OXA-46, a New Class D  -Lactamase of Narrow Substrate Specificity Encoded by a blaVIM-1-Containing Integron from a Pseudomonas aeruginosa Clinical Isolate

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2005, p. 1973–1980 0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.5.1973–1980.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 49, No. 5

OXA-46, a New Class D ␤-Lactamase of Narrow Substrate Specificity Encoded by a blaVIM-1-Containing Integron from a Pseudomonas aeruginosa Clinical Isolate Francesco Giuliani,1 Jean-Denis Docquier,1,2 Maria Letizia Riccio,1 Laura Pagani,3 and Gian Maria Rossolini1* Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Universita ` di Siena, I-53100 Siena,1 and Dipartimento di Scienze Morfologiche, Eidologiche e Cliniche, Sezione di Microbiologia, Universita ` di Pavia, I-27100 Pavia,3 Italy, and Centre d’Inge´nierie des Prote´ines & Laboratoire d’Enzymologie, Universite´ de Lie`ge, B-4000 Lie`ge, Belgium2 Received 19 August 2004/Returned for modification 31 October 2004/Accepted 19 January 2005

A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a blaVIM-1 metallo-␤-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D ␤-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of blaOXA-46 in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D ␤-lactamases. inserted into integrons (24). These secondary OXA-type ␤-lactamase (blaOXA) genes have been reported to occur in isolates of several pathogenic species, including members of the family Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., and Burkholderia cepacia, where they can variably contribute to acquired ␤-lactam resistance (4, 20, 24). OXA-type ␤-lactamases are resistance determinants of increasing clinical importance, due to their potential activity on oxyimino-cephalosporins and carbapenems, their overall poor susceptibility to ␤-lactamase inactivators, and the ability that some blaOXA genes exhibit to disseminate among major gramnegative pathogens. Moreover, these enzymes represent interesting models for enzymology and protein chemistry, since their mechanism exhibits notable differences from the mechanisms of other classes of serine-␤-lactamases (13, 22, 29) and since their structure-function relationships are still poorly understood. In this paper, we report on the identification and characterization of OXA-46, a new OXA-type enzyme belonging to the OXA-2 lineage, encoded by a gene cassette inserted in an integron from a multidrug-resistant clinical isolate of P. aeruginosa.

OXA-type ␤-lactamases are a group of structurally related serine enzymes belonging to molecular class D of the Ambler structural classification of ␤-lactamases (3, 24). Enzymes of this class typically exhibit a good hydrolytic activity against oxacillin and related compounds and are usually poorly susceptible to clavulanate, being classified in group 2d of the functional classification of ␤-lactamases (3). Although several OXA-type ␤-lactamases behave as narrow-spectrum oxacillinases, some of them are also capable of degrading extendedspectrum cephalosporins or carbapenems (24, 28). From the structural standpoint, some 60 variants of OXA-type enzymes (http://www.ncbi.nlm.nih.gov/) that are clustered into several different lineages or groups have been described (1, 24, 43). A number of OXA-type ␤-lactamases are encoded by chromosomal genes that appear to be resident in some microbial genomes (such as in those of some Aeromonas spp., of some Shewanella spp., of Ralstonia pickettii, and of Pseudomonas aeruginosa) (12, 15, 27, 31, 32). On the other hand, several OXA-type enzymes are encoded by genes associated with mobile elements, which are often represented by gene cassettes

* Corresponding author. Mailing address: Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Universita` di Siena, Policlinico Santa Maria alle Scotte, 53100 Siena, Italy. Phone: 39 0577 233455. Fax: 39 0577 233334. E-mail: [email protected]

MATERIALS AND METHODS Bacterial strains and genetic vectors. P. aeruginosa PPV-97 is a multidrugresistant clinical isolate producing a VIM-type metallo-␤-lactamase that was





FIG. 1. Structure of the variable region of In80 from P. aeruginosa PPV-97. Gene cassettes are represented by arrows; the 5⬘-CS and 3⬘-CS of the integron are represented by filled boxes. The thin arrows located above the map show the locations of primers used for PCR mapping of the integron variable region, as described in Materials and Methods.

isolated in 1998 from a patient at Pavia University Hospital (35). Escherichia coli DH5␣ (36) was used as the host for recombinant plasmids. E. coli BL21(DE3) (Novagen Inc., Madison, Wis.) was used as the host for overproduction of the OXA-46 enzyme. Plasmid pBC-SK (Stratagene Inc., La Jolla, Calif.) was used as a cloning vector. Plasmid pET-9a (Novagen Inc.) was used as a T7-based expression vector for overexpression of the blaOXA-46 gene. ␤-Lactamase assays. Analytical isoelectric focusing (IEF) for detection of ␤-lactamases was carried out using Ampholine PAGplate (pH range, 3.5 to 9.5) (Amersham Biosciences, Uppsala, Sweden) as described previously (19), using nitrocefin (Oxoid, Basingstoke, United Kingdom) at 0.5 mM as the chromogenic substrate. ␤-Lactamase activity in crude E. coli extracts and during the purification procedure was assayed spectrophotometrically by measuring the hydrolysis of 200 ␮M nitrocefin at 482 nm (change in εM, ⫹15,000 M⫺1 · cm⫺1) in 50 mM sodium phosphate buffer (pH 7.0) at 25°C. DNA analysis and manipulation methodology. Basic procedures for DNA analysis and manipulation were performed as described by Sambrook and Russell (36). Characterization of the variable region of the blaVIM-1-containing integron from PPV-97 was carried out by a PCR mapping and sequencing approach as described previously (34) using primers INT/5CS and INT/3CS, designed on the 5⬘- and 3⬘-end-conserved segments (5⬘-CS and 3⬘-CS, respectively) of class 1 integrons, in combination with primers VIM-DIA/f and VIMDIA/r, designed on conserved regions of blaVIM genes, to obtain partially overlapping PCR products (Fig. 1 and Table 1) (this strategy was adopted since the INT/5CS and INT/3CS primers preferentially amplified the variable region of another integron, which was shorter than that of In80 and did not contain the blaVIM cassette). Nucleotide sequences were determined on both strands directly on PCR products, as described previously (34). Plasmid DNA and total genomic DNA were extracted from P. aeruginosa isolates as described previously (19). Southern blot analysis was carried out directly on dried gels (44) using a 32Plabeled blaVIM-1 probe as described previously (21). Construction of plasmid pBC-OXA-46 was carried out as follows. The blaOXA-46 open reading frame (ORF) was amplified by PCR with primers OXA-46/fwd, which added SacI and NdeI restriction sites at the 5⬘ end of the ORF, and OXA-46/rev, which added a BamHI restriction site after the blaOXA-46 stop codon (Table 1). Amplification was carried out in a 100-␮l volume using 50 pmol of each primer and the Expand PCR system (Roche Biochemicals, Mannheim, Germany), under the conditions recommended by the manufacturer, and the following cycling parameters: initial denaturation at 94°C for 3 min; denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min, repeated for 30 cycles; and a final extension step at 72°C for 10 min. Genomic DNA of PPV-97 (10 ng) was used as

a template. The amplification product was digested with SacI and BamHI and cloned into the plasmid vector pBC-SK digested with the same enzymes, resulting in recombinant plasmid pBC-OXA-46. Construction of the expression plasmid pET-OXA-46 was carried out as follows. Plasmid pBC-OXA-46 was digested with NdeI and BamHI, and the 0.8-kb fragment containing the blaOXA-46 ORF was subcloned into the expression vector pET-9a digested with the same enzymes, resulting in recombinant plasmid pET-OXA-46. The authenticity of the cloned DNA inserts was always verified by confirmatory sequencing. In vitro susceptibility testing. In vitro susceptibility of E. coli DH5␣(pBCOXA-46) was determined by a macrodilution broth method (26) using cationsupplemented Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) and a bacterial inoculum of 106 CFU per tube. Results were recorded after incubation at 37°C for 18 h. Antimicrobial agents were from Sigma Chemical Co. (St. Louis, Mo.) unless otherwise specified. Mezlocillin, ceftazidime, cefepime, and meropenem were from commercial sources. Imipenem was from Merck (Rome, Italy). Production and purification of OXA-46. Plasmid pET-OXA-46 was transformed in E. coli BL21(DE3). The OXA-46 enzyme was purified from E. coli BL21(DE3)(pET-OXA-46) as follows. The strain was grown aerobically in 1 liter of buffered Super Broth (8), containing 50 ␮g/ml kanamycin, at 28°C (at this temperature, ␤-lactamase production was found to be significantly higher than at 37°C in preliminary experiments). Isopropyl-␤-D-thiogalactopyranoside (IPTG) (Sigma Chemicals Co., St. Louis, Mo.) was added to a final concentration of 0.5 mM when the culture reached an A600 of 0.8, and incubation was continued for an additional 18 h. Cells were collected by centrifugation (10,000 ⫻ g for 40 min at 4°C), resuspended in 40 ml of 20 mM triethanolamine-NaOH buffer (pH 7.5) containing 1 mM MgCl2, and disrupted by sonication (10 times, for 30 s each time, at 45 W). Cell debris were removed by centrifugation (10,000 ⫻ g for 60 min at 4°C). The clarified supernatant was loaded (flow rate, 5 ml/min) on an XK 26/20 column packed with DEAE Sepharose Fast Flow (bed volume, 75 ml) (Amersham Biosciences) equilibrated with 20 mM triethanolamine-NaOH buffer (pH 7.5). Under these conditions the enzyme was obtained in the flowthrough. The ␤-lactamase-containing fractions were pooled, desalted with a HiPrep desalting 26/10 column (Amersham Biosciences) against a 20 mM TrisH2SO4 buffer (pH 9.5) (buffer A) and then loaded (flow rate, 2 ml/min) on an HR 16/5 column packed with Source Q (bed volume, 10 ml) (Amersham Biosciences) equilibrated with the same buffer. After the columns were washed with buffer A, the enzyme was eluted (flow rate, 2 ml/min) with a pH gradient obtained by mixing 20 mM Tris-H2SO4 buffer (pH 6.5) with buffer A (from 0 to 100% in 100 ml). The enzyme-containing fractions, eluted at a pH of ⬃8.5, were

TABLE 1. Primers used in this study Primer pair


Product size (bp)







OXA-46/fwd OXA-46/rev



The restriction sites used for cloning of the blaOXA-46 gene are underlined.



VOL. 49, 2005 pooled and stored at ⫺80°C. All the chromatography steps were performed using ¨ kta Purifier platform (Amersham Biosciences). an A Protein analysis techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed by the method of Laemmli (16) using acrylamide concentrations of 12% and 5% (wt/vol) for the resolving and stacking gels, respectively. Electrospray mass spectrometry was carried out as described previously (8), using a Finnigan LTQ mass spectrometer equipped with an ion spray source (Thermo Electron Co., Shaumberg, Ill.). The data were analyzed with the software delivered with the instrument. Size exclusion chromatography to analyze the quaternary structure of the native OXA-46 enzyme was carried out with 0.12 mg of purified protein on a prepacked Superdex 75 HR 10/30 column (Amersham Biosciences) preequilibrated with 100 mM Tris-H2SO4 buffer supplemented with 300 mM K2SO4 (pH 7.0) and eluted with the same buffer at a flow rate of 0.4 ml/min. The column was calibrated with a mixture containing bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa) (Amersham Biosciences), using the same buffer and flow rate conditions. The same experiment was also performed in the presence of 5 mM EDTA (both in the protein sample and in the elution buffer) after preincubation of the enzyme with the various compounds as described below (see inhibition assays). The retention volumes of the various standards were not affected by the presence of EDTA. Protein concentration in solution was assayed by the method of Bradford using a commercial kit (protein assay; Bio-Rad, Richmond, Calif.) and bovine serum albumin as the standard. Theoretical prediction of the leader peptide size was carried out at the SignalP 3.0 server (2). Theoretical calculation of protein molecular mass and pI was carried out using the software available at the ExPASy proteomic server (http://ca.expasy .org/). Multiple amino acid sequence alignments and unrooted tree construction were made with the help of the ClustalX program (42). Determination of kinetic parameters and of the effect of various compounds on enzyme activity. Kinetic parameters were determined by measuring substrate hydrolysis by the purified enzyme using a Cary 100 UV–visible-light spectrophotometer (Varian, Walnut Creek, Calif.). The wavelengths and changes in the extinction coefficients used in the spectrophotometric assays were 260 nm and ⫺7,400 M⫺1 · cm⫺1 for cefazolin, 260 nm and ⫹470 M⫺1 · cm⫺1 for oxacillin, and as described previously for other substrates (10, 18). The steady-state kinetic parameters (Km and kcat) were determined under initial-rate conditions using the Hanes-Woolf plot (38). Km values lower than 20 ␮M were measured as Ki using 0.1 mM nitrocefin as the reporter substrate, as described previously (11). Enzyme reactions for kinetic measurements were carried out in 50 mM sodium phosphate buffer (pH 7.0) at 25°C, in a total volume of 500 ␮l, using an enzyme concentration ranging from 3 to 830 nM. Inhibition by chloride ions was assayed under the same conditions, using 100 ␮M nitrocefin as the reporter substrate, after a 5-min preincubation of the enzyme with various NaCl concentrations (10 to 250 mM). The effect of EDTA and of divalent cations on the OXA-46 activity was assayed, using 100 ␮M nitrocefin as the substrate at 25°C after a 20-min preincubation at 25°C of a 2.2 ␮M enzyme solution containing the various compounds (1 mM for EDTA and 0.5 mM for the divalent cations) in the buffer system used by Paetzel et al. (29) for studying the effect of cations on OXA-10 (100 mM Tris–H2SO4 [pH 7.0] containing 0.3 M K2SO4). The inactivation rates for carbapenems and mechanism-based inactivators (clavulanic acid and tazobactam) were measured at 25°C using 200 ␮M cephalothin as the reporter substrate. Individual inactivation parameters (k⫹2, K, and k⫹3) were calculated as previously described (7). Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank database and assigned the accession number AF317511.

RESULTS Identification of a new integron-encoded OXA-type ␤-lactamase from a P. aeruginosa clinical isolate. P. aeruginosa PPV-97 is a multidrug-resistant clinical isolate from the University Hospital of Pavia (northern Italy) and is one of the first isolates discovered to produce a VIM-type metallo-␤-lactamase at that hospital (35). As previously reported (35), PPV-97 was resistant to most antipseudomonas ␤-lactams (carbenicillin, ticarcillin, mezlocillin, piperacillin, piperacillin-tazobactam, cefsulodin, ceftazidime, cefepime, imipenem, and meropenem), aminoglycosides (gentamicin, tobramycin, netilmicin, and amikacin), and ciprofloxacin.


Analytical IEF analysis of a crude extract of PPV-97, prepared from a culture grown in antibiotic-free medium, revealed three ␤-lactamase bands with pIs of 5.1, 7.8, and 8.7. The pI 5.1 band likely corresponded to the VIM-type enzyme, while the most alkaline band was likely contributed by the resident P. aeruginosa enzymes (AmpC and/or OXA-50) (12). Analysis of the variable region of the blaVIM-containing integron of PPV-97, by means of a PCR mapping and sequencing approach as described in Materials and Methods, revealed an original array of four gene cassettes (Fig. 1), including a blaVIM-1 cassette, two tandemly repeated aacA4 cassettes, and a cassette encoding a new OXA-type ␤-lactamase. This new OXA-type enzyme was assigned the name OXA-46, and the integron was named In80. The blaVIM-1 cassette of In80 was identical to that previously described as found in other integrons from VIM-1-positive clinical isolates from northern Italy (19, 21, 33). The two aacA4 cassettes are identical to each other, except for a silent A3T transversion at position 384 of the gene cassette, and encode an AAC(6⬘)-Ib⬘ aminoglycoside acetyltransferase (17). Plasmid DNA was not detectable in PPV-97, and in a Southern blot experiment, a blaVIM-1 probe yielded a hybridization signal corresponding to the band of chromosomal DNA (data not shown). These results suggested a chromosomal location of In80. The OXA-46 protein presents all the conserved motifs typical of class D enzymes, including S67XXK, S115XV, and K205TG (29), and a predicted 21-amino-acid signal peptide whose removal would yield a mature polypeptide with a calculated molecular mass of 28,583 Da and a pI of 8.7. Comparison of OXA-46 with other class D ␤-lactamases at the level of primary structure revealed that it belongs to the OXA-2 sublineage (or group II according to the classification of Sanschagrin et al. [37]). Within this sublineage, OXA-46 exhibits the highest similarities (92.5% and 91.7% sequence identity, respectively) with an OXA-like enzyme encoded by a plasmid from unidentified bacteria from a wastewater treatment plant in Germany (41) (henceforth indicated as OXA-PMW) and with an OXA-like enzyme from Burkholderia cepacia clinical isolates from Ireland (4) (henceforth indicated as OXA-Bce), which are almost identical to each other (Fig. 2). OXA-46 shares 77.8% amino acid sequence identity with OXA-2, with OXA-46 being shorter by 9 residues at the carboxy terminus, and exhibits identical residues at positions 150 and 164 (Fig. 2), which are mutated in OXA-2 variants (OXA-15 and OXA-32) with extended-spectrum activities (6, 30). OXA-46 exhibits a similar degree of divergence (77.5% sequence identity) with OXA-53, an OXA-2-related extended-spectrum enzyme recently described to occur in a Salmonella enterica clinical isolate (23) that is even more divergent (72% sequence identity) from OXA-20 (25). Overall, members of the OXA-2 lineage appear to be clustered in three major sublineages (Fig. 3). The attC recombination site (59-base element) of the blaOXA-46 gene cassette is 56 bp long and includes all the elements typical of the attC sites (Fig. 4). It appears to be closely related (93% sequence identity and, overall, the same size) to those of the cassettes encoding the most-similar OXAtype enzymes (OXA-PMW and OXA-Bce) and consistently divergent in size and sequence from those of the cassettes




FIG. 2. Amino acid sequence comparison of OXA-46 with OXA-type enzymes representative of the OXA-2 lineage. Sequences of OXA-2 (5) (accession number AJ295229); OXA-3 (37) (accession number L07945); OXA-20 (24) (accession number AJ319747); OXA-53 (23) (accession number AY289608); OXA-Bce, an OXA-type enzyme from Burkholderia cepacia clinical isolates from Ireland (4) (accession number AF371964); and OXA-PMW, an OXA-type enzyme encoded by a plasmid from an unidentified bacterium from a wastewater treatment plant in Germany (41) (accession number AY139598) are shown. Closely related variants of OXA-2 (OXA-15 and OXA-32), OXA-3 (OXA-21), and OXA-20 (OXA-37) were not included in the alignment. Residues that are identical in all sequences are shaded in grey. The conserved motifs of OXA-type enzymes are in white letters on a black background. The structural elements of OXA-2 enzyme (Protein Data Bank accession no. 1K38) are shown above the sequence. ␣, ␣ helices; ␤, ␤ strands.

encoding members belonging to different sublineages of the OXA-2 lineage (Fig. 4). Expression of OXA-46 in E. coli and contribution to ␤-lactam resistance. E. coli DH5␣ transformed with plasmid pBCOXA46, a pBC-SK derivative carrying a copy of the blaOXA-46 gene cloned downstream of the Plac promoter, produced a pI 7.8 ␤-lactamase activity, as determined by analytical IEF (data not shown). Compared to E. coli DH5␣(pBC-SK), DH5␣(pBC-OXA46)

exhibited a decreased susceptibility to penicillins and narrowspectrum cephalosporins, while susceptibility to oxyiminocephalosporins, cefsulodin, aztreonam, and imipenem was unaffected (Table 2). The ampicillin MIC was decreased in the presence of mechanism-based inactivators. Tazobactam was more active than clavulanic acid (Table 2), as already reported for other class D enzymes (3, 24). Overexpression, purification, and properties of OXA-46. The OXA-46 enzyme was overproduced in E. coli using a

FIG. 3. Unrooted tree showing the relationships among OXA-type enzymes representative of the OXA-2 lineage. The names of the enzymes are the same as described in the legend of Fig. 2.


VOL. 49, 2005


FIG. 4. Comparison of the attC recombination sites (59-base elements) of the blaOXA gene cassettes encoding enzymes of the OXA-2 lineage. The sequences are shown from the inverse core site to the core site, as they would appear in the circular cassette. Identical nucleotides in all sequences are shaded in gray. The core and inverse core sites are boxed. The internal 2L and 2R sites (40) are indicated by horizontal arrows. The vertical arrow indicates the recombination point. The stop codons of the blaOXA ORFs are in white letters on a black background. The names of the enzymes and the sequence sources are the same as described in the legend of Fig. 2.

T7-based expression system and was purified from a crude lysate of the strain by means of two anion-exchange chromatography steps. The first step was effective in the removal of most of the contaminating proteins. The second step yielded an enzyme that was ⬎98% pure, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the preparation (data not shown). Overall, the purification procedure yielded approximately 6 mg of purified OXA-46 protein per

TABLE 2. ␤-Lactam susceptibility and ␤-lactamase activity of E. coli DH5␣(pBC-OXA46), producing the OXA-46 enzyme, in comparison with E. coli DH5␣(pBC-SK) MIC (␮g/ml) for: Antibiotic DH5␣(pBC-OXA-46)

Ampicillin Ampicillin ⫹ CLAa Ampicillin ⫹ TZBb Carbenicillin Mezlocillin Cephalothin Cefuroxime Ceftriaxone Cefotaxime Cefsulodin Ceftazidime Cefepime Aztreonam Imipenem

64 8 4 64 32 64 4 0.06 0.03 32 0.12 0.06 0.12 0.12

␤-Lactamase activityc

34 ⫾ 4


4 4 4 4 2 4 4 0.06 0.03 32 0.12 0.03 0.12 0.12 ⬍10

CLA, clavulanic acid at a concentration of 2 ␮g/ml. TZB, tazobactam at a concentration of 4 ␮g/ml. Specific nitrocefin-hydrolyzing activity (nmol/min · mg protein) of crude cell extracts prepared, by sonic disruption, from early-stationary-phase cultures grown in Mueller-Hinton broth. a b c

liter of culture, with a recovery rate of 37% and an overall purification of 366-fold. The pI of the purified protein, evaluated by analytical IEF, was 7.8 ⫾ 0.2, a value somewhat lower than the predicted one but in agreement with that of the ␤-lactamase band detected in the crude extract of E. coli DH5␣(pBC-OXA46) (see above). This also suggested that the pI 7.8 ␤-lactamase band detected in P. aeruginosa PPV-97 corresponded to OXA-46. The protein mass, determined by electrospray mass spectrometry, was 28,582 ⫾ 3 Da, in excellent agreement with the calculated value after removal of the putative 21-amino-acid leader peptide (28,583 Da). The Mr of the native protein, determined by size exclusion chromatography, was estimated to be 50 ⫾ 6 kDa, suggesting that the native protein is found as a dimer. This value was apparently unaffected in the presence of 5 mM EDTA. Kinetic parameters of OXA-46. The kinetic parameters of OXA-46 were determined with several ␤-lactam substrates. The enzyme was able to hydrolyze most penicillins and narrowspectrum cephalosporins, while no hydrolysis was detected with temocillin, oxyimino-cephalosporins, aztreonam, or carbapenems (Table 3). Biphasic kinetics were observed with some substrates, including nitrocefin, carbenicillin, and cefazolin. The parameters for the burst phase could be determined only for the last two compounds (the burst time observed with nitrocefin was shorter than 15 s). With those compounds, the steady-state phase was essentially characterized by a drop of the turnover rate, which was more evident with carbenicillin. The highest acylation efficiencies (kcat/Km values close to 106 M⫺1 · s⫺1) were observed with oxacillin and nitrocefin, kcat/Km values being lower with other penicillins and narrow-spectrum cephalosporins. Turnover rates were low overall (kcat, ⱕ20 s⫺1), except with oxacillin (Table 3).




TABLE 3. Kinetic parameters determined with the purified OXA46 ␤-lactamasef Substratea

kcat (s⫺1)

Km (␮M)

kcat/Km (M⫺1 · s⫺1)

Oxacillin Penicillin G Ampicillin Carbenicillin (V0) Carbenicillin (Vss) Azlocillin Mezlocillin Temocillin Nitrocefin (Vss)e Cephalothin Cefazolin (V0) Cefazolin (Vss) Cefsulodin Cefuroxime Cefotaxime Ceftazidime Cefepime Aztreonam

300 18 6 85 5 11 12 NHb 20 8 17 7 NH NH NH NH NH NH

320 48 20 730 545 46 30 ⬎1,000c 18 23 23 40 ⬎1,000c ⬎1,000c ⬎1,000c ⬎1,000c ⬎1,000c ⬎1,000c

9.4 ⫻ 105 3.8 ⫻ 105 3.0 ⫻ 105 1.2 ⫻ 105 9.2 ⫻ 103 2.4 ⫻ 105 4.0 ⫻ 105 —d 1.1 ⫻ 106 3.5 ⫻ 105 7.4 ⫻ 105 1.7 ⫻ 105 — — — — — —

a Biphasic kinetics were observed with this substrate; the volumes of distribution were calculated either under initial-rate conditions (V0) or under steadystate conditions (Vss). b NH, no hydrolysis detected at a substrate concentration up to 1 mM and an enzyme concentration up to 850 nM. c Measured as an inhibition constant (Ki), as described in Materials and Methods. d —, not calculated. e Biphasic kinetics were observed with nitrocefin, but only kinetic parameters under steady- state conditions could be measured. f Km and kcat values are the means of three different measurements. The standard deviation was always lower than 10%.

Competition experiments with nitrocefin showed that, among the substrates that were not hydrolyzed, temocillin, cefsulodin, oxyimino-cephalosporins, and aztreonam were apparently not recognized by the enzyme (Table 3). Clavulanic acid was a poor inactivator of OXA-46, while tazobactam exhibited an acylation efficiency 2 orders of magnitude higher (Table 4), in agreement with susceptibility data (see above). Carbapenems efficiently inactivated OXA-46, showing an acylation efficiency even higher than that of tazobactam. In inactivation experiments, a steady state was observed with all compounds, and deacylation rates were measurable (Table 4). Like other OXA-type ␤-lactamases, OXA-46 was inhibited by NaCl (50% inhibitory concentration, 230 ⫾ 20 mM). However, the NaCl susceptibility of OXA-46 appeared to be lower overall than those observed with other enzymes of this class (24). Effect of EDTA and metal ions on OXA-46’s activity. The effect of EDTA and of divalent metal ions on the activity of OXA-46 was investigated under the same experimental condi-

TABLE 4. Inactivation parameters of OXA-46 with mechanismbased inactivators and carbapenemsa Compound

k⫹2 (s⫺1) ⫺3

K (nM)

Clavulanic acid ⬎8.8 ⫻ 10 ⬎11,000 Tazobactam ⬎1.2 ⫻ 10⫺2 ⬎110 Imipenem 5.8 ⫻ 10⫺2 75 Meropenem ⬎1.1 ⫻ 10⫺2 ⬎11 a

k⫹2/K (M⫺1 · s⫺1)

k⫹3 (s⫺1)

8.0 ⫻ 10 1.1 ⫻ 105 7.4 ⫻ 105 1.0 ⫻ 106

6.0 ⫻ 10⫺3 5.3 ⫻ 10⫺3 6.9 ⫻ 10⫺3 7.1 ⫻ 10⫺3

The standard deviation was always lower than 10%.


tions used in previous studies of class D ␤-lactamases (9, 29). The activity of OXA-46 was apparently unaffected by 1 mM EDTA or by 0.5 mM divalent cations (Mg2⫹, Zn2⫹, Mn2⫹, Cd2⫹, and Cu2⫹), except for a slight decrease observed with Ca2⫹ (residual activity, 85%). DISCUSSION The recombination system based on integrons and mobile gene cassettes plays a major role in the dissemination of antimicrobial resistance genes among bacterial pathogens (14). Since this system was recognized (39), the number of integronborne gene cassettes carrying resistance determinants described in the scientific literature has steadily increased (8). Class D ␤-lactamase genes are among the resistance determinants that can be associated with mobile gene cassettes (8, 24). This was also the case for OXA-46, the new class D enzyme described in this paper, which was found to be encoded by a gene cassette inserted in a class 1 integron from a P. aeruginosa clinical isolate. The integron also carried a blaVIM-1 metallo␤-lactamase gene cassette and two tandemly inserted aacA4 aminoglycoside acetyltransferase gene cassettes. This integron was also detected in other VIM-1-producing P. aeruginosa clinical isolates from various Italian hospitals (unpublished results). OXA-46 is a new member of the OXA-2 lineage which exhibits a narrow substrate specificity, limited to penicillins and narrow-spectrum cephalosporins, that is similar to the specificities of several other narrow-spectrum oxacillinases (3, 24). Given these functional features and the repertoire of ␤-lactamases present in P. aeruginosa PPV-97 (also including a VIM-1 metallo-enzyme), it is hardly conceivable that OXA-46 could provide a significant contribution to the ␤-lactam resistance phenotype of that strain. Most likely, the acquisition of OXA-46 was antecedent to that of the metallo-enzyme (as also suggested by the relative positions of the two ␤-lactamase gene cassettes in the integron) and could reflect a previously acquired resistance mechanism against antipseudomonas penicillins. Within the OXA-2 lineage, at least three different sublineages could be identified. OXA-46 belongs to one of these lineages, as does another integron-encoded class D enzyme with two minor variants, one of which was recently described to occur in B. cepacia clinical isolates (4) and the other of which is encoded by a plasmid from unidentified bacteria from a wastewater treatment plant (41). Another sublineage includes OXA-2 (and its close allelic variants OXA-15 and OXA-32), OXA-3 (and its close allelic variant OXA-21), and OXA-53, while the third lineage includes OXA-20 (and its close allelic variant OXA-37). At the genetic level, the similarity between members of each lineage does not appear to be limited to the coding sequences but extends to the attC recombination sites of the gene cassettes, suggesting a common ancestry for these blaOXA cassettes. Conversely, a substantial diversity (both in sequence and in length) is observed among the attC recombination sites of the gene cassettes encoding members of different sublineages, pointing to different phylogenies of those cassettes. OXA-46 was apparently a dimeric enzyme, and its quaternary structure was not affected by the presence of EDTA.


VOL. 49, 2005

Moreover, neither EDTA nor divalent metal ions affected the activity of the enzyme. This behavior was different from that observed with OXA-10 (29) and OXA-29 (10) and suggests a considerable heterogeneity in the effect of divalent cations on the quaternary structure and function of class D enzymes. Compared to other class D enzymes, most of which have been reported to be fully inhibited by a NaCl concentration of 100 mM (24), another peculiar feature of OXA-46 was represented by its relatively low susceptibility to NaCl inhibition. In conclusion, the biochemical characterization of OXA-46, a new member of the OXA-2 lineage, further underscores the notion that considerable functional and structural diversity can be encountered among class D ␤-lactamases. Additional investigation of enzymes of this class, which exhibit an increasing clinical importance, will be important to clarify their structurefunction relationships which remain poorly understood. ACKNOWLEDGMENTS This work was supported in part by a grant from the European Union (6th PCRD, LSHM-CT-2003-503335). J.-D.D. is a postdoctoral fellow of the Belgian Fonds National de la Recherche Scientifique. REFERENCES 1. Barlow, M., and B. G. Hall. 2002. Phylogenetic analysis shows that the OXA ␤-lactamase genes have been on plasmids for millions of years. J. Mol. Evol. 55:314–321. 2. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783–795. 3. Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A functional classification scheme for ␤-lactamases and its correlation with molecular structure. Antimicrob. Agents Chemother. 39:1211–1233. 4. Crowley, D., M. Daly, B. Lucey, P. Shine, J. J. Collins, B. Cryan, J. E. Moore, P. Murphy, G. Buckley, and S. Fanning. 2002. Molecular epidemiology of cystic fibrosis-linked Burkholderia cepacia complex isolates from three national referral centres in Ireland. J. Appl. Microbiol. 92:992–1004. 5. Dale, J. W., D. Godwin, D. Mossakowska, P. Stephenson, and S. Wall. 1985. Sequence of the OXA2 ␤-lactamase: comparison with other penicillin-reactive enzymes. FEBS Lett. 191:39–44. 6. Danel, F., L. M. C. Hall, D. Gur, and D. M. Livermore. 1997. OXA-15, an extended-spectrum variant of OXA-2 ␤-lactamase, isolated from a Pseudomonas aeruginosa strain. Antimicrob. Agents Chemother. 41:785–790. 7. De Meester, F., B. Joris, G. Reckinger, C. Bellefroid-Bourguignon, J. M. Fre`re, and S. G. Waley. 1987. Automated analysis of enzyme inactivation phenomena. Application to ␤-lactamases and DD-peptidases. Biochem. Pharmacol. 36:2393–2403. 8. Docquier, J.-D., F. Pantanella, F. Giuliani, M. C. Thaller, G. Amicosante, M. Galleni, J.-M. Fre`re, K. Bush, and G. M. Rossolini. 2002. CAU-1, a subclass B3 metallo-␤-lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome. Antimicrob. Agents Chemother. 46:1823–1830. 9. Fluit, A. C., and F. J. Schmitz. 2004. Resistance integrons and super-integrons. Clin. Microbiol. Infect. 10:272–288. 10. Franceschini, N., L. Boschi, S. Pollini, R. Herman, M. Perilli, M. Galleni, J.-M. Fre`re, G. Amicosante, and G. M. Rossolini. 2001. Characterization of OXA-29 from Legionella (Fluoribacter) gormanii: molecular class D ␤-lactamase with unusual properties. Antimicrob. Agents Chemother. 45:3509– 3516. 11. Franceschini, N., B. Caravelli, J.-D. Docquier, M. Galleni, J.-M. Fre`re, G. Amicosante, and G. M. Rossolini. 2000. Purification and biochemical characterization of the VIM-1 metallo-␤-lactamase. Antimicrob. Agents Chemother. 44:3003–3007. 12. Girlich, D., T. Naas, and P. Nordmann. 2004. Biochemical characterization of the naturally occurring oxacillinase OXA-50 of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 48:2043–2048. 13. Golemi, D., L. Maveyraud, S. Vakulenko, J. P. Samama, and S. Mobashery. 2001. Critical involvement of a carbamylated lysine in catalytic function of class D ␤-lactamases. Proc. Natl. Acad. Sci. USA 98:14280–14285. 14. Hall, R. M. 1997. Mobile gene cassettes and integrons: moving antibiotic resistance genes in gram-negative bacteria. Ciba Found. Symp. 207:192–202. 15. He´ritier, C., L. Poirel, and P. Nordmann. 2004. Genetic and biochemical characterization of a chromosome-encoded carbapenem-hydrolyzing Ambler class D ␤-lactamase from Shewanella algae. Antimicrob. Agents Chemother. 48:1670–1675.


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