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Antimicrobial Agents and Chemotherapy, February 2008, p. 551-556, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.01145-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic and Biochemical Characterization of CAD-1, a Chromosomally Encoded New Class A Penicillinase from Carnobacterium divergens

Djalal Meziane-Cherif,^1, Dominique Decré,² E. Arne Høiby,³ Patrice Courvalin,^1* and Bruno Périchon¹

Unité des Agents Antibactériens, Institut Pasteur, 75724 Paris Cedex 15,¹ Hôpital Saint-Antoine, 184, Rue Fbg. St. Antoine, 75012 Paris, France,² Department of Bacteriology, National Institute of Public Health, P.O. Box 4404, NO-0403 Oslo, Norway³

Received 30 August 2007/ Returned for modification 24 October 2007/ Accepted 28 November 2007

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Carnobacterium divergens clinical isolates BM4489 and BM4490 were resistant to penicillins but remained susceptible to combinations of amoxicillin-clavulanic acid and piperacillin-tazobactam. Cloning and sequencing of the responsible determinant from BM4489 revealed a coding sequence of 912 bp encoding a class A β-lactamase named CAD-1. The bla_CAD-1 gene was assigned to a chromosomal location in the two strains that had distinct pulsed-field gel electrophoresis patterns. CAD-1 shared 53% and 42% identity with β-lactamases from Bacillus cereus and Staphylococcus aureus, respectively. Alignment of CAD-1 with other class A β-lactamases indicated the presence of 25 out of the 26 isofunctional amino acids in class A β-lactamases. Escherichia coli harboring bla_CAD-1 exhibited resistance to penams (benzylpenicillin and amoxicillin) and remained susceptible to amoxicillin in combination with clavulanic acid. Mature CAD-1 consisted of a 34.4-kDa polypeptide. Kinetic analysis indicated that CAD-1 exhibited a narrow substrate profile, hydrolyzing benzylpenicillin, ampicillin, and piperacillin with catalytic efficiencies of 6,600, 3,200, and 2,900 mM^–1 s^–1, respectively. The enzyme did not interact with oxyiminocephalosporins, imipenem, or aztreonam. CAD-1 was inhibited by tazobactam (50% inhibitory concentration [IC₅₀] = 0.27 µM), clavulanic acid (IC₅₀ = 4.7 µM), and sulbactam (IC₅₀ = 43.5 µM). The bla_CAD-1 gene is likely to have been acquired by BM4489 and BM4490 as part of a mobile genetic element, since it was not found in the susceptible type strain CIP 101029 and was adjacent to a gene for a resolvase.

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Antibiotic degradation by β-lactamases and alterations in penicillin-binding proteins are the major mechanisms by which gram-positive bacteria express resistance to β-lactams. β-Lactamases are widely distributed in bacteria, in particular in gram-negative species (21). By contrast, only a few have been found in gram-positive bacteria, including Staphylococcus (18), Streptomyces lividans (15), Enterococcus faecalis (24), Bacillus spp. (41), some clostridia (4), Mycobacterium tuberculosis (17), Actinomadura strain R39 (26), and various Nocardia spp. (19). No β-lactamases have been characterized for lactic acid bacteria. With the exception of the class B enzyme from Bacillus cereus, all β-lactamases produced by gram-positive genera belong to the A class and mainly to group 2a as defined by K. Bush (11). These enzymes contain serine at the active site, have a molecular mass of approximately 30 kDa, and usually have greater penicillinase than cephalosporinase activity (23).

Lactic acid bacteria are the largest group of microorganisms associated with humans (42). They are associated with mucosal surfaces, particularly the gastrointestinal tract, and are also present in food-related habitats, including plant, wine, milk, and meat environments. However, there is increasing evidence that lactic acid bacteria can act as opportunistic pathogens (1, 36). Several Lactobacillus species, in particular L. casei, L. plantarum, L. rhamnosus, and L. acidophilus groups, have been implicated in human diseases, including septicemia, rheumatic and vascular diseases, meningitis, lung abscesses, endocarditis, peritonitis, and urinary tract infections (2, 13).

The Carnobacterium genus has been proposed to accommodate the species Lactobacillus divergens and certain so-called atypical lactobacilli isolated from poultry meat, seafood, and cheese (8). Carnobacterium spp. are used in the food industry because of the ability of some strains to prevent food contamination, in particular due to Listeria monocytogenes, by production of bacteriocins (35). To the best of our knowledge, no clinical human infections due to Carnobacterium spp. have been reported to date.

We describe the genetic location and biochemical characterization of β-lactamase-related penicillin resistance in two clinical isolates of Carnobacterium divergens.

(An initial report of this work was presented at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy [25a]).

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Strains, plasmids, and growth conditions. C. divergens BM4489 was isolated in April 1996 from the blood of a newborn delivered by caesarean section and treated with ampicillin in Akerhus County, Norway. Strain BM4490 was isolated in January 1997 from a febrile lymphoma patient treated with penicillin G in Vestfold County, Norway. Species identification was performed by sequencing PCR products obtained using the primer pairs 8FPL-806R and 515FPL-13B, complementary to consensus sequences in rrs encoding 16S rRNA (31, 32). The C. divergens type strain CIP 101029 was used as a control. Escherichia coli TB1 was used as the host in cloning experiments. E. coli BL21(DE3)pLys (Novagen, Madison, WI) and BL21-CodonPlus (DE3)-RIPL (Stratagene, La Jolla, CA) were used with the pET28a(+) expression vector (Novagen, Madison, WI). All strains were grown on brain heart infusion broth or agar (Difco Laboratories, Detroit, MI) at 37°C.

Susceptibility testing. Antibiotic susceptibility was tested by disk diffusion on Mueller-Hinton (MH) agar according to the standards of the Comité de l'Antibiogramme de la Société Française de Microbiologie (9). MICs of antimicrobial agents were determined by using E-test (AB Biodisk, Combourg, France) on MH agar.

DNA preparation and transformation. Total (34) and plasmid (6) DNA was isolated as described previously. Amplification of DNA was performed in a 9700 thermal cycler (Perkin Elmer Cetus, Notwalk, CT) with Pfu DNA polymerase (Stratagene, La Jolla, CA) as recommended by the manufacturer. The PCR products were purified using the QIA-quick PCR purification kit (Qiagen, Inc., Chatsworth, CA).

Recombinant DNA techniques. Cleavage of DNA with the restriction endonuclease EcoRI (Invitrogen Corporation, Cergy Pontoise, France) and ligation with T4 DNA ligase (Amersham Pharmacia Biotech, Uppsala, Sweden) were performed by using standard methods (34).

Plasmid construction. Plasmid pAT513 was constructed as follows. Total DNA from C. divergens BM4489 was digested with EcoRI and ligated with pBGS18 DNA cleaved similarly. Clones were selected on brain heart infusion agar containing kanamycin (20 mg/liter) and ampicillin (50 mg/liter).

DNA sequence determination and analysis. Plasmid DNA was labeled using a dye-labeled ddNTP Terminator Cycle sequencing kit (Beckman Coulter UK, Ltd.) and sequenced with a CEQ 2000 automated sequencer (Beckman). The sequence was analyzed using the GCG sequence analysis software package (version 10.1; Genetics Computer Group, Madison, WI). Comparison with known genes and proteins was carried out using BlastN and BlastX, available at the National Center for Biotechnology Information website (http://www.ncbi.nih.gov/BLAST). Multiple sequence alignment of the deduced peptide sequences was carried out using ClustalW at the European Bioinformatics Institute website (http://www.ebi.ac.uk).

Pulsed-field gel electrophoresis. Genomic DNA embedded in agarose plugs was digested for 3 h at 37°C with 0.01 U of I-CeuI, an intron-encoded endonuclease specific for rRNA genes (20), or overnight at 27°C with 25 U of SmaI. Fragments were separated on agarose gel (1.2% for I-CeuI and 0.8% for SmaI digestion) with a contour-clamped homogeneous electric field DR III system (Bio-Rad Laboratories, Hercules, CA) under the following conditions: total migration, 24 h for I-CeuI or 27 h for SmaI; initial pulse, 60 s for I-CeuI or 5 s for SmaI; final pulse, 120 s for I-CeuI or 35 s for SmaI; voltage, 6 V/cm; included angle, 120°; and temperature, 14°C. Fragments generated by I-CeuI were transferred to a nylon membrane and hybridized (i) to a -³²P-labeled 16S rRNA (rrs) probe obtained by amplification of an internal portion of the rrs gene (12) and (ii) to a bla_CAD-1-specific probe obtained by PCR with the Pen1 (5'-TGGTTGTCGGAGCCGGAGCT-3') and Pen2 (5'-GAGTCCCGTATGAACCAGCT-3') primers.

Production and purification of CAD-1. The deoxyoligonucleotides peniprot4 (5'-CTCCTCGAGATTGAAATCATCACTAACAAT-3') and peniprot5 (5'-GGTCGTCTCCCATGAAAAATAGAAAGTACG-3'), containing XhoI and BsmBI restriction sites (underlined), were used to amplify the bla_CAD-1 gene from pAT513 with Pfu DNA polymerase. The PCR product was cloned in the pCR-blunt vector, sequenced, and subcloned under the control of the T7 promoter in pET28a(+) previously digested with NcoI and XhoI enzymes, producing pAT514 (pET28a(+)bla_CAD-1).

The CAD-1 enzyme engineered to have a C-terminal His₆ tag was produced from E. coli BL21-CodonPlus (DE3)-RIPL harboring plasmid pAT514 (pET28a(+)bla_CAD-1) in high-cell-density medium (HDM) using multiple-microfermentor technology (10). The strain was grown in 80 ml of HDM containing kanamycin (30 µg/ml) at 37°C to an A₆₀₀ of 39, at which point isopropyl-1-thio-β-D-galactopyranoside was added to a final concentration of 0.5 mM. Induction was carried out overnight at 16°C to a final A₆₀₀ of 69. The cells were harvested by centrifugation and used immediately or stored at –80°C. The cell pellet was resuspended (4 ml/g of wet cells) in buffer A (20 mM sodium phosphate, pH 7.4, containing 0.5 M NaCl and 20 mM imidazole) and disrupted by sonication (five times for 30 s at 60 W). Cellular debris was removed by centrifugation (15,000 x g, 1 h, 4°C). The supernatant was collected, filtered (0.22 µm), and applied to a HisTrap Fast Flow column (GE Healthcare, Uppsala, Sweden) (5 ml, 5.0-ml/min flow rate) previously equilibrated with buffer A. The enzyme was eluted with a linear gradient of 20 to 500 mM imidazole over 50 ml. Fractions (1 ml each) containing recombinant CAD-1 were analyzed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The fractions containing β-lactamase activity were further purified by size exclusion chromatography using a Hiload 16/60 Superdex 75 colunm (GE Healthcare) equilibrated with 50 mM sodium phosphate buffer containing 0.15 M NaCl, pH 7.0, and eluted in the same buffer at a flow rate of 1 ml/min. The pure fractions were pooled, dialyzed overnight against 50 mM sodium phosphate (pH 7.4), and concentrated to approximately 10 mg/ml with a centriprep 30 concentrator (Amicon). The enzyme concentration was determined on the basis of the A₂₈₀ value assuming an value of 24,870 cm^–1 M^–1 as calculated by the ProtParam program of the ExPASy Proteomics server (http://www.expasy.org). The purified enzyme was stored at 4°C.

Determination of the kinetic parameters of CAD-1. Purified CAD-1 was used for determination of kinetic parameters (k_cat and K_m). Assays were performed at 25°C in 100 mM sodium phosphate (pH 7.0) in a total volume of 0.5 ml. The initial rates of hydrolysis were determined with an Uvikon UV931 spectrophotometer (Kontron Instruments, Saint-Quentin-en-Yvelines, France). The wavelengths and changes in extinction coefficients of β-lactams were as previously described (33). The antibiotics were provided as powders by Sigma-Aldrich (benzylpenicillin, ampicillin, piperacillin, carbenicillin, oxacillin, and cephalothin), Glaxo-Smith-Kline (clavulanic acid, cephaloridine, cefuroxime, and ceftazidime), Bristol-Myers-Squibb (cefepime), Merck Sharp and Dohme-Chibret (cefotaxime and imipenem), Pfizer (sulbactam), Wyeth Laboratories (tazobactam), and Sanofi-Aventis (aztreonam). The enzyme concentration in the reaction mixture was in the range of 10 to 10³ nM. The steady-state kinetic parameters were determined using the Hanes-Woolf plot (14). The 50% inhibitory concentrations (IC₅₀) were determined as the β-lactamase inhibitor concentrations that reduced the hydrolysis rate of 100 µM nitrocefin by 50% when the enzyme was preincubated with various concentrations of inhibitor for 30 min at 25°C before addition of the substrate (27).

Nucleotide sequence accession number. The sequence of the bla_CAD-1 gene and of the flanking regions has been deposited in the GenBank data library under accession number AY650920.

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Strains and susceptibility testing. Identification of C. divergens BM4489 and BM4490 was confirmed by partial sequencing of the rrs gene for 16S rRNA (31, 32). The two strains had distinct patterns following contour-clamped homogeneous electric-field gel electrophoresis of SmaI-digested total DNA (data not shown).

They were resistant to benzylpenicillin, ampicillin, amoxicillin, ticarcillin, and piperacillin but proved susceptible to oxacillin and to the combinations amoxicillin-clavulanic acid and piperacillin-tazobactam (Table 1). Both strains hydrolyzed nitrocefin, suggesting involvement of a β-lactamase in resistance. The MICs varied greatly with the inoculum size, an observation consistent with the fact that β-lactamases are predominantly extracellular enzymes in gram-positive species. The isolates were susceptible to chloramphenicol, tetracycline, rifampin, erythromycin, pristinamycin, and the glycopeptides and had a diminished susceptibility to lincomycin.

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TABLE 1. MICs of β-lactams for C. divergens and E. coli strains with or without CAD-1

Cloning and sequencing of the bla_CAD-1 gene. Fragments obtained after digestion of C. divergens BM4489 total DNA with EcoRI were cloned in pBGS18 cleaved with the same enzyme into E. coli TB1, and transformants were screened on agar containing kanamycin plus ampicillin. Plasmid pAT513 carried an insert of 4,107 bp that was sequenced and found to contain two open reading frames, ORF1 and ORF2, 912 bp and 579 bp in size, respectively. The sequence deduced from ORF1 was 304 amino acids long and shared similarity with class A β-lactamases. The highest degrees of identity were with β-lactamases from B. cereus (53%; accession number NP_978847); Bacillus mycoïdes (51%; accession number P28018); Bacillus anthracis (50%; accession number NP_844879); Bacillus subtilis (50%; accession number NP_389761); Bacillus weihenstephanensis (47%; accession number ZP_01186593); Staphylococcus aureus (42%; accession number ABB82608); and Nocardia farcinica (38%; accession number AAB81957). The putative β-lactamase gene, named bla_CAD-1, specified a protein with a calculated mass of 33,550 Da. Within ORF1, a putative GGAGG ribosome-binding site was found 6 bp upstream from the ATG initiation codon. The bla_CAD-1 gene had a G+C content of 34%, similar to that of C. divergens (33 to 35%) (16).

The deduced sequence of ORF2 exhibited 60% and 45% identity with resolvases of Listeria welshimeri and B. cereus, respectively, suggesting that bla_CAD-1 could be part of a mobile element.

Primers Pen1 and Pen2, designed from the sequence of bla_CAD-1 of BM4489, allowed amplification of a 716-bp fragment from BM4490 DNA with a sequence identical to that in BM4489. No PCR product was obtained using total DNA of CIP 101029 as a potential template.

The bla_CAD-1 gene was assigned to a chromosomal fragment of ca. 300 kb in BM4489 and BM4490 by pulsed-field gel electrophoresis of total DNA digested with I-CeuI followed by successive hybridization with 16S rRNA (rrs) and bla_CAD-1 probes (Fig. 1).

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FIG. 1. Genetic location of blaCAD-1 by contour-clamped homogeneous electric field gel electrophoresis of total DNA of C. divergens BM4489 and BM4490 digested with I-CeuI and by Southern hybridization with an [-32P]-labeled 16S rRNA (rrs) probe (A) or a blaCAD-1 probe (B).

Structural properties of CAD-1. Comparative analysis of CAD-1 sequence allowed detection of motifs present in all class A β-lactamases. The first one, the Ser-Thr-Tyr-Lys tetrad containing the active-site serine, was found at positions 70 to 73 (Ser⁷⁰ according to ABL numbering [3]). The second element, Ser-Asp-Asn, was at ABL positions 130 to 132; the third, Lys-Ser-Gly, at positions 234 to 236; and the fourth element, Glu-Thr-Glu-Leu-Asn, at positions 166 to 170. Furthermore, CAD-1 had a single amino acid (Tyr in place of Leu at ABL position 207) that differed among the 26 residues that are isofunctionally conserved in class A β-lactamases (23). This Tyr residue is present in class A NmcA and IMI-1 β-lactamases from Enterobacter cloacae (30) and in SmeI from Serratia marcescens (25), which hydrolyze carbapenems, but its role in hydrolysis is not clearly established.

Using the SignalP program (5), the enzyme was predicted to have a signal peptide for translocation with a cleavage site at residue 24 (Fig. 2). Edman degradation of the purified protein yielded the AILKPS sequence, confirming the position of the signal peptidase action. However, CAD-1 lacks the N-terminal cysteine residue present in all membrane-bound β-lactamase lipoproteins of gram-positive bacteria and to which the lipid is covalently linked (38). It is therefore not clear if this β-lactamase exists as a free protein or a membrane-bound protein.

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FIG. 2. Sequence alignment of CAD-1 with other class A β-lactamases. Sequences of B. cereus (accession number NP_978847), B. mycoïdes (accession number P28018), B. anthracis (accession number NP_844879), B. subtilis (accession number NP_389761), B. weihenstephanensis (accession number ZP_01186593), S. aureus (accession number ABB82608), B. licheniformis (accession number CAA71115), and N. farcinica (accession number AAB81957) are shown. The numbering below the sequences is according to the work of Ambler (3). The arrow indicates the position of the signal peptidase action. The asterisks indicate residues that are involved in the catalytic mechanism and/or in substrate binding.

Purification and characterization of CAD-1 β-lactamase. Initial attemps to produce carboxy-terminus His₆-tagged CAD-1 from E. coli BL21(DE3)pLys harboring plasmid pAT514 (pET28(a)+bla_CAD-1) were unsuccessful. SDS-PAGE analysis revealed that CAD-1 was very weakly expressed (ca. <1% of total proteins), which was possibly the consequence of the low G+C content (34%) of the bla_CAD-1 gene. However, using E. coli BL21-CodonPlus (DE3)-RIPL, large quantities of the enzyme were produced. This strain rescues expression of heterologous genes from organisms that have either AT- or GC-rich genomes. CAD-1 was purified from crude lysates by nickel affinity chromatography followed by size exclusion chromatography. The protein was >98% pure, as judged by SDS-PAGE analysis (data not shown). Overall, the procedure yielded approximately 30 mg of purified CAD-1 from 80 ml of HDM using a microfermentor.

Kinetic parameters indicated that the CAD-1 β-lactamase has a narrow substrate profile that includes benzylpenicillin, ampicillin, piperacillin, and carbenicillin (Table 2). As with all class A β-lactamases, benzylpenicillin was the best substrate, with a high turnover rate (k_cat, 178 s^–1) and, consequently, a high catalytic efficiency (k_cat/K_m, 6,600 mM^–1 s^–1), being hydrolyzed two- and sevenfold more efficiently than piperacillin and carbenicillin, respectively. However, the penicillinase activity of CAD-1 was about 13-fold lower than that of TEM-1 class A β-lactamase (23). Oxacillin, which was a poor substrate, was hydrolyzed 275-fold less efficiently than benzylpenicillin. CAD-1 had a very weak activity on early-generation cephalosporins, with k_cat turnover rates of 0.56 and 0.1 s^–1 for cephaloridine and cephalothin, respectively. No hydrolysis of extended-spectrum cephalosporins (cefuroxime, cefoxitin, cefotaxime, ceftazidime, and cefepime) and of aztreonam was detected even at a high enzyme concentration (1 µM). This might be due to the conservation of Asp¹⁷⁹ and Ala²³⁷ residues in CAD-1, since it has been shown that loss of electrostatic interaction between Asp¹⁷⁹ and Arg¹⁶⁴ in TEM-1-derived β-lactamases leads to easier access to the active site of bulky extended-spectrum β-lactams (28) and the substitution Ala²³⁷Gly is characteristic of an increased affinity for aztreonam (7). However, Asp¹⁷⁹ and Arg¹⁶⁴ might not be, by themselves, responsible for the lack of extended-spectrum cephalosporinase activity, since Actinomadura R39 and Bacillus licheniformis enzymes, which possess the Asp¹⁷⁹ and Arg¹⁶⁴ residues, have significant catalytic activity on cefotaxime (22). CAD-1 activity did not account for the resistance phenotype of C. divergens, which includes extended-spectrum β-lactams (Table 1). As for other gram-positive organisms, this may be due to various penicillin-binding protein affinities. An absence of imipenem hydrolysis could result from the lack of cysteine residues in CAD-1, since a striking characteristic of class A carbapenemases is the presence of a disulfide bond between positions 69 and 238 (29, 37, 39).

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TABLE 2. Kinetic parameters of purified CAD-1 β-lactamase for hydrolysis of some β-lactamsa

CAD-1 was inhibited by all three β-lactamase inhibitors tested. Tazobactam was the strongest inhibitor, with an IC₅₀ of 0.27 µM, followed by clavulanic acid with an IC₅₀ of 4.7 µM. Sulbactam was the least potent, with an IC₅₀ of 43.5 µM. This hierarchy was in agreement with the susceptibility pattern of C. divergens BM4489 and of E. coli BL21 carrying the cloned bla_CAD-1 gene (Table 1). The strains were resistant to most penicillins, whose antimicrobial activities were restored by clavulanic acid and tazobactam. However, clavulanic acid was a relatively poor inhibitor of CAD-1, with a 60-fold higher IC₅₀ than that against TEM-1 (23). Class A β-lactamases with amino acid substitutions at Arg²⁴⁴, Asn²⁷⁶, Arg²⁷⁵, Met⁶⁹, Met¹⁸², and Trp¹⁶⁵ have been reported to be resistant to clavulanate and other mechanism-based inhibitors (40). Therefore, the amino acid changes in CAD-1 compared to TEM-1, i.e., Met⁶⁹Ala, Trp¹⁶⁵Phe, Met¹⁸²Thr, Arg²⁷⁵Asn, or Asn²⁷⁶Asp, may explain the low susceptibility of the enzyme to clavulanic acid.

The bla_CAD-1 gene was not found in the β-lactam-susceptible type strain CIP 101029 and is therefore not species specific. In addition, in the two strains studied, the resistance gene was adjacent to that for a resolvase, suggesting that both genes could be part of a mobile genetic element. Since the base composition of bla_CAD-1 (G+C content, 34%) was similar to that of the C. divergens chromosome (G+C content, 33 to 35%) (16), the resistance determinant may have been acquired from a species phylogenetically close to its current host or, alternatively, a long time ago.

There is current concern over the possible dissemination of resistance from bacteria used in food production or as probiotics for human bacterial pathogens in the digestive tract, either directly or indirectly through commensals. Lactic acid bacteria, such as C. divergens, could act as a reservoir of antibiotic resistance determinants and should thus be carefully examined for resistance to antimicrobials and for putative transferability.

 
D.M.-C. was supported by a Novartis postdoctoral fellowship.

We thank J. Belallou for advice on CAD-1 production and P. E. Reynolds for reading of the manuscript.

 
* Corresponding author. Mailing address: Unité des Agents Antibactériens, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33) (1) 45 68 83 20. Fax: (33) (1) 45 68 83 19. E-mail: pcourval{at}pasteur.fr

Published ahead of print on 10 December 2007.

Present address: Laboratoire de Biochimie Appliquée, Faculté des Sciences, Université Ferhat Abbas, 19000 Sétif, Algeria.

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Antimicrobial Agents and Chemotherapy, February 2008, p. 551-556, Vol. 52, No. 2
0066-4804/08/$08.00+0     doi:10.1128/AAC.01145-07
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