E)..


introDuCtion
Acinetobacter baumannii is a non-fermentative Gramnegative bacillus widely recognized as an opportunistic nosocomial pathogen.It has assumed high clinical importance in the last two decades due to its frequent association with healthcare infections (healthcare-associated infections -HAI ), most of which with unfavorable prognosis and expression of comprehensive antimicrobial resistance mechanisms to antibiotics (ATBs) (11,25,101,116,118,121) .
The risk factors comprise invasive procedures such as mechanical ventilation, central venous catheter or urinary catheter as well as the prior use of broad-spectrum ATBs (14,29,42,76,85,101,117,118) .
In many cases, recurrent outbreaks of nosocomial infection are favored by the intrinsic factors of the species such as the following: i ) tolerance to desiccation ; ii ) viability and growth in a wide temperature and pH range; iii ) the multidrug resistance, which contributes to the spread of these isolates among patients Oxacillinase (OXA)-producing Acinetobacter baumannii in Brazil: clinical and environmental impact and therapeutic options at hospital environment (8,25,53,85) .Furthermore, the epidemiology of the infection by A. baumannii is often complex and the coexistence of epidemic and endemic infections in a given unit promotes the widespread use of broad-spectrum antibiotics for prolonged periods, hence promoting selective pressure on hospital microbiota and selecting resistant bacterial strains (10,29,82,86,90,98,118) .
One of the most striking features of Acinetobacter species is its extraordinary ability to develop multiple resistance mechanisms against major classes of commercially available antibiotics.In fact, A. baumannii can easily express resistance to the broad spectrum beta-lactam (third generation cephalosporins, carboxypenicillins and carbapenems) and to aminoglycosides by the production of a variety of hydrolytic enzymes, namely beta-lactamases and transferases, which inactivate this class of antibacterial agents.Additionally, most strains can express high levels of resistance to fluoroquinolones (29,118) ( Figure 1).
The widespread use of ATBs has contributed to the emergence of multi-resistant bacteria which are associated with nosocomial infections and high morbidity and mortality rates (82,98) .This problem is exacerbated by the failure to develop new antibiotics (7,148) .
The multiple resistance mechanisms in A. baumannii may have an intrinsic and/or acquired origin, including the following: i) loss of membrane permeability; ii) ATB efflux; iii ) change in the target binding site; iv ) production of enzymes (beta-lactamases, methylases and transferases); vi) alternative metabolic routes (39,86,101) .
Carbapenems have been regarded as drugs of choice for the treatment of infection by MR A. baumannii.In this regard , the Antimicrobial Surveillance Program (SENTRY), Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) and Surveillance and Control of Pathogens of Epidemiological Importance (SCOPE) have reported that carbapenem resistance in A. baumannii has increased considerably in Latin America, mainly in Brazil, Argentina and Chile (Table ) .

Acinetobacter baumannii and production of oxacilinase type carbapenemase: clinical and environmental significance
Oxacillinase type carbapenemases (OXA) belonging to Class D (Ambler classification) have been reported in Acinetobacter species worldwide, particularly in hospital environment.In Brazil, OXA-producing strains have been associated with outbreaks of nosocomial infection (4,22,24,86) .
Class D differs from other classes of enzymes due to the fact that it contains serine in the active site.Initially, the main species belonging to the Pseudomonadaceae family, particularly the Pseudomonas aeruginosa species, showed resistance to some types of non-carbapenem beta-lactams, mediated by the action of these enzymes.However, the first OXA-type carbapenemase was characterized from a clinical strain of MR A. baumannii isolated from a patient in Edinburgh, Scotland, in 1985 (126,138) .
Unfortunately, OXA-23-producing A. baumannii isolates have been detected in hospital sewage in large cities such as Porto Alegre (27) and in urban rivers from the state of São Paulo (109) , which entails the possibility of spread of hospital strains to the environment, hence a serious public health problem.
In Brazil, the high rate of resistance to imipenem in A. baumannii was initially attributed to the production of metallo-beta-lactamase (imipenemase type -IMP -1) (33,131) .However, following a global trend , the production of OXA -23 began to be reported in several medical centers, contributing to the endemicity of multiple clones (85,139) , which are commonly associated with outbreaks of nosocomial infection (4,18,24,84,85,96,139) .
The hydrolysis of carbapenems by OXA-23 enzyme contributes greatly to the emergence of resistant strains (63) .The contamination is associated with risk factors such as previous antibiotic treatment, ICU admission, immunosuppression and severe underlying diseases (14,15,29,42,76,117,118) .
The treatment of infection by carbapenem resistant A. baumannii is hampered by the lack of therapeutic options.An additional feature of OXA-23 positive strains has been the resistance expression to other classes of antibiotics such as aminoglycosides and fluoroquinolones (153) (Figure 1).ATB agents exhibiting activity against OXA-23 producing A. baumannii are restricted to the use of polymyxin and ampicillin/sulbactam (67,107,161) .Nevertheless, strains resistant to both antibiotics have already been identified (129) , corroborating the emergence of MDR phenotypes and/or XDR (75) .
OXA-58 enzyme confers resistance to carbapenems, cephalosporins and monobactams of third and fourth generations.OXA-58 producing isolates have shown a MR profile for aminoglycosides, fluoroquinolones and sulfonamides as well as intermediate resistance to tigecycline.Moreover, they are only sensitive to rifampicin, tetracycline, colistin, polymyxin B and , in some cases, ampicillin/sulbactam (12,20,49,122) .
The first cases of infection by OXA-58-producing A. baumannii have been recently identified in the states of São Paulo and Rio de Janeiro, Brazil (5,28,87) .
The first report of OXA-72 occurred in Thailand in 2004 (GenBank Accession no.AY739646 ).In 2004, it was subsequently identified in Taiwan (72,73) and other Asian countries such as China (153) and Korea South (65) .In Europe, OXA-72 isolates have been found in France (8) , Spain (17) and Croatia (30) .In Brazil, this type of isolate has been recently reported in sporadic cases in the states of São Paulo and Rio de Janeiro (5,86,155) and more recently in Recife (personal communication , Professor Marcia Maria Camargo de Morais, Instituto de Ciências Biológicas, Universidade de Pernambuco) .
This OXA confers resistance to third and fourth generation cephalosporins as well as carbapenems.Nonetheless, OXA-72 isolates have shown a MR profile to fluoroquinolones and aminoglycosides.In most cases, they are only sensitive to polymyxin B and colistin (17,73,155) .

Acinetobacter baumannii and production of OXA-143 and 231: new genetic event emerging in Brazil
A new OXA denominated OXA-143 was announced by Higgins et al. in 2009.The bla OXA-143 gene was identified in a clinical isolate of MR A. baumannii recovered in Brazil and collected in an unspecified hospital during a multicenter study (51) .The bla OXA-143 gene relates to other OXA enzymes from the group OXA-24/40-like and OXA-182, accounting for over 80% similarity (61,117,123) .This new genetic event so far has referred exclusively to A. baumannii in Brazil.As there are no studies assessing the figurE 2 -Current overview of the spread of OXA -type carbapenemase producing A. baumannii isolates in Brazilian states (4-6, 18, 19, 24, 28, 40, 41, 52, 84-87, 96, 97, 139, 155, 156) States filled in gray indicate the occurrence of OXA-producing strains.OXA: oxacillinase.
Oxacillinase (OXA)-producing Acinetobacter baumannii in Brazil: clinical and environmental impact and therapeutic options impact of this new genetic resistance determinant, our group has conducted a multicenter study in public hospitals from the state of São Paulo and Minas Gerais, describing a high prevalence of OXA -143 producing A. baumannii isolates, which could reflect a new phenomenon with endemic features (6,87,88) .These data were corroborated in another study by Mostachio et al. (97) .During the year 2011, a case of OXA-143 producing A. baumannii appeared in the state of Rio de Janeiro and a new allelic variant of the bla OXA-143 gene was found in the state of Paraná (40,41,156) .In the 51st Interscience Conference on Antimicrobial Agents and Chemotherapy ( ICAAC ), held in Chicago, USA , in September 2011, Cayo et al. claimed that the presence of OXA-143 producing A. baumannii in Brazil dates from 1995 (21) .Recently, OXA-143 producing A. baumannii strains have been isolated in Juiz de Fora, Minas Gerais (108) .

Identification of bla OXA genotypes and molecular epidemiology
Unlike other carbapenemases identified in Gram-negative bacteria, which can be screened by phenotypic methods using enzyme inhibitors such as ethylenediaminetetraacetic acid ( EDTA), phenyl boronic acid and thiol derivatives , the detection of OXA-type carbapenemase in A. baumannii samples is made by molecular biology.Initially, Woodford et al. developed a polymerase chain reaction (PCR ) multiplex for the identification of genes encoding the major OXAs (bla OXA-51 -, bla OXA-23 -, bla OXA-40ebla OXA-58 -like) (158) .Subsequently, Higgins et al. published a paper in which the inclusion of primers for the identification of new variant bla OXA-143 was standardized in the multiplex PCR reaction (52) .More recently, Mostachio et al. published a multiplex PCR method for the screening of genotypes associated with the production of OXA type carbapenemase and metallo-betalactamases in A. baumannii (96) .
To facilitate the epidemiological study of OXA-producing A. baumannii, there is a consensus for the identification of endemic clones spread internationally, which is given by the MLST typing.There are two MLST schemes available for A. baumannii, which contain information about primers, PCR conditions, sequencing and a sequence database (SD) for comparative analysis and identification of SDs and CCs.One of the schemes was described by Bartual (9) , whose software was developed by Keith Jolley and is hosted at the University of Oxford, UK (http://pubmlst.org/abaumannii/) .This scheme includes alleles gltA, gyrB, gdhB, recA, cpn60, gpi e rpoD (56) .A second scheme has a database hosted at the Pasteur Institute (www.pasteur.fr/ mlst) and includes alleles cpn60, fusA, gltA, pyrG, recA, rplB e rpoB (45) .
Using the software eburst (http://eburst.mlst.net/), the current database comprises 38 different STs of A. baumannii isolates in Brazil, which reflects the genetic diversity of strains with bla OXA genes (45) .

Therapeutic options for the treatment of infections caused by Acinetobacter baumannii ampicillin -sulbactam
This compound is a combination of a beta-lactam and a beta-lactamase inhibitor (Figure 3).Betalactamase inhibitors are beta-lactam analogues with limited antibacterial activity and act competitively inhibiting the activity of the beta-lactamase enzyme (68) .Generally, these inhibitors are used in association with beta-lactams, promoting the restoration of their activity (44) .The ampicillin sulbactam is associated with ampicillin in a fixed ratio 1:2, optimizing its activity spectrum.

Imipenem
Imipenem is an ATB that belongs to the class of beta-lactams, more specifically the carbapenem subclass (Figure 3).This drug inhibits cell wall synthesis by binding beta-lactam with penicillin-binding proteins( PBPs ), which catalyze the synthesis of peptidoglycan present in the bacterial cell wall by means of a transglycosylation and transpeptidation reaction (74,136) .
Carbapenems, conversely, are more efficient and stable to degradation by a broad spectrum of beta-lactamases, exhibiting high antimicrobial activity against almost all Gram-negative bacteria, including MR fermenters (44) .
Studies on the synergistic effect against MR A. baumannii have been conducted with the use of imipenem in combination with lipopeptides, glycylcyclines , aminoglycosides, aztreonam, rifampicin, and even beta-lactams such as ampicillinsulbactam (62,81,89,114,119,125,132,140,141,152,154) .

Polymyxin B
Polymyxin B (PB) is an ATB belonging to the class of lipopeptides (Figure 3), which act primarily on the cell wall of Gram-negative bacteria, leading to a rapid change in the permeability of the cytoplasmic membrane, which may ultimate cause cell death (32) .This drug has demonstrated significant in vitro antimicrobial activity against Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Acinetobacter spp.and Pseudomonas aeruginosa (34,35,92) .Currently, it is an important ATB option against MR microorganisms (34,35,159) , including OXA producing A. baumannii.
In recent years, a few studies have confirmed that polymyxin B has a synergistic potential when used in combination with carbapenems (89,112,152,154) and vancomycin (89) as well as partially synergistic potential with rifampicin (89,154) .

Vancomycin
Vancomycin belongs to the glycopeptide class (Figure 3), whose action mechanism is the inhibition of peptidoglycan synthesis in the bacteria cell wall in the late phase, preventing the incorporation of peptidoglycans into the growing cell wall by binding the end portion of D-Alanyl-D-Alanine with the pentapeptide side chain (57,130) .This drug is widely applied in the treatment of infections caused by methicillin resistant Staphylococcus aureus (MRSA) and serious infections by Gram-positive microorganisms in patients who are hypersensitive to penicillin (44,57,93,120) .
Gordon and Wareham tested a combination of antimicrobials that are not conventionally applied in the treatment of infections by Acinetobacter baumannii, a glycopeptide (vancomycin), which is used for the treatment of Gram-positive, and one lipopeptide (colistin), used to treat Gram -negative infections.Therefore, this combination showed synergism when tested in MR A. baumannii strains (42,89) , which is clinically remarkable insofar as many nosocomial patients suffer from polymicrobial infections by Gram-positive bacteria such as Enterococcus spp.and coagulase-negative staphylococcus (157 ) as well as Gramnegative bacteria such as A. baumannii, Klebsiella pneumoniae and Escherichia coli (47,101) .

Tigecycline
Tigecycline is a broad-spectrum antimicrobial drug from the Glycylcycline class, semisynthetic derivative of minocycline (Figure 3), representing the first ATB from this class available for clinical use (105,111) .It inhibits the bacterial protein synthesis by binding to the 30s subunit of the ribosome (26) .Due to its broad spectrum, it has good performance for both Gram -positive and gram-negative (S. aureus, Enterococcus spp., S. pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, Peptostreptococcus and Clostridium spp.), including enterobacteria and Bacteroides spp. (105,111) Some studies have assessed the use of tigecycline in vitro against MR A. baumannii, thus revealing a bacteriostatic activity (79,110) .
Other studies have described a synergistic effect on the clinical use against A. baumannii, which is due to the combination of tigecycline with other ATBs such as carbapenems, aminoglycosides, minocycline, lipopeptides, quinolones and beta-lactamase inhibitors (71,94,119,125,140,141,145) .

Rifampicin
Rifampicin is an ATB belonging to the ansamycin class that was introduced in clinical practice in the 1970s (2) (Figure 3).The action mechanism of this drug consists in the inhibition of ribonucleic acid (RNA) synthesis by attacking the β subunit of RNA polymerase.This drug has a broad spectrum and is applied in the treatment for tuberculosis, which is caused by M. tuberculosis microorganism (2,74,128) .Some studies indicate a synergistic effect against A. baumannii by associating colistin and rifampicin (132) as well as a partially synergistic effect by combining PB (89,154) , tigecycline (26,119,123) or colistin (70) .

Minocycline
Minocycline belongs to the tetracycline class (Figure 3), broadspectrum bacteriostatic ATBS, including anaerobic, Gram-positive and Gram -negative bacteria as well as other microorganisms such as Rickettsia, Chlamydia, Plasmodium spp.and Mycoplasma pneumoniae (39,102) .
The action mechanism of this drug is associated with the inhibition of protein synthesis by binding to the 30S subunit of the bacterial ribosome preventing aminoacyl -tRNA binding (13,39) .
The drug minocycline has not been closely related to the study of the synergistic effect on the treatment of A. baumannii.Tan et al. demonstrated that the association of deminocycline and colistin offers synergistic potential (144) as well as minocycline/ meropenem (70) and minocycline/ cefoperazone -sulbactam (115) .

Assessment of synergistic effect in Brazil
Few studies have evaluated the synergistic potential of antibiotic combinations against endemic A. baumannii strains in Brazil.A study carried out by Kiffer et al. and another investigation by Guelfi et al. indicated that approximately 50% of the isolates tested in vitro responded partially to the synergistic effect obtained by the combined use of meropenem/polymyxin B and meropenem/sulbactam.It is particularly worth noting that these investigations did not identify carbapenemase producing strains (47,61) .

ConCLuSion
The spread of MR OXA producing Acinetobacter baumannii in Brazil is a serious public health problem.The emergence of these strains is associated with high rates of resistance to ATBs commonly used in clinical practice, which increasingly hinders the choice of drugs with in vitro activity employed in the treatment of HAIs.The identification of OXA phenotype and genotype is of utmost importance for a suitable patient management, preventing the introduction and spread of outbreaks and establishing a differential therapeutic approach that preferably includes the combined use of antibacterial agents.

figurE 3 -
figurE 3 -Chemical structures of the major antimicrobial compounds applied in the clinical treatment of Acinetobacter baumannii

tABLE -
Antimicrobial resistance indexes in A. baumannii isolates reported by SENTRY, MYSTIC and SCOPE