Acinetobacter baumannii is a Gram-negative non-fermentative coccobacillus that has gained growing notoriety as a nosocomial pathogen, showing patterns of increasing resistance to antimicrobial drugs and disinfectants. These organisms are implicated in a diverse array of infections, especially in intensive care units (ICUs) associated with countless outbreaks.1,2 Classically, carbapenems plays a crucial role in the treatment of serious nosocomial infections due to A. baumannii, but this scenario has changed substantially in the last decade, with an increasing prevalence of carbapenem-resistant and multidrug-resistant isolates.3

Carbapenem resistance in A. baumannii is mainly due to the presence of β-lactamases (class B metallo-β-lactamases – MBL, or class D OXA-type β-lactamases – oxacillinases – OXA), to the loss of outer membrane proteins,4 and to altered penicillin-binding proteins (PBP).5 OXA-type β-lactamases are chromosomal enzymes that in A. baumannii can be intrinsic (e.g., OXA-51-like) or, more frequently, acquired (e.g., OXA-23-like, OXA-24-like, OXA-58-like).6 Outbreaks of OXA-23-producing A. baumannii have been reported in some Brazilian hospitals. Moreover, a new oxacillinase, OXA-143-like, was recently described in this country.3,7–9

Only few studies concerning the molecular basis of carbapenem resistance in A. baumannii, as well as the clinical impact of these resistant strains on patient outcomes, have been reported, despite the growing importance of these strains in ICUs of Brazilian hospitals.3,7,8

Thus, the aim of this study was to investigate the genetic determinants of carbapenem resistance and the clonal relatedness of carbapenem-resistant A. baumannii (CRAb) strains isolated from patients admitted to a Brazilian intensive care unit.

The blaOXA-51 gene was detected in all studied strains, whereas blaOXA-23 was found in 22 (51.2%) and blaOXA-143was detected in eight (18.6%) isolates. Of the 17 strains susceptible to imipenem in the confirmatory phenotypic tests, i.e., agar dilution method, two presented blaOXA-23 and one presented blaOXA-143. Table 3 depicts the main clinical and microbiological characteristics of the 56 patients harboring (colonized or infected by) OXA producing CRAb strains. Other resistance-encoding genes such as VIM and IMP-like were not found among the tested strains. Finally, two imipenem-resistant strains did not display any of the acquired oxacillinases or metallo-β-lactamases during the investigation.

Carbapenem-resistant A. baumannii has emerged over the last decades as an important infection-causing microorganism in intensive care patients.1,2 Despite being considered opportunistic, many authors underline the negative impacts stemming from CRAb infections, probably due to the delay to initiate appropriate antimicrobial therapy.14 However, in spite of the high prevalence of this microorganism in hospitals around the world, especially in Brazil, little is known about the relationship between the presence of resistance genes and the outcome of CRAb infected patients.

CRAb is an important microorganism that colonizes and infects patients in our intensive care unit. During this study CRAb was isolated from ∼10% of the patients and the main determinant of carbapenem resistance among A. baumannii isolates in this population was the production of oxacillinases OXA-23 and OXA-143. Moreover, a polyclonal pattern was observed among the studied strains.

This study presents limitations that are inherent to retrospective analysis and the quality of data depended on the clinical records. Patient enrollment occurred during a limited period of time, with small sample of patients evaluated. Additionally, molecular tests were done in ∼77% of cases (43/54) and even though carbapenem resistance was an inclusion criteria for the study, only ∼60% of A. baumannii samples had this resistance confirmed by microdilution tests. The disk diffusion is a method of screening, with potential limitations as inadequate drug concentration and lability of the drug on the disk. Thus, transportation and disk storage in less than ideal conditions may interfere in the results.15

In a study conducted in Brazil showed that none of the evaluated brands distributed in Brazil presented satisfactory performance.15 It is worth mentioning that some strains even presenting a sensible profile, but with elevated minimum inhibitory concentration, had already genetic mutations (e.g. OXA 23) related to resistance. In this case series only enzymatic mechanisms of resistance were investigated.

Patients colonized or infected by CRAb strains presented a high hospital mortality rate (59%), although mortality could not be attributed only to the presence of CRAb. This feature, however, was not the focus of our study. Recently, Lemos et al. (2013) observed in a meta-analysis that CRAb patients had an increased mortality risk (OR-2.22; 95% CI: 1.66–2.98). These authors included 16 studies and most of them referred to infections due to CRAb, mainly bacteremia. Only one study considered infected and colonized patients. Nevertheless, most patients also presented a severe underlying illness and received inappropriate empirical antimicrobial treatment.16

Treatment options for CRAb infections are scarce and generally employ polymyxins or tigecycline.17,18 In our study, 76.7% of the strains were susceptible to tigecyline, a lower percentage than the 97.1% found by Rossi et al. in samples from Latin America.19 Regarding polymyxins (polymyxin B and E), resistance to polymyxin B was observed in three (7%) of the analyzed strains in this study (C> 4μg/mL, CLSI 2011). SENTRY – 2006 found 2.1% resistance to polymyxin in Latin America.20 This finding is worrisome since polymyxins are regarded as the major therapy options for treating CRAB infections, despite their toxicity.21 Another important issue is the variable therapeutic effectiveness of polymyxin on CRAB (50–80%).18,22

In the present study, we found that the blaOXA-51 gene was present in all strains studied, confirming the A. baumannii species. OXA-51 has weak carbapenemase activity, but potentially increases the minimum inhibitory concentration (MIC) when overproduced.23 Carbapenem resistance in A. baumannii frequently results from the production of acquired oxacillinases, such as OXA-23, OXA-24, and OXA-58, besides the less frequent OXA-143 and OXA-72.24 The production of the carbapenemase OXA-23 has been frequently identified in CRAB strains from patients admitted to Brazilian hospitals.25 In this study, OXA-23 was the most commonly acquired carbapenemase (51.2%), followed by OXA-143, which was identified in eight strains (18.6%). It is worth noting that OXA-143 was described in 2009 by Higgins et al., and has so far only been found in Brazilian hospitals.3,7,8

In our study, almost all CRAB strains presented at least one acquired oxacillinase (OXA-23 or OXA-143), and only two resistant strains did not display any of the investigated enzyme mechanisms. In this case, other resistance mechanisms may be involved, such as efflux pumps, changes in outer membrane proteins (OMPs) or changes in affinity or expression of penicillin-binding proteins (PBPs).1

Among the analyzed strains, 15 distinct clones were identified in six main related clusters, which characterizes a polyclonal distribution but includes groupings with >90% similarity. This fact is probably related to cross-transmission. CRAb strain was first identified in our institution in 2003, and since then there has been an exponential increase in number of cases. HC/UFMG is a hospital of high complexity and public reference center. Thus, the hospital always receives many patients who are possibly colonized/infected with CRAb from many other services.

In the present study, three out of the four CRAb patients with the K clone ultimately passed away. Moreover, the blaOXA-23 gene was identified in all these CRAb strains. Due to the small sample of patients and the heterogeneity of their clinical and microbiological presentations, associations regarding the production of oxacillinases and patient mortality could not be performed in this study.

Despite the limitations, this study showed that CRAb colonized or infected patients presented a high frequency of comorbidities, with long hospital stays and increased hospital morbidity. The strains studied showed a wide genetic diversity with polyclonal dissemination pattern. The most frequent resistance mechanism was the production of carbapenemases, notably OXA-23, but with cases of OXA-143 expression. Although the production of metallo-β-lactamases was not identified, other mechanisms may be involved, as there were resistant strains that did not express any gene related to carbapenemases. Strains drug resistance profile was discordant in 40% of cases when using disk diffusion and agar dilution methods. We also need to underscore that polymyxin B resistance was higher than described in literature and this may be a challenge for patient management, due to the limitations of therapeutic options. In our view, prevention for MDR infection is the key for better assisting patients in intensive care unit and antimicrobial susceptibility tests need to present more rigorous quality control. The increasing of polymixin resistance also warrants a better approach.

Foundation for Research Support of the State of São Paulo (FAPESP), Higher Education Personnel Improvement Coordination (CAPES) and Nacional Council of Scientific and Technological Development (CNPq).

The authors declare no conflicts of interest.