Carbapenem-resistant Acinetobacter baumannii in Brazil : susceptibility profile and diversity of oxacillinases Acinetobacter baumannii resistente aos carbapenêmicos no Brasil : perfil de suscetibilidade e diversidade de oxacilinases

Introduction: The Acinetobacter calcoaceticus-baumannii (ABC) complex includes five species, and the A. baumannii is the most important of them because it carries mechanisms of carbapenems resistance, especially the oxacillinases. Objectives: The objectives of this study were to identify the species of the ABC complex, to evaluate the susceptibility profile and to investigate the presence of oxacillinases in carbapenems-resistant isolates from four Brazilian States. Methods: In the study period, 92 isolates from Rio Grande do Sul (RS), Rio de Janeiro (RJ), Paraná (PR) and São Paulo (SP) were collected. The isolates were identified by matrixassisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS) and sequencing of gyrB gene. Evaluation of susceptibility was performed by disk diffusion and broth microdilution. The presence of oxacillinases was performed by in-house multiplex polymerase chain reaction (PCR). Results: Ninety-one (99%) isolates were identified as A. baumannii by MALDI-TOF and sequencing. The majority of isolates (56; 61%) showed resistance to the six antimicrobial agents tested. Three isolates were resistant to polymyxin B [minimum inhibitory concentration (MIC) ≥ 4 μg/ml). Eighty (87%) isolates were positive to OXA-23-like, and twelve (13%) isolates to OXA-24-like. Conclusion: Our findings confirm the knowledge about the dissemination of the bla OXA-23 gene in Brazil and suggest the recent emergence and spread of bla OXA-24 gene, since it was identified in three of the four sampled states.


introDuCtion
The Acinetobacter baumannii-calcoaceticus (ABC) complex includes the species A. baumannii, A. calcoaceticus, A. pittii, A. dijkshoorniae and A. nosocomialis (1,2) .The conventional methods used in the routine laboratory are unable to distinguish between ABC species.Recently, the mass spectrometry matrixassisted laser desorption ionization-time of fight mass spectrometry (MALDI-TOF MS) was implemented in clinical microbiology laboratory for identification of specie level (3) .The ABC complex has become increasingly important due to the carbapenems resistance mainly associated with production of carbapenemases.This isolates have been reported in many regions of the world associated with high morbidity and mortality (4) .
The most prevalent carbapenemase in A. baumannii are OXA-carbapenemases, and the less frequently metallo-betalactamases.The OXA-carbapenemases already identified in the ABC complex are divided into 6 subfamilies: OXA-51-like, OXA-23like, OXA-24-like, OXA-58-like, OXA-143 and OXA-235.In Brazil, strains producing enzymes of all these families have already been reported, except the OXA-235 (5,6) .The aim of this study was to evaluate the susceptibility profile and to determine the prevalence of oxacillinases in carbapenems-resistant ABC isolates from four Brazilian states: Rio Grande do Sul (RS), Rio de Janeiro (RJ), Paraná (PR), and São Paulo (SP).

Identification of species of the ABC complex
The isolates were first identified by the Vitek ® II system (BioMérieux, France).MALDI-TOF MS system (Bruker Biotyper ® system -version 3.1) and gyrB gene sequencing were performed to confirm the identification at the specie level.To perform MALDI-TOF, a bacterial colony was placed in the polymeric matrix and inserted into the machine.The results were interpreted according to the manufacturers' recommendations.PCR amplification of gyrB was performed with a Applied Biosystems ® Veriti ® 96-Well Fast Thermal Cycler by using PCR reaction 5X Phusion HF buffer each one containing deoxynucleoside triphosphates 1.0 µl, primers at a concentration of 10 pmol/µl, deoxyribonucleic acid (DNA) 2.6 µl and 0.5 µl Phusion DNA polymerase (Perkin-Elmer) in a total volume of 50 µl (7) .A total of 35 amplification cycles were performed with denaturation of DNA template at 98ºC for 10 s, annealing at 57ºC for 30 s and extension at 72ºC for 10 min.Amplified products were purified by using QIAquick ® (Qiagen) for sequencing.The sequences produced were compared with those available on the GenBank and subsequently aligned using the BioEdit software version 7.1.3.

Susceptibility profile
The antimicrobial susceptibility profile was performed by disk diffusion for amikacin, Ampicillin/sulbactam, cefepime, ceftazidime, piperacillin/tazobactam, gentamicin (Oxoid ® ) and by broth microdilution to polymyxin B and tigecycline.The results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) 2016, except for tigecycline wich we used the European Committee on Antimicrobial Susceptibility Testing (EUCAST).The chi-square test or Fisher's exact test was used to statistical analyses with significance at 5% (p ≤ 0.05).

Identification of species of the ABC complex
According to gyrB gene sequencing, all isolates were identified as A. baumannii.MALDI-TOF Bruker ® correctly identified 91 (99%) isolates (score > 2.0) as A. baumannii.One isolate (1%) had inconclusive result.

Oxacillinases genes
Bla OXA-51-like was identified in all isolates; 80 (87%) of the isolates presented bla OXA-23-like .It was also possible to identify 12 (13%) isolates producing bla OXA-24-like from Rio Grande do Sul (RS), Paraná (PR) and São Paulo (SP).The bla OXA-58-like and bla OXA-143-like genes were not detected (Table ).The main enzyme associated with carbapenem resistance in A. baumannii isolates is OXA-23 (5) .In this study, the four Brazilian states confirmed the high prevalence of OXA-23 associate with carbapenems resistance.This finding corroborate data already reported in a study that evaluated the spread of OXA-23 in five geographic regions in Brazil, in which the prevalence of carbapenems-resistant OXA-23-positive A. baumannii was 94.2% (12) .
In addition, 12 (13%) isolates were identified as OXA-24-likeproducing strains from three states [Rio Grande do Sul (RS), Paraná (PR), and São Paulo (SP)] suggesting the beginning of spread of this gene in Brazil.To date, there are few reports of OXA-24/40-like in Brazil, but they are all associated with the multidrug resistance phenotype (13,14) .
In conclusion, we observed high rates of antimicrobial resistance and dissemination of OXA-23 in all evaluated states.Furthermore it should be noted that this report evidences the dissemination of the enzyme OXA-24-like in different Brazilian states, suggesting the emergence of this novel type of enzyme in A. baumannii strain in our country.

DiSCuSSion
The species prevalence and the resistance profile of the ABC complex may vary according to the geographic region.However, A. baumannii is the most common specie associated with nosocomial infections and high resistance to carbapenems worldwide (9) .In this study, all isolates were confirmed as A. baumannii by the methods used (MALDI-TOF and gyrB sequencing) and MALDI-TOF proved to be a quick and easy method for the identification of A. baumannii compared to the molecular method, obtaining high agreement score, as already reported (10) .
In our study, two isolates of A. baumannii presented polymyxin MIC of 8 mg/ml and one isolate showed MIC of 4 mg/ml which are considered resistant to polymyxin.Despite the low prevalence of polymyxin resistance in our study, phenotypes related to polymyxin resistance are worrisome as they may impair antimicrobial therapy (11) .On the other hand, high rates of antimicrobial resistance were observed in the four states and this situation highlights the few therapeutic options to treat severe infections cause of A. baumannii (2,4) .