First detection of metallo-β-lactamases in nosocomial isolates of Pseudomonas aeruginosa in Alagoas , Brazil Primeira detecção de metalobetalactamases em isolados nosocomiais de Pseudomonas aeruginosa em Alagoas , Brasil

Introduction: Pseudomonas aeruginosa is a leading cause of opportunistic infections in humans, and the choice of effective antimicrobial agents to control this bacterium has been limited, mainly due to its ability to produce metallo-β-lactamases (MβL), enzymes capable of inactivating many antimicrobials through hydrolysis. Objective: This study aimed to detect the presence of multidrug-resistant (MDR) P. aeruginosa strains and the MβL-encoding genes (blaSPM, blaIMP and blaVIM) in nosocomial isolates in Maceió (AL). Methods: The isolates were collected from four public institutions/hospitals in Maceió, and cultures were identified by conventional methods. Antibiotic susceptibility was determined by the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI), and polymerase chain reaction (PCR) was used to identify the presence of the MβL-encoding genes blaSPM, blaIMP and blaVIM. Results: Forty-three strains of P. aeruginosa were MDR among 85 identified nosocomial isolates (50.6%), 79.1% and 20% of which were resistant to carbapenem (imipenem and meropenem) and aztreonam, respectively. PCR was performed in susceptible or resistant isolates and we identified nine (20.9%) MDR strains with blaSPM gene, whereas only one strain had blaIMP and none blaVIM positive was found. Conclusion: Production of MβL is an important mechanism of resistance to carbapenems and other β-lactams among P. aeruginosa strains in the evaluated samples. We reported the first identification of MβL-encoding genes in P. aeruginosa from nosocomial environments in Maceió, a new insight for the epidemiology of MβL in the Northeastern region of Brazil.


intRoDuCtion
Pseudomonas aeruginosa is a Gram-negative bacterium and one of the most common causes of human infections.In Brazilian hospitals, it is the first cause of pneumonia (1) and it often presents an antibiotic resistance profile that makes its eradication difficult.The indiscriminate use of antibiotics has contributed to the selection and subsequent spread of strains resistant to carbapenems (mainly imipenem and meropenem), considered an important therapeutic option for multiresistant P. aeruginosa, limiting therapy options and compromising the patient treatment (2) .

oBjECtivE
The aim of this study was to detect the presence of MDR P. aeruginosa strains and the MβL-encoding blaSPM, blaIMP and blaVIM genes in nosocomial isolates from four public institutions/hospitals in Maceió (Alagoas), using disk-diffusion and polymerase chain reaction (PCR) approaches, respectively.

Collection and identification of bacterial isolates
Isolates of Pseudomonas aeruginosa were recovered from patients admitted into four hospitals in the state of Alagoas, Brazil, from August to July 2009.The isolates were previously identified by conventional procedures at Santa Casa de Misericórdia and Centro de Patologia Médica e Laboratorial (CPML), responsible for bacterial identification of samples from the three major public hospitals in Maceió: Hospital Geral do Estado Professor Osvaldo Brandão Vilela, Hospital Escola Dr. Hélvio Alto and Maternidade Escola Santa Mônica.After bacterial identification (morphological characteristics, pyoverdin and pyocyanin production, oxidase and Gram stain), P. aeruginosa strains were re-isolated on Mueller-Hinton agar (Difco) for the next laboratorial steps.

Antimicrobial susceptibility testing
The resistance profile of each isolate was evaluated by Kirby Bauer's disk diffusion method as described by the Clinical and Laboratory Standards Institute (CLSI) (16) , with five classes of antimicrobial agents such as carbapenems (imipenem [IMP], meropenem [MEM]), monobactam (aztreonam [ATM]), flourquinolones (ciprofloxacin [CIP], levofloxacin [LVX]), aminoglycosides (gentamicin [GEN], amikacin [AMC]) and cephalosporins (ceftazidime [CAZ]), in duplicates.For the interpretation of susceptibility test results, all isolates that were resistant to two or more classes of antibiotics were considered MDR, according to CLSI (15) .P. aeruginosa ATCC 27853 was used as the quality control strain.

RESuLtS anD DiSCuSSion
Isolates of MDR P. aeruginosa and the prevalence of MβL genes were evaluated in clinical specimens recovered from patients admitted into institutions /hospitals of Maceió.Eightyfive P. aeruginosa isolates were obtained from different clinical specimens, mainly urine (29.4%), tracheal aspirates (28.2%), and wounds (25.9%), as well as bloodstream, catheter tips, sputum and other sources (16.5%).The highest detected rate of P. aeruginosa resistance was to meropenem (34.1%), followed by gentamicin (31.8%); and the lowest was to aztreonam (20%).Among the total screened isolates, 43 (50.6%) were MDR and 79.1% of them were resistant to at least one tested carbapenem (60.5% to imipenem and 69.8% to meropenem).Resistant and susceptible isolates of P. aeruginosa were evaluated, and only one of them was positive for blaIMP gene (Figure 1A); nine isolates with the MDR phenotype were positive for blaSPM gene (20.9%)(Figure 1B), but blaVIM was absent in all.The Pa130, a blaIMP-positive strain, was MDR and resistant to all tested antibiotics, including carbapenems.The highest prevalence of SPM-producing strains was observed among clinical isolates from urine tract infections (five), whereas eight showed resistance to imipenem and meropenem, except for one sample that was only resistant to meropenem.The resistance profile of the 10 MβL-positive strains (SPM and IMP), with additional information of clinical specimens, is depicted in Table.P. aeruginosa is highly adapted to the hospital environment and the emergence of MDR isolates, mainly due to the production of MβLs, has been associated with high mortality rates among infected patients.Endorsing this fact, Zavascki et al. (18) confirmed the mortality rate of 51% among the patients infected with metalloenzyme-producing isolates, against 32% in the patients infected with metalloenzyme-nonproducing isolates in Porto Alegre (RS).Likewise, Marra et al. (19) reported a high mortality rate (86%) in patients with bloodstream infection by P. aeruginosa MβL-positive strains (São Paulo, Brazil).Such data suggest that the spread control of the MβL-positive isolates can substantially reduce the number of post-infection deaths.In this context, associated with the increase of bacterial resistance and immunocompromised individuals, it is important to implement a regular monitoring of antimicrobial susceptibility of nosocomial P. aeruginosa isolates to establish protocols for prophylaxis and empirical treatment.Higher resistance to many antimicrobials in P. aeruginosa MβL-producing isolates was observed more frequently than in those MβL-nonproducing.
After the evaluation of different cases from many hospitals in Rio de Janeiro, Nouér et al. (22) identified antibiotics usage as the major risk factor associated with acquisition of multidrug resistance when P. aeruginosa expresses the blaSPM-1 gene, mainly the administration of quinolones.Perez et al. (23) recovered seventy-nine MβL-producing P. aeruginosa (86.8%) from patients of Porto Alegre that showed the ability to produce biofilm in vitro, what can represent a new problem.Hence, we can observe different epidemiological data in Brazil, mainly about blaVIM prevalence.
In the northeastern states, 2% (4/198) of the P. aeruginosa strains recovered from patients in João Pessoa and 7.71% (24/311) from those in Fortaleza were classified as metallo-β-lactamase producers by phenotypic methods (11,14) , whereas PCR was carried out for the detection of MβL-encoding genes in Recife (13) .Poirel et al. (12) identified eleven carbapenem-resistant P. aeruginosa isolates from hospitals of Recife, and the VIM, IMP and SPM screening through PCR only detected the blaSPM-1 gene.The genetic elements surrounding the blaSPM-1 gene in these strains were sequenced and described as possibly involved in the MβL dissemination.
The antibiotic susceptibility profile associated to MβLs is generally characterized by resistance to imipenem, meropenem and most β-lactams, with the exception of aztreonam (9,12,13,24) .Among the isolates of P. aeruginosa obtained in this survey, Pa130 was positive for the blaIMP gene and also resistant to imipenem, meropenem and aztreonam, unlike most of the previously described MβL samples.In the study by Yan et al. (25) , 90% of Klebsiella pneumonia isolates were resistant to aztreonam, with a resistant profile similar to that of the Pa130 sample, what might be due to other mechanisms, such as the production of β-lactamases or AmpC oxacillinases (25,26) .These researchers draw attention to metalloenzymes in carbapenem-susceptible strains or with lowlevel resistance phenotypes.
In our research, the bla gene-positive samples were obtained from the neurological intensive care unit (ICU), the general ICU and the oncology unit at three major hospitals in Maceió (Table ), and the application of specific techniques will be further evaluated in order to discuss the clonality of the strains.
The emergence of different mechanisms of resistance in P. aeruginosa is an important public health problem, particularly due to the expression of MβLs by nosocomial isolates, encoded by genes often connected to mobile elements, which facilitate their spread (27) .Here we report the identification of genes encoding metalloenzymes SPM and IMP in strains resistant to carbapenems and other β-lactams, recovered from hospitals of Alagoas.Since meropenem and imipenem are the most active and potent β-lactams against Gram-negative bacteria, besides being currently the only carbapenems commercially available in Brazil, this resistance profile represents a challenge to any treatment.
The adequate identification of strains with MβL-encoding genes is important to guide therapeutic choices in public and private hospitals and can give support to better treatments against these infections.Thus, control measures should be implemented to prevent further spread of metallo-β-lactamase-producing strains.

aCknowLEDgMEntS
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de Alagoas (Fapeal), Maceió, Brazil.The authors thank Flávia Soares de Lima, from Centro de Patologia e Medicina Laboratorial (CPML), for technical assistance.
First detection of metallo-β-lactamases in nosocomial isolates of Pseudomonas aeruginosa in Alagoas, Brazil

taBLE −
Resistance profile of MβL-positive Pseudomonas aeruginosa strains from Alagoas