Emergence of an IMP-like metallo-enzyme in an Acinetobacter baumannii clinical strain from a Brazilian teaching hospital

Multidrug-resistant Acinetobacter spp. constitute a serious cause of nosocomial infection in Brazilian hospitals Gales et al 1986, Levin et al 1999. Typically, carbapenems remain as the widest spectrum therapeutic option for treatment of such infections. However, resistance to these antimicrobial agents has increased, resulting in the use of potentially more toxic agents such as the polymyxins (Levin et al., 1999). Although the high carbapenem resistance rates have been reported among Acinetobacter spp. isolated in Brazil, very little is known about their mechanisms of resistance Gales et al 1986, Costa et al 2000; Gales et al., 2002).The knowledge of such mechanisms could contribute to a better understanding of the emergence of resistance and the epidemiological scenario, which could guide the infection control services in implementing effective interventions to avoid the spread of carbapenem-resistant isolates between patients or organisms species.

This report documents the first appearance of an IMP-like metallo-β-lactamase found in a clinical isolate of A. baumannii from a Brazilian teaching hospital. The strain A3227 was isolated from a quantitative tracheal secretion sample in a male patient, who developed nosocomial pneumonia during his hospitalization at the Hospital São Paulo (São Paulo, Brazil). This hospital is a 600-bed teaching medical center, where carbapenem resistance among Acinetobacter spp. isolates has reached rates of approximately 10.0%.

The minimum inhibitory concentrations (MICs) were determined using broth microdilution method according to the National Committee for Clinical Laboratory Standards (NCCLS, 2000). The hydrolysis of carbapenems was evaluated using a bioassay described by Gots (1945). This test utilizes Micrococcus luteus ATCC 9341 (yellow pigmented) as an indicator of antimicrobial hydrolysis by the challenge strains. Briefly, using a multi-prong inoculator, the clinical strain A3227, the positive and negative control strains were plated on Mueller-Hinton agar plates containing approximately 109 cfu/ml of M. luteus ATCC 9341 and meropenem at concentrations of 0.06 or 0.12 μg/mL. The positive control strains were the IMP-1-producing Pseudomonas aeruginosa (PSA319) and A. baumannii (ACB 17-4), while the negative control strains were Escherichia coli (ATCC 25922) and Enterococcus faecalis (ATCC 29212). An investigational Etest strip (MBL strip; AB BIODISK, Solna, Sweden) was used as a screening test for the detection of metallo-enzyme producing isolates, as well as a disk-approximation test using 2-mercaptopropionic acid (2-MPA) or EDTA Arakawa et al 2000, Walsh et al 1900.

Isoelectric focusing (IEF) was performed utilizing crude β-lactamase extracts prepared by freeze-thaw lysis of bacterial cultures grown to exponential phase in tryptic soy broth. Isoelectric focusing tests utilized a Multiphore II electrophoresis system in ampholine-polyacrylamide gels, pl 3.5-9.5 (Pharmacia Biotech, Sweden). Gels were run for 90 minutes at a potential difference of 1500 V, 30 mA and 30 W, and stained with 0.5 mM nitrocefin (Microbiology Systems, Cockeysville, MD). Preparations of TEM-1, TEM-4, SHV-1, SHV-3, SHV-4, and SHV-5 enzymes were included as IEF standards.

To determine whether these strains were carrying imp gene derivatives, PCR was performed on total DNA after boiling the bacterial cells using IMP-1 (imp1, sense: 5′-CTACCGCAGCAGAGTCTTTGC-3′, antisense: 5′-GAACAACCAGTTTTGCCTTACC-3′) and IMP-2 (imp2, sense: 5′-TGCCGCGGGAGCGCGTTTG-3′, antisense: 5′-GCCCTTTAACAGCCTGTTCCC-3′) primers Osano et al 1994, Riccio et al 2000. The amplification PCR conditions were as follows: 35 cycles of denaturation at 94°C for 1.0 minute, annealing at 61°C for 1.0 minute, and amplification at 72°C for 1 minute. PCR products were purified using the Concert Rapid PCR Purification System (Gybco, USA). DNA sequence analysis was performed using Big Dye terminator cycle sequencing chemistry for ABI BioPrism 377/310 (Applied Biosystems, California, USA).

To examine whether the carbapenem resistance was transferable, mating experiments were carried out using Escherichia coli K12 (streptomycin-resistant, MIC ≥ 1024 μg/ml) and P. aeruginosa ATCC 27853 mutant (streptomycin-resistant, MIC ≥ 1024 μg/ml) as recipients. Overnight cultures were mixed together and plated onto selective media containing streptomycin 1000 μg/ml, ampicillin 60 μg/ml, and imipenem 24 μg/ml.

The strain A 3227 was highly resistant to imipenem and meropenem (MIC, ≥ 32 μg/ml), extended-spectrum cephalosporins (ceftazidime MIC, > 256 μg/ml; cefepime MIC, 128 μg/ml). In contrast, this isolate was susceptible to aztreonam (MIC, 4 μg/ml), gentamicin (MIC, 1 μg/ml) and ciprofloxacin (MIC, 0.12 μg/ml). This isolate exhibited intermediate resistance to ampicillin/sulbactam (MIC, 16 μg/ml) and amikacin (MIC, 32 μg/ml).

The growth of the M. luteus ATCC 9341 was routinely inhibited at 0.12 μg of meropenem. Thus, the growth of M. luteus ATCC 9341 around A3227 colonies on 0.12 μg/ml meropenem agar plate indicated carbapenem inactivation. Using the Etest MBL strip, the A3227 strain was considered to be a metallo-β-lactamase producer since there was a 64-fold reduction of the imipenem MIC in the presence of EDTA. A enhanced zone of inhibition was also observed between the 2-MPA disk and both ceftazidime and imipenem disks, confirming that A3227 was a possible metallo-β-lactamase producer.

Two bands focusing at isoelectric points (pls) of 5.4 and 8.6 were detected on the IEF gel. Among Acinetobacter spp. isolates, enzymes with pls 5.4 have been described as derivatives of blaTEM- or blaPER-genes and have not been capable of hydrolyzing carbapenems Da Silva et al 1999, Bou and Martínez-Beltrán 2000. Thus, the pl 8.6 enzyme might have been responsible for the carbapenem hydrolysis.

The PCR utilizing blaIMP-2 primers failed to produce any products; however, a 580-bp product was amplified utilizing blaIMP-1 primers. A total of 508-bp were sequenced, showing 100.0% of homology to blaIMP-6. However, the N-terminal region could not be amplified or sequenced yet. Transfer of carbapenem resistance to E. coli K12 or P. aeruginosa ATCC 27853 mutant was not detected despite several attempts.

There have been increasing number of reports, especially from the Asia-Pacific region, of gram-negative organisms that carry the transferable carbapenem resistance gene blaIMP. Most of these isolates produce the metallo-β-lactamase IMP-1 (Osano et al., 1994). Variants of IMP-1, such as IMP-2 through -9, have been described in a variety of species in diverse parts of the world Da Silva et al 1999, Chu et al 2001, Yano et al 2001, Gibb et al 2002. Among these enzymes, differences in the carbapenem and penicillin hydrolysis activity have been observed. Commonly, these enzymes have no activity against monobactams, a feature noted for the strain A3227 Chu et al 2001, Yano et al 2001.

Recently, a new metallo-β-lactamase (SPM-1) was detected from a P. aeruginosa strain isolated at the same Brazilian institution. The N-terminal sequence from SPM-1 was completely different from that described for IMP-derivative enzymes (Walsh et al., in press). It has been suggested that metallo-β-lactamases-producing isolates have emerged as a consequence of the selective pressure exerted by carbapenems. However, the exact reason for their emergence is not completely understood. To determine the sequence of the N-terminal of the IMP-like metallo-β-lactamase described in this report (A. baumannii A3227), cloning experiments are underway and new transference attempts using transformation techniques have been carried out. Only after completing these procedures, will we be able to conclude if this index strain (A. baumannii A3227) carries a new variant of the blaIMP gene and if the gene was inserted into the bacterial chromosome or carried by a plasmid. Finally, expanded surveillance studies are necessary to evaluate the prevalence of these emerging IMP- or SPM-producing isolates in the Brazilian hospitals as a component of already existing carbapenem-resistant strains of non-fermentative gram-negative bacilli. Dissemination of these novel metallo-enzymes could have disastrous consequences for therapy of serious nosocomial infections especially for those caused by AmpC-producing isolates since most of them are resistant to polymyxins. (Gales et al 2001)