Emergence of polymyxin B-resistant Acinetobacter baumannii in hospitals in Rio de Janeiro Emergência de Acinetobacter baumannii resistente a polimixina B em hospitais do Rio de Janeiro

Introduction: Acinetobacter baumannii has been considered a prevalent pathogen in hospitals, raising concern in the medical community due to its broad spectrum of antimicrobial resistance. Since it is a subject that arouses much interest, it has been increasingly studied. Due to the emergence of multidrug-resistant (MDR) Gram-negative bacteria, the use of polymyxins was reestablished. The polymyxins have been considered the only option for the treatment of severe infections caused by MDR A. baumannii. Objective: To investigate the susceptibility profile of A. baumannii to polymyxin B. Material and method: 92 clinical isolates from two public hospitals in the Rio de Janeiro city were studied using broth microdilution method. Results: Most of the isolates were resistant to polymyxin B, 81.5% (n = 75), and minimum inhibitory concentration (MIC) values ranged between 4-64 mcg/ml. Conclusion: These results are a concern since currently the polymyxins have been considered the most effective therapeutic option against MDR isolates of A. baumannii.


introDuCtion
Acinetobacter baumannii has been considered a prevalent pathogen in hospitals, raising concerns for the medical community due to its broad-spectrum for antimicrobial resistance (1) .It is an opportunistic pathogen, with a high incidence in immunocompromised individuals, particularly those who have had prolonged hospitalization.
The World Health Organization (WHO) identified the antimicrobial resistance as one of the three major human health problems (2) .In this context, A. baumannii is among the most common and dangerous multidrug-resistant (MDR) pathogens (3,4) , because of that, this subject has been increasingly studied (5) .
Due to high levels of MDR Gram-negative bacteria, the use of polymyxins was reestablished, especially in cases of carbapenem-resistant isolates.The polymyxins are cyclic peptides positively charged that interact with the lipid A component of the lipopolysaccharides (LPS) present in the outer membrane of Gram-negative bacteria (6) .On the other hand, this class of antimicrobials has serious adverse effects, which are mainly nephrotoxicity (mainly acute renal failure) and neurotoxicity.Other effects are also described, such as, for example, allergies, fever and eosinophilia (7) .In general, resistance to polymyxins is still uncommon among nonfermenting Gram-negative microorganisms, although reports of A. baumannii -resistance to these agents have increasingly been described (8,9) .Currently, polymyxins have been considered the only option for the treatment of severe infections caused by MDR A. baumannii (10)(11)(12)(13) .

oBjECtivE
This study aimed to investigate the susceptibility profile of 92 clinical isolates of Acinetobacter baumannii from two public hospitals of the Rio de Janeiro city between 2010 and 2011.

Bacterial identification
All isolates were previously identified in hospitals studied by automated VITEK 2 system (bioMérieux Inc., Hazcwood, Mo.), using cards for identification of Gram-negative (GN, reference 21341).In the laboratory, the isolates were tested for their purity and their morphotinctorial characteristics were observed by Gram staining technique.The isolates were also subjected to the following biochemical tests: motility, citrate utilization test (Simmons), indole production, oxidation-fermentation glucose in Hugh-Leifson medium, cytochrome oxidase production and growth at 42ºC, according to Murray et al. (2010) (14) .The confirmation of identification was performed by amplifying and sequencing of the rpoB gene, which encodes the β subunit of ribonucleic acid (RNA) polymerase, considering an identity level of 99%-100% (15) .Isolates were stored in Tryptone Soy Broth (TSB) (Difco ® ), containing 20% glycerol (v/v) and kept in the freezer at -20ºC and -70ºC.

Determination of the minimum inhibitory concentration (MIC) for polymyxin B by broth microdilution method
The isolates were evaluated by the broth microdilution method using the protocol and the breakpoints recommended by the Clinical and Laboratory Standards Institute (CLSI) (16) .The following antimicrobial concentrations were chosen for testing: 64 µg/ml, 32 µg/ml, 16 µg/ml, 8 µg/ml, 4 µg/ml, 2 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.25 µg/ml and 0.125 µg/ml.After isolates growth at 37ºC for 24 hours in Nutrient Agar medium, bacterial suspensions were carried out in sterile saline 0.85% compatible to 0.5 McFarland standard (1.8 × 10 8 CFU/ml), which were subsequently diluted in the range of 1:100 in cation-adjusted Mueller Hinton broth (CAMH) medium pH 7.3, to obtain a final cell concentration of 1.8 × 10 6 CFU/ml.Using enzymelinked immunosorbent assay (ELISA) microplate plate, 50 µl of polymyxin B were added into the wells at different concentrations followed by 50 µl of bacterial suspensions.Bacterial inoculation in the wells was performed in the range of up to 30 minutes, and the plates were homogenized on the bench in rotational movement and incubated for 24 hours at 37ºC.The reading was performed by visual inspection, in which the first concentration which showed no bacterial growth (medium turbidity) was assumed as the MIC.Some wells of the plate were used as sterility controls of the antimicrobial and the bacterial growth; the reference strain Escherichia coli ATCC 25922 was used as test control.

Bacterial identification
Conventional biochemical tests were performed for all isolates from the two public hospitals in Rio de Janeiro city.These tests were important at the first stage of confirmation of identification.To confirm the identification of these isolates to the species-level, it was necessary to perform a molecular identification by polymerase chain reaction (PCR) amplification and sequencing of rpoB gene.This methodology enables the species-level identification of isolates, and all belonged to Acinetobacter baumannii specie (similarity 99%-100%).As a result, all 92 isolates that were previously subjected to biochemical and genotypic tests were identified as Acinetobacter baumannii.

Distribution of isolates
Among the 92 A. baumannii isolates studied, most were from the hospital 1 (81.5%;n = 75).The remaining isolates originated from patients from hospital 2.
The A. baumannii isolates were obtained from different specimen types.Among those collected at hospital 1, the majority were from urine (22.7%; n = 17), followed by blood and catheter (18.7%; n = 14, each) and tracheal aspirates (16%; n = 12), fewer in number were originated from wound and bronchoalveolar lavage.The origin of the specimen was not identified in 1.3% (n=1) of the isolates collected in this hospital (Figure 1).About the hospital 2, the clinical specimen was not identified in 35.3% (n=6) of the studied isolates (Figure 2).

Determination of MIC
Polymyxin MIC of the isolates was determined using the microdilution broth method (16) .By this method, we observed a small number of susceptible isolates to this antimicrobial (18.5%; n = 17).Among the susceptible isolates, two presented MIC of 1 µg/ml and the other 15 presented MIC of 2 µg/ml.Most of the isolates were polymyxin B-resistant (81.5%; n = 75), showing MIC values between 4-64 µg/ml (Figure 3).About the susceptible isolates to polymyxin B (n = 17), only four were present in hospital 2. Among the polymyxin B-resistant isolates (n = 75), it could be noted that most of them were collected from urine (18.8%; n = 15).collected in hospitals is of great importance, since this microorganism is associated with several outbreaks in Brazil and worldwide (20,21) .
The species-level identification of isolates among Acinetobacter species is often problematic.Currently, Acinetobacter species are identified by molecular techniques (22,23) such as sequencing the gene encoding the subunit 16S RNA ribosomal (16S rRNA), which is one of the most common method used for bacterial identification (24) .However, the main limitation is because this gene is so preserved, which does not enables, in case of Acinetobacter, differentiating all species (25) .Other genes that encode proteins such as rpoB gene, which have been used to identify isolates of Acinetobacter spp., allow to differentiate most species of this microorganism (26) .
In our study, we observed a large number of isolates of A. baumannii polymyxin B-resistant, which differs from some studies in Iran and Mexico, that show isolates with 100% of susceptibility to polymyxin B (26) .In some studies, polymyxin B was the only antimicrobial that showed efficacy against A. baumanni (27)(28)(29) .Moreover, in isolates collected in Latin America in 2001, this antimicrobial showed excellent activity, since 96,4% of the isolates were susceptible to it.In this study, only six isolates were classified as polymyxin-resistant, they were from three different Brazilian hospitals; only one of them was also carbapenem-resistant (27) .
Despite these reports on high susceptibility of isolates to polymyxins, in the past few years the intensive use of polymyxins has led to the selection of A. baumannii isolates resistant to these antibiotics; the resistance rates of 40.7% in Spain and 30.6% in Korea were already reported (8,9) .More recently, isolates of A. baumannii polymyxin B-resistant were also recovered in Iran and the United States (30,31) .Some studies have predicted the increased resistance to polymyxin, as observed in our work.Rolain et al. (2011) (32) reported that resistance rates would increase once the use of antimicrobial become more common, for example, in the treatment of infection caused by A. baumannii carbapenem-resistant, leading to the development of resistance by adjustment to selective pressure exerted by the antimicrobial (32,33) .
It is evident that the rational use of antibiotics is essential to prevent outbreaks of MDR A. baumannii infections, since the emergence of this specie is usually associated with selective pressure of prolonged use of broad-spectrum antimicrobials (34) .Carbapenems are still among the drugs of choice for the treatment of infections caused by A. baumannii, however increasing number of resistance reports of this microorganism

DiSCuSSion
Acinetobacter species are commonly associated with hospitalacquired infections or healthcare-associated infections (HAI), especially in developing countries (17,18) .The A. baumannii specie has become a major problem in the nineties and, over the years, this organism has become an important HAI-causing agent in the world (19) .In this regard, the study of A. baumannii isolates to these antibiotics has become a great concern for the medical community (35,36) .As an alternative to the use of carbapenems, polymyxins B and E have been used as the most effective therapy for treatment of serious infections caused by MDR A. baumannii, even with reports of isolates of this pathogen resistant to these antimicrobials (37) .

ConCLuSion
In this study, by evaluating the MIC for polymyxin B, we observed high percentages of resistance among the studied isolates (81.5%; n = 75), which increasingly limits the therapeutic options available for the treatment of infections caused by A. baumannii.Therefore, strict infection control measures to prevent the emergence and spread of such isolates should be adopted.

aCknowLEDgEMEntS
The Instituto Nacional de Controle de Qualidade em Saúde (INCQS) for financial and structural support.The Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) for financial support through the granting of Master Scholarships for Gabrielle Limeira Genteluci and Daniela Betzler Cardoso Gomes.The Programa de Pós-Graduação em Vigilância Sanitária (PPGVG) at INCQS/Fiocruz.Karyne Rangel Carvalho is a fellow of PNPD at Capes.

figurE 1 −figurE 2 −
figurE 1 − Percentage of 75 Acinetobacter baumannii isolates from hospital 1 according to the source of specimens