Prevalence of Metallo-ββ-Lactamases Producing Acinetobacter baumannii in aMoroccan Hospital

Objective. To determine the prevalence of metallo beta-lactamases (MBL) among carbapenem resistant strains of Acinetobacter baumannii in our hospital. Methodology. During a period of 12 months (January–December 2010), 47 isolates of Acinetobacter baumannii were collected from different clinical specimens of in-patients. Antimicrobial susceptibility was determined and interpreted using the disk diffusion method according to the Antibiogram Committee of the French Society for Microbiology guidelines. Imipenem nonsusceptible isolates were further screened for production of MBL. Results. All Acinetobacter baumannii’ isolates were resistant to ticarcillin, ticarcilline/clavulanate, piperacillin, piperacillin/tazobactam, gentamicin, tobramycin, and cipro�oxacin, except an isolate that was sensitive to ceazidime and cefepime. In addition to that, amikacin and trimethoprim/sulfamethoxazole were, respectively, sensitive by 59.5% and 53%. Among 57,4% (27/47) imipenem non-susceptible isolates of Acinetobacter baumannii, 74% (20/27) were found to be MBL producers. Conclusion. Although the rate of imipenem nonsusceptible isolates of Acinetobacter baumanni seems to remain stable in 2005 (57%) and 2010 (57,5%), the prevalence of MBL producer strain is increasing (38% in 2005 versus 75% in 2010). e �ndings strongly suggest that there is a need to track the detection of MBL producers; moreover, a judicious use of carbapenems is necessary to prevent further spread of these organisms.


Introduction
Acinetobacter baumannii is a typical nosocomial pathogen causing infections and a high mortality oen among patients hospitalized in the Intensive Care Unit.Acinetobacter baumannii is intrinsically less susceptible to antibiotics than Enterobacteriaceae is; moreover, it has propensity to acquire resistance.e resistance of Acinetobacter baumannii to carbapenem is now a major worldwide issue [1][2][3][4].A mechanism of this resistance is being characterized by the production of a speci�c enzyme called metallo beta-lactamases (MBL).ese enzymes belong to Ambler class B -lactamases based on their amino acid sequence homology and to group 3 according to the Bush classi�cation based on their substrate pro�les (imipenem hydrolysis.)ese enzymes are inhibited by ethylene diamine tetra-acetic acid (EDTA) [1].e rapid detection of MBL positive isolates is necessary to control infection and to prevent their dissemination.PCR method was initially of simple use in detecting MBL-producing isolates but became more difficult with the increased number of types of MBLs; moreover, it is expensive for daily routine application in developing countries' laboratories as it is the case in Morocco.In 2010, the aim of this study was �rst to determine the prevalence of MBL among carbapenem resistant strains of Acinetobacter baumannii in our hospital, and second to compare this rate to the one found in 2005 [5].

Methodology
is study was conducted in the specialty hospital of Rabat, a 322 bedded tertiary care teaching hospital including Intensive Care, Neurology, Neurosurgery, Otolaryngology, and Ophthalmology Departments.Between January and December 2010, 47 nonduplicate Acinetobacter baumannii were isolated and identi�ed based on a battery of biochemical tests of which the galleries 20 NE API (BioMerieux SA, Marcy-l'Etoile, France).ese were taken from various clinical specimens of in-patients.Antimicrobial susceptibility of all isolate was determined using the disk diffusion method according to the Antibiogram Committee of the French Society for Microbiology (CA-SFM), guidelines [4].e panel of antimicrobial agents tested were as follows: ticarcillin (75 g), ticarcillin/clavulanate (75/10 g), piperacillin (75 g), piperacillin/tazobactam (75/10 g), ceazidim (30 g), cefepim (30 g), imipenem (10 g) gentamicin (15 g), amikacin (30 g), tobramycin (10 g), cipro�oxacin (5 g), trimethoprim/sulfaméthoxazole (1,25/23,75 g), and colistin (50 g).e source of the Mueller-Hinton agar and the antibiotics discs is the same as both of them belong to the Oxoid Ltd Company.Intermediately susceptible strains were accepted as resistant.Pseudomonas aeruginosa ATCC 27853 was used as the control strain for susceptibility testing.

Detection of MBL Production
All strains of Acinetobacter baumannii non-susceptible to imipenem (diameter < 24 mm) were screened for MBL production by a phenotypic method as described by Yong et al. [4].In this test, organisms were inoculated in plates of Mueller-Hinton agar, as recommended by CA-SFM [6].A solution was prepared by dissolving 186.1 g of disodium EDTA.2H2O in 1,000 mL of distilled water and adjusting it to pH 8.0 by using NaOH.e mixture was sterilized by autoclaving.Two 10 g imipenem disks were placed on the plate, and 750 mg of a 0.5 M EDTA solution was added to one of them.From 18 to 24 hours of incubation in the air at 37 ∘ C, the inhibition zones with imipenem-EDTA disks were ≤14 mm for the MBL-negative isolates, while they were ≥17 mm for the MBL-positive isolates.All the MBL positive isolates were repeatedly checked for reproducibility.

Discussion
e present study revealed a high proportion of imipenem resistance among Acinetobacter baumannii' isolates in our hospital.Although this rate seems to remain stable by 57% and 57,5%, respectively, in 2005 and 2010.Over all, this rate of resistance was higher than those reported from other Moroccan studies (23,8% [6] and 42,6% [7]).Within the last �ve years, the increasing number of carbapenem resistant Acinetobacter baumannii is of major importance in the context of resistance to -Lactams.Indeed, between 1998 and 2004, this rate of resistance in Europe, North America, South America, and Asia ranged between 0% and 40% [8].But in most studies aer 2005, the rate of resistance was greater than 50%.e higher rates of resistance were found in Iran (63%) [2], Italy (62,5%) [9], China (55,6%) [10], Turkey (53,7%) [11], and Korea (51%) [12].e source of Acinetobacter baumannii in our hospital was and still is in the intensive care unit (76% in 2005 versus 82% in 2010) especially from respiratory samples being taken from patients mechanically ventilated (62% for 2005 and 69% for 2010).e same distribution was reported by Peymani et al. [2] and Lee et al. [13].is suggested that the invasive devices such as tracheal tubes are important reservoirs involved in Acinetobacter baumannii's transmission.
In 1988, �apan reported the �rst plasmid-mediated MBL (IMP-1) in Pseudomonas aeruginosa [14].Later this enzyme and its variants were detected in different Gram negative bacilli in different countries.In our present study, 74% of imipenem nonsensitive strains of Acinetobacter baumannii were MBL producers.is proportion was higher than the one found in 2005 in our institution (38% in 2005 versus 75% in 2010) and higher than those reported in other studies such as in Korea 14,2% [13], India 14,8% [15], and Iran 49% [2].However, in two recent studies made in Pakistan by Kaleem et al. [16] and by Irfan et al. [17], the frequency of MBL Acinetobacter baumannii is higher by 84% and 96%, respectively.is emergence is a serious epidemiological risk for at least two reasons.First of all, the MBL does not confer resistance to carbapenem only, but to all -Lactams and other classes of antibiotics such as aminoglycoside and �uoroquinolone.Second of all, the genes encoding these enzymes spread easily on plasmids, by that causing nosocomial infections and outbreaks with a mortality rates range from 25% to 75% [1].ese results indicate that the available choices for appropriate treatments for infection, caused by Acinetobacter baumannii, are currently limited.In vitro, studies reveal that tigecyclin, and colistin are the only antibacterial agents with consistent activity against MBLproducing strains [1].Random controlled trials are required in order to evaluate the available therapeutic regimens, including treatment combinations.

Conclusion
e prevalence of MBL Acinetobacter baumannii is possibly increasing because of clonal and horizontal dissemination of resistance in our hospital.Respiratory and urine samples collected from Intensive Care Unit patients were found to be the main sources of MBL-producing isolates.Early detection and infection control practices are the best defenses against these organisms; therefore, systematic surveillance to detect MBL producers is necessary.Last, but not least, a judicious use of carbapenems is essential to prevent the spread of these organisms.