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Antimicrobial Agents and Chemotherapy, December 2004, p. 4693-4702, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4693-4702.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Integron Carrying a Novel Metallo-ß-Lactamase Gene, blaIMP-16, and a Fused Form of Aminoglycoside-Resistant Gene aac(6')-30/aac(6')-Ib': Report from the SENTRY Antimicrobial Surveillance Program

Rodrigo E. Mendes,1,2* Mark A. Toleman,2 Julival Ribeiro,3 Helio S. Sader,1,4 Ronald N. Jones,4,5 and Timothy R. Walsh2

Disciplina de Doenças Infecciosas e Parasitárias, Universidade Federal de São Paulo, São Paulo,1 Hospital de Base do Distrito Federal, Brasília, Brazil,3 Department of Pathology & Microbiology, University of Bristol, Bristol, United Kingdom,2 The JONES Group/JMI Laboratories, North Liberty, Iowa,4 Tufts University School of Medicine, Boston, Massachusetts5

Received 19 April 2004/ Returned for modification 26 June 2004/ Accepted 4 September 2004


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ABSTRACT
 
Since January 2002 Pseudomonas sp. strains resistant to carbapenems and ceftazidime have been routinely screened as part of the SENTRY Antimicrobial Surveillance Program for metallo-ß-lactamase production, and their resistance determinants have been analyzed. Pseudomonas aeruginosa index strain 101-4704, which harbors a novel blaIMP variant, blaIMP-16, was isolated in April 2002 from a 60-year-old man in Brasília, Brazil. blaIMP-16 was found on the chromosome of the P. aeruginosa index strain, and the deduced amino acid sequence (IMP-16) showed the greatest identities to IMP-11 (90.3%) and IMP-8 (89.5%). Sequence analysis revealed that blaIMP-16 was associated with a class 1 integron, which also encoded aminoglycoside-modifying enzymes. Downstream of blaIMP-16 resided an open reading frame, which consisted of a new aminoglycoside-modifying gene, namely, aac(6')-30, which was fused with aac(6')-Ib'. The amino acid sequence of the aac(6')-30 putative protein showed the most identity (52.7%) to the sequence of AAC(6')-29b described previously. The fourth gene cassette constituted aadA1. The steady-state kinetics of IMP-16 demonstrated that the enzyme preferred cephalosporins and carbapenems to penicillins. The main functional difference observed among the kinetic values for IMP-16 compared to those for other IMPs was a lack of cefoxitin hydrolysis and a lower kcat/Km value for imipenem (0.36 µM–1 · s–1). This report further emphasizes the spread of metallo-ß-lactamase genes and their close association with various aminoglycoside resistance genes.


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INTRODUCTION
 
The carbapenems represent important therapeutic options for serious infections caused by Pseudomonas aeruginosa, especially multidrug-resistant strains. The activities of these compounds are mainly related to their rapid permeation across bacterial membranes and their stabilities against most ß-lactamases (3). However, as with other antimicrobial agents, carbapenem-resistant P. aeruginosa strains continue to be isolated with an increasing frequency from nosocomial infection sources. Trends have been observed that demonstrate a decrease in carbapenem susceptibility among P. aeruginosa strains isolated from hospitalized patients in Latin America (from 83.0 to 64.4% between the years 1997 and 2001 [P < 0.001; odds ratio = 2.70; 95% confidence interval = 1.88 to 3.89]) (2).

The mechanisms responsible for carbapenem resistance include decreased outer membrane permeability (porin mutations), up-regulation of multidrug efflux pumps, substantial production of a chromosomal ampC ß-lactamase or a class D ß-lactamase (often accompanied by porin changes), and production of a class B ß-lactamase (1, 22). The class B enzymes, or metallo-ß-lactamases (MßLs), are zinc-dependent enzymes that catalyze the hydrolysis of a broad range of ß-lactams, including carbapenems, using zinc ions as metal cofactors (44). To date, three types of mobile MßL genes have been reported. In 1994, the IMP-type MßL gene was first reported in Japan (29), and since then, IMP MßL-producing strains have been reported from many different countries worldwide (4, 6, 7, 13, 46, 51). The VIM-type enzymes were first reported from Italy in 1999, and strains producing these enzymes have now been reported from European countries as well as Asia and the Americas (21, 25, 35, 47, 50). Some studies have indicated the presence of MßL genes among multidrug-resistant strains in Brazil (10, 31), and this evidence was later confirmed by the report of a P. aeruginosa strain producing a new MßL subclass, namely, SPM-1 (26, 48).

Most mobile MßL genes are part of a gene cassette consisting of a single gene and a downstream recombination site, known as a 59-base element (59-be) (36). The genes are usually associated with class 1 and 3 integrons (5), apart from SPM-1, for which the genetic environment has recently been described (34). Among MßL genes, the class 1 integron is the most commonly encountered and consists of a 5' conserved sequence (5'-CS), which constitutes an intI1 gene coding for an integrase; a recombination site, attI1; a promoter; and usually, a 3'-CS that possesses the qacE{Delta}1 and sul1 genes (9). Integrons are able to capture gene cassettes by a site-specific recombination event between two recombination sites, one in the integron and one in the cassette. Both recombination sites confer mobility due to their recognition by the integrase that catalyzes the integration of the gene cassette between attI1 in the integron and the 59-be in the gene cassette. Although recombination between different sites has been documented, it occurs at a very low frequency (30, 36).

In the present study, we describe the blaIMP-16 variant, the kinetic parameters for IMP-16, and the genetic context of the blaIMP-16-carrying integron, which also included a fused form of aminoglycoside-modifying resistance gene aac(6')-30/aac(6')-Ib', found in a P. aeruginosa strain (strain 101-4704) isolated from the Latin American component of the SENTRY Antimicrobial Surveillance Program.


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MATERIALS AND METHODS
 
Clinical context of carbapenem-hydrolyzing P. aeruginosa index strain (strain 101-4704). The SENTRY Program was established in 1997 to monitor the most important pathogens and antimicrobial resistance patterns of the organisms causing nosocomial and community-acquired infections via a broad network of sentinel hospitals distributed by geographic location and bed capacity. As part of that study, a P. aeruginosa index strain (strain 101-4704) was isolated from a 60-year-old man with bronchogenic carcinoma who was first admitted to the Hospital de Base do Distrito Federal, Brasília, Brazil, in May 2001. A pneumonectomy of the right lung was performed in July 2001, and in August 2001 the patient developed severe pneumonia and required mechanical ventilation. An empyema was also diagnosed, and drainage was performed. The patient's symptoms improved, but he had three other episodes of pneumonia and remained in the intensive care unit for 151 days.

Several pathogens were recovered from respiratory specimens, including P. aeruginosa, an Acinetobacter sp., and Stenotrophomonas maltophilia, but all blood cultures were negative. When the patient was in the intensive care unit he received meropenem at 1 g every 8 h (for 54 days), amikacin at 500 mg every 12 h (q12h; for a total of 42 days), trimethoprim-sulfamethoxazole at 800 mg every 6 h (for 16 days), ciprofloxacin at 400 mg q12h (for 14 days), and vancomycin at 1 g q12h (for 21 days). The patient was subsequently discharged. In April 2002, he was readmitted with cough and dyspnea. Pulmonary secretions collected during a bronchoscopy yielded the P. aeruginosa index strain, strain 101-4704. No antimicrobial was administered to the patient, and he was discharged in May 2002. Strain 101-4704 was resistant to several ß-lactams, including meropenem and imipenem (MICs > 16 µg/ml), but was susceptible to aztreonam, piperacillin, and piperacillin-tazobactam. The microorganism was also resistant to most aminoglycosides, including gentamicin, tobramycin, and netilmicin, but remained susceptible to amikacin and the fluoroquinolones.

Susceptibility testing. The susceptibilities of all isolates collected in the SENTRY Program were tested by the reference broth microdilution method described by the National Committee for Clinical Laboratory Standards (NCCLS) (27). The aminoglycoside resistance profiles of Escherichia coli DH5{alpha} harboring recombinant plasmids were assayed by MIC determinations by either the reference agar dilution method or Etest (AB Biodisk, Solna, Sweden) methodology, according to the guidelines of NCCLS and the manufacturer, respectively. Antimicrobial agents were obtained from the respective manufacturers; and quality control was performed by concurrent testing of E. coli ATCC 25922, P. aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212.

Phenotypic detection of ß-lactamase enzymes. Isolates were initially screened for the production of MßLs by the modified disk approximation test. Briefly, a 100-mm-diameter Mueller-Hinton agar plate was inoculated with a 0.5 McFarland standard suspension from a fresh overnight culture. Imipenem, meropenem, and ceftazidime disks were strategically aligned around disks that contained either EDTA (750 µg) or thiolactic acid (0.3 µl) as MßL inhibitors. The test result was read after 20 h of incubation at 35°C. Any appearance of either an elongated or a phantom zone between the carbapenems and/or ceftazidime and either one of the disks containing an MßL inhibitor was considered a positive test result. IMP-2-producing Acinetobacter baumannii 54/97 was used as a positive control. MßL Etest strips were used to confirm the disk approximation test results. In addition, ceftazidime-ceftazidime-clavulanic acid and cefepime-cefepime-clavulanic acid ESBL Etest strips were used to evaluate the organisms for the production of extended-spectrum ß-lactamases (ESBLs).

Bacterial strains, plasmids, conjugation, and transformation. The bacterial strains and plasmids used in this study are described in Table 1. Plasmid DNA from P. aeruginosa 101-4704 was extracted with a plasmid DNA Midi kit (Qiagen, Chatsworth, Calif.). The transfer of ß-lactam resistance markers from strain 101-4704 into E. coli DH5{alpha} and a rifampin-resistant (Rifr) mutant of P. aeruginosa pA01 was performed with a Gene Pulser apparatus (Bio-Rad, Watford, United Kingdom) that was set at 2.5 kV, 25 µF, and 400 {Omega}. The transfer of resistance to Rifr mutant E. coli K-12 and P. aeruginosa pAO1 was also performed by conjugation experiments, as described previously (40). Strains DH5{alpha}, pAO1, and K-12 harboring the possible plasmids of strain 101-4704 were selected by plating the strains onto nutrient agar plates containing ceftazidime (10 µg/ml) or ceftazidime and rifampin (500 µg/ml).


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  		TABLE 1. Bacterial strains and plasmids used in this study

Analytical IEF. The ß-lactamase extract from P. aeruginosa strain 101-4704 was obtained by cell lysis with a BugBuster apparatus (Novagen, Nottingham, United Kingdom), and isoelectric focusing (IEF) was performed with a NOVEX apparatus (Invitrogen, Paisley, United Kingdom). The focused ß-lactamases were detected by overlaying the gel with nitrocefin solution (150 µM; Microbiology Systems, Cockeysville, Md.). The ß-lactamase pIs were estimated by linear regression, obtained by comparison to the pIs of reference proteins by using a standard IEF marker containing proteins with a pI range of 4.5 to 9.5 (Bio-Rad).

PCR and DNA sequencing. The SENTRY Program strains with positive MßL phenotypic test results were screened for MßL genes by standard PCRs (40) with Extensor Hi-Fidelity PCR Master Mix (ABgene, Surrey, United Kingdom) and primers targeting conserved regions of blaVIM, blaIMP, and blaSPM. Additional primers designed to target the 5'-CS and 3'-CS regions of the class 1 integron were used to amplify the blaIMP-16-containing integron resident in P. aeruginosa 101-4704. These primers yielded PCR products, and both strands were sequenced on a Perkin-Elmer system 377 DNA sequencer (Advanced Biotechnology Centre, London, United Kingdom). The DNA sequences were found to overlap, and these were assembled to produce a contiguous sequence of 4,333 bp.

Recombinant DNA methodology. The blaIMP-16 and aminoglycoside resistance genes were amplified by PCR. Primers were designed to amplify specific individual genes or sets of genes that could subsequently be cloned into pPCRScriptCam SK(+) (Stratagene Cloning Systems, La Jolla, Calif.). blaIMP-16 and the downstream region were amplified with primer set Int1-1F-aacA4FR. The fused-form gene aac(6')-30/aac(6')-Ib' was amplified with primer set IMP-16FF-aadA1FR. Additionally, aac(6')-30 and aac(6')-Ib' were separately amplified with primer sets IMP-16FF-aacA4FR and aacA30FF-aadA1FR, respectively (Fig. 1). The ribosome-binding site and the stop codon were included in order to allow gene expression. This technique yielded several subclones of the original integron that were subsequently screened by PCR with primer set M13F-M13R, and the presence of the insertion and its orientation were confirmed by sequencing. Because XL10-Gold Kan ultracompetent E. coli cells are intrinsically resistant to streptomycin due to a chromosomal mutation, the recombinant plasmids were transferred into E. coli DH5{alpha}, and their respective antimicrobial resistance profiles were evaluated.



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  		FIG. 1. Schematic representation of the class 1 integron-containing blaIMP-16 gene cassette from clinical isolate P. aeruginosa 101-4704. Boxes, inserted genes; arrows, transcriptional orientations; black circles, 59-be's; white circle, attI1 recombination site; lines, DNA of the inserts contained within recombinant plasmids pREM-1, pREM-2, pREM-3, and pREM-4; arrowheads, primer positions and their orientations; M, start codon; asterisk, the location of the stop codon for the particular gene.

ß-Lactamase purification. A single colony of E. coli harboring recombinant plasmid pREM-4 was grown overnight in 10 ml of nutrient broth containing ceftazidime (10 µg/ml) and chloramphenicol (30 µg/ml) at 37°C. The cells were harvested by centrifugation (4,000 x g for 10 min at 4°C) and then added to 4 liters of terrific broth (12% tryptone, 20% yeast extract, 0.4% glycerol, 0.17 M monopotassium phosphate, 0.72 M dipotassium phosphate) for aerobic growth in an orbital shaker for 24 h at 37°C. The cells were again harvested, as described above, and were resuspended in 100 ml of 10 mM HEPES buffer containing 50 µM ZnCl2 (pH 7.5). The periplasm preparation was obtained by the addition of 400 µl of lysozyme solution (20 mg/ml; Sigma-Aldrich, Poole, United Kingdom) and 400 µl of calcium chloride (147 mg/ml). Cells debris was removed by centrifugation (9,000 x g for 20 min at 4°C), the supernatant was loaded onto a Q-Sepharose (quaternary ammonium) HR 16/50 column (Amersham Pharmacia Biotech, Uppsala, Sweden), and then the proteins were eluted with a linear NaCl gradient (0 to 1 M) at a flow rate of 2 ml/min. Fractions containing ß-lactamase activity were pooled, concentrated, and then injected onto a Superdex 75 HR 10/30 column (Amersham Pharmacia Biotech) that had previously been equilibrated with HEPES buffer containing 50 µM ZnCl2 and 0.2 M NaCl. Proteins were eluted at a flow rate of 0.7 ml/min. During the purification procedure the presence of ß-lactamase activity was monitored with nitrocefin solution.

Protein electrophoretic technique. The enzyme that was obtained was submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Mini-Protein II apparatus (Bio-Rad) in order to confirm its purity.

Kinetic measurements. The reactions for kinetic measurements were performed at 20°C with 20 µl of enzyme in 1 ml of HEPES buffer containing 50 µM ZnCl2 (pH 7.0). Hydrolysis was measured by observing the changes in absorbance due to the opening of the ß-lactam ring at a range of concentrations in a Lambda 35 spectrophotometer (Perkin-Elmer, Cambridge, United Kingdom). The steady-state kinetic parameters Km (in micromolar units) and kcat (per second) were deduced from the initial rates of hydrolysis by using the Hanes-Woolf plot (41). The extinction coefficients and wavelengths for each antimicrobial agent evaluated were those described previously (26).

Computer sequence analysis. The nucleotide sequences were compared by using software available over the Internet (http://www.ebi.ac.uk/fasta33/). The nucleotide sequences and their deduced protein products, alignments, and phylogenetic relationships were determined with the Lasergene software package (DNASTAR, Madison, Wis.). The putative cleavage site of the signal sequence of IMP-16 was identified by computer analysis with software available at the Center for Biological Sequence Analysis website (http://www.cbs.dtu.dk).

Nucleotide sequence accession number. The nucleotide sequence of the blaIMP-16-containing integron described in this paper has been submitted to the EMBL/GenBank/DDBJ sequence databases and assigned accession number AJ584652.


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RESULTS
 
Genetic environment of blaIMP-16. The blaIMP-16 gene was located in a class 1 integron that contained a 5'-CS composed of integrase gene intI1, the attI1 recombination site, and a promoter region (Fig. 2). The P1 promoter (–35 [TGGACA]; –10 [TAAGCT]) was identified and was found to contain two hexamers separated by 17 bp, followed by a second promoter, P2 (–35 [TTGTTA]; –10 [TACAGT]), 82 bp downstream of P1. The fused disinfectant determinant and sulfonamide gene qacE{Delta}1/sul1 was found at the 3'-CS end. The integron consisted of four antimicrobial resistance gene cassettes (Fig. 1). Located at the first position downstream of the 5'-CS was the MßL blaIMP-16, which was flanked by typical features of a gene cassette, namely, a core site (GTTACGC), an inverse core site (TTCTAAC), and a 59-be (Fig. 2). This 59-be was 133 bp in length and showed the greatest identity to the 59-be from IMP-11, which has been found in P. aeruginosa (EMBL/GenBank/DDBJ accession no. AB074437) and A. baumannii (EMBL/GenBank/DDBJ accession no. AB074436) strains isolated in Japan. The blaIMP-16 59-be sequence differed by 11 of 132 bp (90.6% identity) from the blaIMP-11 59-be sequence.




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  		FIG. 2. DNA and amino acid sequences of the blaIMP-16-carrying integron and its gene. The start and stop codons of the ORFs are indicated by horizontal arrows and asterisks, respectively. The corresponding predicted protein translation is reported below the DNA sequence. The double slashes represent the putative cleavage site of the signal sequence of IMP-16. The conserved 7-bp core sites located at the cassette boundaries and the 7-bp inverse core sites located at the left end of each 59-be are boxed. The recombination crossover sites are indicated by vertical arrows. The 59-bes are in italic; and their conserved sequences are highlighted in boldface and are labeled 1L, 2L, 2R, and 1R. The entire sequence has been assigned EMBL/GenBank/DDBJ accession number AJ584652.

blaIMP-16 sequence analysis and its deduced protein sequence. blaIMP-16 encoded a putative protein of 246 amino acids and presented a G+C content of 38.5%. The N terminus of the protein showed features typical of bacterial signal peptides that target proteins to the periplasmic space, and the most likely cleavage site was identified between the alanine and glycine residues (Fig. 2). This produced a mature protein of 25,266 Da with a theoretical pI of 6.5. The results of the analytical IEF experiment gave a pI that was in accordance with the theoretical pI of blaIMP-16 (data not shown). IMP-16 showed six unique amino acid differences compared to the sequences of the other IMP variant, namely, E50D, V74F, T88A, T122S, V183L, and K252R, and displayed the greatest identities to IMP-11 (90.3%) and IMP-8 (89.5%), with 21 and 24 amino acid differences at the level of the mature protein, respectively (Fig. 3). The MßL active site implicated in the binding of zinc ions, as well as residues known to not tolerate substitutions, were conserved, as previously described for all the other IMP-type enzymes (Fig. 3).



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  		FIG. 3. Amino acid alignment of the IMP-16 protein sequence with those of IMP-type enzymes. Differences in the amino acid sequences are noted by insertion of a single letter representing the amino acid change within that particular sequence. Asterisks under the IMP-16 sequence represent amino acids involved in the coordination of zinc ions, and residues known to not tolerate substitutions are underlined. References for each sequence are as follows: IMP-1 (20), IMP-2 (37), IMP-3 (15), IMP-4 (4), IMP-5 (7), IMP-6 (52), IMP-7 (13), IMP-8 (51), IMP-9 (EMBL/GenBank accession no. AY033653), IMP-10 (16), IMP-11 (EMBL/GenBank accession no. AB074437), IMP-12 (8), IMP-13 (46), and IMP-16 (this study). Numbering is according to the scheme for class B ß-lactamases (12).

Antimicrobial resistance pattern of E. coli DH5{alpha} harboring recombinant plasmid pREM-4. blaIMP-16 was amplified by PCR and ligated into a vector, pPCRScriptCam SK(+), in order to create recombinant plasmid pREM-4, which was expressed in E. coli DH5{alpha} (Fig. 1). E. coli DH5{alpha} harboring recombinant plasmid pREM-4 showed an antimicrobial resistance profile consistent with that observed for the index strain, strain 101-4704. It showed some degree of resistance to benzylpenicillin, ampicillin, amoxicillin-clavulanate, ceftazidime, and cefotaxime and decreased susceptibility to cefepime, piperacillin, piperacillin-tazobactam, imipenem, and meropenem. The index strain as well as E. coli DH5{alpha}(pREM-4) remained highly susceptible to the monobactam aztreonam (Table 2).


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  		TABLE 2. Antimicrobial susceptibility profiles of blaIMP-16-carrying clinical isolate P. aeruginosa 101-4704; E. coli DH5{alpha} harboring recombinant plasmid pREM-1, pREM-2, pREM-3, or pREM-4; and the E. coli DH5{alpha} recipient strain

MßL resistance marker transfer experiments. Conjugation experiments between rifampin-susceptible (Rifs) strain P. aeruginosa 101-4704 and Rifr strain E. coli K-12, as well as P. aeruginosa 101-4704 and Rifr strain P. aeruginosa pA01, did not yield transconjugants. Despite repeated attempts, analysis of plasmid DNA from P. aeruginosa 101-4704 did not identify any plasmid, and transformation experiments were unsuccessful. As a ß-lactam resistance marker was not transferred to the recipient strains, even in the conjugation experiments, blaIMP-16 is likely encoded by the chromosome and is not carried on a plasmid.

Genetic context of fused gene aac(6')-30/aac(6')-Ib'. An open reading frame (ORF) of 984 bp was detected immediately downstream of blaIMP-16. The ORF was preceded by a ribosome-binding site and potentially encoded a protein of 36.7 kDa. This ORF consisted of a novel gene cassette, namely, aac(6')-30 fused with the aac(6')-Ib' gene. aac(6')-30 was also flanked by typical features, but it presented a shortened 59-be of 19 bp, including the core and inverse core sites (Fig. 2). aac(6')-Ib' had a core site with a 1-bp mismatch, an A residue (in boldface) instead of the usual G residue (ATTAGGC) and an inverse core site (GCCTAAC), and the translation could start at the GTG codon located 19 bp downstream from its core site or at either one of the ATG codons located farther downstream (Fig. 2) (11, 28).

aac(6')-30/aac(6')-Ib' sequence analysis and its deduced protein sequence. The deduced amino acid sequence of the aac(6')-30 product possessed the highest similarity (52.7%) to previously described protein AAC(6')-29b (EMBL/GenBank/DDBJ accession no. AAK26254) (Fig. 4) (32). Except for the substitution aspartate-171->valine, AAC(6')-Ib' contained a sequence homologous to the previously described product of aac(6')-Ib' (EMBL/GenBank/DDBJ accession no. AAA25685), which encodes the aminoglycoside 6'-N-aminoglycoside acetyltransferase. This gene was characterized by a leucine-90->serine substitution and specified a type II enzyme that conferred resistance to gentamicin but not to amikacin (19).



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  		FIG. 4. Comparison of the deduced amino acid sequences of AAC(6')-30 with those members of the AAC(6') family. Differences in the amino acid sequences are noted by insertion of a single letter representing the amino acid change within that particular sequence. Motifs A, B, C, and D are conserved among all members of the AAC(6') family, while motifs E, F, and G are conserved among most of the members of the AAC(6') subfamily. References for each sequence are as follows: AAC(6')-Ic (43); AAC(6')-If (45); AAC(6')-Ig (18); AAC(6')-Ih and AAC(6')-Ij (17); AAC(6')-Ik (38); AAC(6')-Ir, AAC(6')-Is, AAC(6')-It, AAC(6')-Iu, AAC(6')-Iv, AAC(6')-Ix, and AAC(6')-Iw (39); AAC(6')-Iy (23); AAC(6')-29a and AAC(6')-29b (32); and AAC(6')-30 (this study).

IMP-16 purification and kinetics. The IMP-16 enzyme was overproduced and purified from E. coli(pREM-4) by fast-performance liquid chromatography, followed by a gel permeation chromatography step. The yield of the IMP-16 purified protein preparation was 0.3 µM, which appeared to contain a single band just under the band at 28.8 kDa, and it was estimated to be >95% pure (data not shown). Steady-state kinetics demonstrated that IMP-16 was able to hydrolyze several ß-lactams, including penicillins, narrow- to expanded-spectrum cephalosporins, and carbapenems. Kinetic values showed that the cephalosporins and the carbapenems were the best substrates (kcat/Km ratios, ≥0.15 µM–1 · s–1), while penicillins were uniformly poorer substrates (kcat/Km ratios, ≤0.13 µM–1 · s–1) (Table 3). No hydrolysis of cefoxitin or aztreonam was observed.


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  		TABLE 3. Kinetic parameters of purified IMP-1, IMP-2, IMP-12, and IMP-16

Expression of aminoglycoside-modifying genes in E. coli DH5{alpha}. The P. aeruginosa index strain was resistant to kanamycin, tobramycin, gentamicin, and netilmicin but was susceptible to amikacin and isepamicin (Table 2). In order to evaluate the functional status of AAC(6')-30 and AAC(6')-Ib', their respective genes, as well as the fused form, were amplified by PCR and ligated into the pPCRScriptCam SK(+) vector, creating recombinant plasmids pREM-1, pREM-2, and pREM-3 (Fig. 1). E. coli harboring pREM-1 [AAC(6')-30] showed decreased susceptibilities to amikacin, kanamycin, tobramycin, and neomycin but remained susceptible to gentamicin, sisomicin, isepamicin, and netilmicin. E. coli harboring pREM-2 [aac(6')-30/aac(6')-Ib'] showed decreased susceptibilities to all aminoglycosides tested, apart from isepamicin. Strikingly, E. coli harboring pREM-2 [AAC(6')-Ib'] did not confer the expected AAC(6')-II phenotype, since it remained susceptible to gentamicin. Increases in the MICs of kanamycin, tobramycin, and sisomicin were observed (Table 2).


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DISCUSSION
 
Multidrug-resistant Pseudomonas sp. isolates continue to be a growing concern, particularly for institutions where carbapenems are readily used. Many hospitals in Brazil routinely use carbapenems and expanded-spectrum cephalosporins to treat infections caused by gram-negative organisms, which provides selective pressure for the development of resistance. P. aeruginosa 101-4704 remained susceptible to amikacin, aztreonam, piperacillin, and fluoroquinolones, yet it displayed high-level resistance to most other aminoglycosides and ß-lactams (including carbapenems), a resistance phenotype that is becoming common in Brazil.

The kinetics of IMP-16 showed that the enzyme hydrolyzes ß-lactams less efficiently than IMP-1 does (Table 3). However, similar to other IMP variants, IMP-16 demonstrated an overall preference for cephalosporins and carbapenems rather than penicillins. The poor hydrolytic activity of IMP-16 toward piperacillin (kcat/Km ratio, 0.09 µM–1 · s–1) was consistent with the relatively low increase in the piperacillin MIC for E. coli harboring recombinant plasmid pREM-4. The kcat/Km ratios of IMP-16 for penicillins showed very similar results (0.09 to 0.13 µM–1 · s–1, which may suggest that the structure of the C-6 side chain, which varies among penicillins, did not affect the hydrolytic activity of IMP-16.

The kinetic data also revealed a lower kcat/Km value for imipenem (0.36 µM–1 · s–1), essentially due to a higher Km value. Other IMP variants, such as IMP-3 and IMP-12, also showed similar kcat/Km ratios (0.08 and 0.26 µM–1 · s–1, respectively), apparently due to the S262G substitution (8, 15). This amino acid mutation was not present in IMP-16, and no other amino acid changes were present in IMP-3, IMP-12, and IMP-16 which were absent from the other IMP variants with higher levels of imipenem hydrolysis. However, IMP-16 showed six unique amino acid substitutions (Fig. 3), which included V74F, located seven residues after the IMP loop site, and T122S, just adjacent to the zinc ligand Asp120. These changes may have contributed to the IMP-16 hydrolytic activity.

The 5' region of the contiguous gene aac(6')-30/aac(6')-Ib', located downstream from blaIMP-16, was associated with a shortened 59-be. To date, there have been only three cases in which a complete cassette was also found in an alternative form with a shorter 59-be (30). aac(6')-30 is the second case in which the complete version of the gene has not yet been found (33). It appears likely that the creation of the short aac(6')-30 59-be occurred through misreading of the 2L 59-be conserved sequence for the natural core sequence during an excision event, as described previously (49). Given the short 59-be, it is possible that the aac(6')-30 and aac(6')-Ib' genes were fused and may move as a single unit on excision.

The E. coli strain harboring recombinant plasmid pREM-1 [AAC(6')-30] did not demonstrate an obvious resistance profile; however, a slight increase in aminoglycoside MICs was observed for the strain, suggesting that its complete form may be functional and expresses an AAC(6')-I phenotype. This phenotype can be inferred because (i) this class of proteins is more active against tobramycin and amikacin than gentamicin (42), and (ii) AAC(6')-30 revealed a large number of the same conserved residues present in all related members of the AAC(6') family (motifs A, B, E, and F) (Fig. 4). However, like AAC(6')-29a and AAC(6')-29b, AAC(6')-30 did not contain the G conserved motif, commonly present in most of the AAC(6') subfamily members (24), probably due to a truncation event in the C-terminal region of these proteins (Fig. 4) (32).

Comparison of the MICs for E. coli harboring the three recombinant plasmids showed that pREM-2 [AAC(6')-30/AAC(6')-Ib'] conferred broad aminoglycoside-modifying enzyme activity, similar to that of the index strain (strain 101-4704), and a resistance profile similar to that of strain 101-4704. The MICs for E. coli harboring plasmid pREM-2 were between two- and fourfold higher than those observed for E. coli harboring plasmid pREM-1 [AAC(6')-30] and E. coli harboring plasmid pREM-3 [AAC(6')-Ib']. The phenotype expressed by the strain harboring recombinant plasmid pREM-2 may be characterized as an AAC(6')-II type with an additional decreased susceptibility to amikacin.

Although it is still not clear, the phenotype expressed by the strain harboring recombinant plasmid pREM-2 may be due to the expression of the fused enzyme [AAC(6')-30/AAC(6')-Ib'] alone or even expression of the fused enzyme accompanied by the additional expression of AAC(6')-Ib', since aac(6')-Ib' contains its own promoter and ribosome-binding site, which are essential for transcription and translation. Thus, the aminoglycoside resistance profile may be maximized due to the expression of both proteins, leading to a synergistic effect. However, the gentamicin and amikacin MICs for the strain harboring recombinant plasmid pREM-2 were fourfold higher than those for the strain harboring pREM-1 and pREM-3, suggesting the activity of a unique protein rather than the additive effects of two enzymes.

The association of mobile MßL genes with aminoglycoside resistance genes has become very common. blaIMP, blaVIM, and the recently discovered gene blaGIM-1 (M. Castanheira, R. E. Mendes, F. Schmitz, M. A. Toleman, R. N. Jones, and T. R. Walsh, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-669, p. 76, 2003) all represent MßL genes associated with aminoglycoside-modifying enzymes. The exception among the MßL genes is blaSPM-1, which does not appear to be associated with an integron (34, 48). In the majority of cases the aminoglycoside resistance genes appear to be functional and confer significant resistance. Their mobilization with ß-lactamase genes that confer broad-spectrum ß-lactam resistance and the fact that both classes of enzymes cannot be neutralized by clinically available enzyme inhibitors result in a situation of great concern regarding the treatment of infections caused by multidrug-resistant gram-negative bacilli.


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ACKNOWLEDGMENTS
 
We thank Douglas J. Biedenbach, Lalitagauri M. Deshpande, Ricardo Guzman, and Elen Simaan for excellent technical support. We are also grateful to Alan M. Simm for important advice during the purification and analysis of IMP-16.

The SENTRY Antimicrobial Surveillance Program is funded by an educational and research grant from Bristol-Myers Squibb. Mark A. Toleman was supported in part by EU grant LSHM-CT-2003-503335.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratório Especial de Microbiologia Clínica, Division of Infectious Diseases, Universidade Federal de São Paulo, Rua Leandro Dupret, 188 São Paulo, SP 04025-010, Brazil. Phone: (55-11) 5081-2819. Fax: (55-11) 5571-5180. E-mail: rodrigo.mendes{at}lemc.com.br. Back


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Antimicrobial Agents and Chemotherapy, December 2004, p. 4693-4702, Vol. 48, No. 12
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.12.4693-4702.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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