Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cédex, France
Received 28 January 2002; returned 12 July 2002; revised 25 July 2002; accepted 29 October 2002
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Abstract |
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Keywords: class B ß-lactamase, Flavobacterium johnsoniae, carbapenem
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Introduction |
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Following an extensive phylogenetic study, the taxonomy of the CytophagaFlavobacteriumBacteroides group of the eubacterial branch has recently been modified, resulting in amendment to the genera Cytophaga, Flavobacterium and Flexibacter and nomenclature change.15 Flavobacterium johnsoniae (formerly Cytophaga johnsonae) is an environmental bacterium that can cause skin lesions in fish and is a plant pathogen.16,17 In this study, we have cloned and characterized a class B carbapenem-hydrolysing ß-lactamase from F. johnsoniae, in the process identifying a novel member of the highly divergent subclass B1 of class B enzymes.
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Materials and methods |
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F. johnsoniae reference strain CIP100931 was from the Institut Pasteur collection in Paris (France). Escherichia coli DH10B was used for cloning and protein expression experiments. The F. johnsoniae strain was cultured either in Trypticase soy (TS) broth (Becton Dickinson, Le-Pont-de-Claix, France) or on TS-containing agar for 48 h at 30°C in an aerobic atmosphere.
Antimicrobial agents and MICs
The antimicrobial agents used in this study were obtained in the form of standard laboratory powders and were dissolved in water and used immediately. The agents and their sources were as described previously.18 MICs of ß-lactams were determined by an agar dilution technique using MuellerHinton agar (Sanofi-Diagnostics Pasteur, Marnes-La-Coquette, France) with an inoculum of 104 cfu per spot.19
DNA techniques
Whole-cell DNA of F. johnsoniae CIP100931 was extracted as described previously.20 All enzymes used in cloning experiments were from Amersham Pharmacia Biotech (Orsay, France). Fragments generated by Sau3AI partial digestion of genomic DNA were ligated into the BamHI site of the pBK-CMV phagemid (Stratagene, Amsterdam, The Netherlands), as previously described.21 Recombinant clones were selected on ampicillin- (30 mg/L) and kanamycin- (30 mg/L) containing TS agar. Recombinant plasmid DNA was obtained from 100 mL TS broth cultures grown overnight in the presence of ampicillin (20 mg/L) at 37°C. Plasmid DNA was recovered by passage through Qiagen columns (Qiagen, Courtaboeuf, France). Plasmid mapping was achieved by analysis of fragments generated by single- and double-restriction endonuclease digestion.21 Fragment sizes were estimated by comparison with a 1 kb DNA ladder (Amersham Pharmacia Biotech).
Using whole-cell DNA of F. johnsoniae CIP100931 as template, PCR experiments were carried out with primers annealing at the extremities of the blaJOHN-1 gene (primer JoA, 5'-GCCGCGGTTTCAAATAGTTTGGG-3'; primer JoB, 5'-GGCAGATTTTTGAGCCAAATACTG-3').22 Then, Southern blotting and hybridization analyses were carried out using a 0.8% agarose electrophoresis gel22 containing unrestricted whole-cell DNA of F. johnsoniae CIP100931, which was transferred onto a nylon membrane and hybridized with a PCR-generated internal fragment of blaJOHN-1 using primers JoA and JoB. Visualization of hybridization was by the ECL non-radioactive labelling and detection kit, as described by the manufacturer (Amersham Pharmacia Biotech).
The cloned DNA fragment of recombinant plasmid pJOHN-1 was sequenced on both strands, using an Applied Biosystems sequencer (ABI 377). The nucleotide and the deduced protein sequences were analysed with software available over the Internet, as described previously.9 The nucleotide sequence and deduced ß-lactamase amino acid sequence reported in this work have been assigned to the GenBank and EMBL databases under the accession no. AY028464.
ß-Lactamase purification
A culture of E. coli DH10B harbouring recombinant plasmid pJOHN-1 was incubated overnight at 37°C in 4 L of TS broth containing ampicillin (100 mg/L). Bacterial cells were pelleted, resuspended in 60 mL of 20 mM TrisHCl buffer (pH 8.0), incubated at 4°C for 2 h with lysozyme (1 mg/mL) (Sigma) and DNase I (1 mg/mL), disrupted by sonification (three times at 30 W for 2 min using a Vibra Cell 75022 Phospholyser, Bioblock, Illkirch, France) and centrifuged at 48 000g for 1 h at 4°C. The supernatant was ultracentrifuged at 100 000g for 1 h at 4°C and then dialysed overnight against 20 mM TrisHCl (pH 8.0). This extract was loaded onto a pre-equilibrated Q-Sepharose column (Amersham Pharmacia Biotech) in the same TrisHCl buffer and eluted with that buffer. The enzyme recovered in the flow-through was dialysed overnight at 4°C against 50 mM phosphate buffer (pH 7.0) and then loaded onto a pre-equilibrated S-Sepharose column (Amersham Pharmacia Biotech). The fractions with the highest ß-lactamase activity (nitrocefin test, Oxoid, Dardilly, France) eluted at 300 mM NaCl (gradient 0 to 500 mM) in the phosphate buffer (pH 7.0). Fractions containing the ß-lactamase activity were pooled, dialysed overnight against 150 mM NaCl and concentrated with a Vivaspin 10 000 column (Sartorius, Göttingen, Germany). The concentrated ß-lactamase extract was loaded onto a 1.6 x 47 cm gel filtration column packed with Superdex 75 (Amersham Pharmacia Biotech) equilibrated with 50 mM phosphate buffer (pH 7.0) and eluted with 150 mM NaCl. The fraction containing the ß-lactamase activity was dialysed overnight against 50 mM phosphate buffer (pH 7.0) containing 50 µM ZnCl2 and then concentrated 10-fold with a Vivaspin 10 000 column. The specific activities of the ß-lactamase in the crude cell extract and in the purified preparation were compared using 100 µM imipenem as substrate, as previously described.9 Purity of the enzyme was estimated by SDSPAGE analysis.22
IEF analysis and determination of relative molecular mass
Purified enzyme from E. coli DH10B (pJOHN-1) and a ß-lactamase-containing crude extract from a 100 mL culture of F. johnsoniae CIP100931 were subjected to analytical isoelectric focusing (IEF) analysis on a pH 3.59.5 ampholine-containing polyacrylamide gel (Ampholine PAG plate, Amersham Pharmacia Biotech), as described previously.18 The relative molecular mass of the purified ß-lactamase was estimated using the same gel filtration column as described for ß-lactamase purification.9
Kinetic parameters of JOHN-1 ß-lactamase
Kinetic data using the JOHN-1 ß-lactamase were obtained at 30°C in 100 mM sodium phosphate (pH 7.0) containing 50 µM ZnCl2. kcat and Km values were determined with an ULTROSPEC 2000 spectrophotometer (Amersham Pharmacia Biotech), as previously described.10,23 To investigate the effects of potential inhibitors, the enzyme was pre-incubated in various concentrations of EDTA and clavulanic acid for 3 min at 30°C before testing the rate of imipenem hydrolysis. Fifty per cent inhibitory concentrations (IC50 values) were determined for EDTA and clavulanic acid and results were expressed in micromolar units.
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Results and discussion |
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Twelve ampicillin-resistant E. coli DH10B clones harbouring recombinant plasmids with inserts of 610 kb were obtained after cloning Sau3AI-restricted DNA from the F. johnsoniae reference strain. Among them, E. coli DH10B (pJOHN-1) harboured a recombinant plasmid with one of the smallest inserts (6.2 kb) and was retained for further analysis.
DNA sequence analysis of the 6201 bp insert of pJOHN-1 revealed an open reading frame (ORF) of 746 bp named blaJOHN-1, encoding a predicted 248 amino acid pre-protein. A 19-amino-acid leader sequence and putative cleavage site (after the SerLeuGly) were identified by computer analysis (Figure 1).24 The overall G+C content of blaJOHN-1 was 33%, which is close to that expected for Flavobacterium genes (3338%).15 The deduced protein shares significant identity with Ambler class B ß-lactamases (Figure 1).25 Three hundred and thirty-six base pairs upstream of the initiation codon for blaJOHN-1 there is a possible promoter sequence (5'-TAATTTTC-3') that is the same as those identified upstream of the gldD and gldE genes involved in gliding motility of F. johnsoniae.26 It agrees with the consensus promoter sequence proposedfor B. fragilis, another member of the CaptocytophagaFlavobacteriumBacteroides branch of eubacteria.26
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PCR experiments with primers annealing at the extremities of blaJOHN-1 and whole-cell DNA of F. johnsoniae CIP100931 as template, followed by sequencing of the PCR products identified the origin of the blaJOHN-1 gene. Using a PCR-prepared probe internal to blaJOHN-1 and whole-cell DNA of F. johnsoniae CIP100931 as template, a hybridization signal was obtained at the position of chromosome DNA migration (data not shown), indicating a likely chromosomal location of this gene. No plasmid DNA was detected in F. johnsoniae CIP100931 (data not shown).
Susceptibility testing
MICs of ß-lactams for F. johnsoniae CIP100931 showed that the bacterium is resistant to amino- and carboxy-penicillins, to narrow- and extended-spectrum cephalosporins, and to the monobactam aztreonam, and has reduced susceptibility (but remains susceptible) to carbapenems (Table 1). Interestingly, the ß-lactamase inhibitor clavulanic acid was found to have a significant antibacterial activity in its own right (Table 1). F. johnsoniae CIP100931 is less resistant to carbapenems than C. indologenes and C. meningosepticum.911 E. coli DH10B (pJOHN-1) is resistant to amoxicillin, ticarcillin, to some narrow-spectrum cephalosporins, such as cefalothin and cephamycins (cefoxitin and moxalactam), and has decreased susceptibility to extended-spectrum cephalosporins and carbapenems.
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Biochemical properties of ß-lactamase JOHN-1
A preliminary experiment using a crude cell extract of F. johnsoniae CIP100931 and imipenem (100 µM) as substrate revealed a carbapenem-hydrolysing ß-lactamase activity in that strain (data not shown). IEF analysis showed that E. coli DH10B (pJOHN-1) produces a ß-lactamase with a pI value of 9.0, similar to that found in a crude cell extract of F. johnsoniae CIP100931, although in this latter case, the pI value could only be estimated to be between 8.8 and 9 (data not shown). These pI values agree with the calculated pI value of mature JOHN-1 ß-lactamase (9.1). The specific activity of the purified JOHN-1 ß-lactamase was estimated to be 2.73 µmol/min/mg of protein, determined with 100 µM imipenem as substrate, with a 153-fold purification factor. Enzyme purity was estimated to be 90% by SDSPAGE analysis (data not shown). The mature protein expressed in E. coli has a relative molecular mass, determined experimentally, of 26 kDa (data not shown), which corresponds to the calculated molecular mass of the mature protein (26 kDa). As found for most metallo-ß-lactamases, except L-1 from S. maltophilia, JOHN-1 is a monomeric enzyme.
Kinetic analysis of JOHN-1 revealed that this enzyme has a broad substrate profile that includes carbapenems, as is the case for most metallo-ß-lactamases.1 Like other metallo-ß-lactamases, its substrate profile does not include the monobactam aztreonam.27 The overall catalytic activity of JOHN-1 for all ß-lactams was found to be lower than that reported for BlaB.12 As compared with IND-like enzymes (IND-2),11 JOHN-1 has a 10-fold lower hydrolytic activity against carbapenems, but has similar and low catalytic activity against ceftazidime and cefepime (low kcat values) and low affinity (high Km values) for these substrates. Overall, JOHN-1 is an enzyme with lower hydrolytic activity than other metallo-ß-lactamases.
A comparison of MIC values of ß-lactams for F. johnsoniae CIP100931 (Table 1) and the kinetic parameters of JOHN-1 (Table 2) indicates that expression of the JOHN-1 ß-lactamase does not explain the entire ß-lactam resistance profile of this strain. For example, F. johnsoniae CIP100931 is resistant to ceftazidime, cefepime and aztreonam, whereas the JOHN-1 ß-lactamase has poor or no catalytic activity with these compounds. Other ß-lactam resistance mechanisms such as impermeability, efflux and penicillin-binding affinity may contribute to the ß-lactam resistance phenotype observed for F. johnsoniae CIP100931.
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Conclusion
This study identifies a novel bacterial species for which ß-lactam resistance is mediated at least in part by an Ambler class B metallo-ß-lactamase. The JOHN-1 ß-lactamase belongs to the B1 subclass of metallo-enzymes, which includes Bc-II from B. cereus and CcrA from B. fragilis. Tertiary structures are available for Bc-II and CcrA.31,32 It would be interesting to investigate biochemical/structural relationships of JOHN-1 in comparison with these enzymes, given that Bc-II and CcrA use different reaction pathways since they are monozinc- and dizinc-dependent, respectively.33
Whereas the JOHN-1 ß-lactamase is not related to the plasmid-mediated carbapenem-hydrolysing IMP- and VIM-like ß-lactamases, it may be added to the list of class B ß-lactamases found in the environmental reservoir.
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Acknowledgements |
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Footnotes |
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References |
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2 . Walsh, T. R., Hall, L., Assinder, S. J., Nichols, W. W., Cartwright, S. J., MacGowan, A. P. et al. (1994). Sequence analysis of the L-1 metallo ß-lactamase from Xanthomonas maltophilia. Biochimica Biophysica Acta 1218, 199201.[ISI][Medline]
3 . Lim, H. M., Pene, J. J. & Shaw, R. (1988). Cloning, nucleotide sequence, and expression of the Bacillus cereus 5/B/6 ß-lactamase II structural gene. Journal of Bacteriology 170, 28738.[ISI][Medline]
4 . Podglajen, I., Breuil, J., Casin, I. & Collatz, E. (1995). Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content. Journal of Bacteriology 177, 52705.[Abstract]
5 . Rasmussen, B. A., Gluzman, Y. & Tally, F. P. (1990). Cloning and sequencing of the class B ß-lactamase gene (ccrA) from Bacteroides fragilis TAL3636. Antimicrobial Agents and Chemotherapy 34, 15902.[ISI][Medline]
6 . Massidda, O., Rossolini, G. M. & Satta, G. (1991). The Aeromonas hydrophila cphA gene: molecular heterogeneity among metallo-ß-lactamases. Journal of Bacteriology 173, 46117.[ISI][Medline]
7
.
Boschi, L., Mercuri, P. S., Riccio, M. L., Amicosante, G., Galleni, M., Frère, J.-M. et al. (2000). The Legionella (Fluoribacter) gormanii metallo-ß-lactamase: a new member of the high divergent lineage of molecular subclass B3 ß-lactamases. Antimicrobial Agents and Chemotherapy 44, 153843.
8
.
Rossolini, G. M., Condemi, M. A., Pantanella, F., Docquier, J. D., Amicosante, G. & Thaller, M. C. (2001). Metallo-ß-lactamase producers in environmental microbiota: new molecular class B enzyme in Janthinobacterium lividum. Antimicrobial Agents and Chemotherapy 45, 83744.
9
.
Bellais, S., Aubert, D., Naas, T. & Nordmann, P. (2000). Molecular and biochemical heterogeneity of class B carbapenem-hydrolyzing ß-lactamases in Chryseobacterium meningosepticum. Antimicrobial Agents and Chemotherapy 44, 187886.
10 . Bellais, S., Léotard, S., Poirel, L., Naas, T. & Nordmann, P. (1999). Molecular characterization of a carbapenem-hydrolyzing ß-lactamase from Chryseobacterium (Flavobacterium) indologenes. FEMS Microbiology Letters 171, 12732.[CrossRef][ISI][Medline]
11
.
Bellais, S., Poirel, L., Léotard, S., Naas, T. & Nordmann, P. (2000). Genetic diversity of carbapenem-hydrolyzing metallo-beta-lactamases from Chryseobacterium (Flavobacterium) indologenes. Antimicrobial Agents and Chemotherapy 44, 302834.
12 . Rossolini, G. M., Franceschini, N., Riccio, M. L., Mercuri, P. S., Perilli, M., Galleni, M. et al. (1998). Characterization and sequence of the Chryseobacterium (Flavobacterium) meningosepticum carbapenemase: a new molecular class B ß-lactamase showing a broad substrate profile. Biochemical Journal 332, 14552.[ISI][Medline]
13
.
Lauretti, L., Riccio, M. L., Mazzariol, A., Cornaglia, G., Amicosante, G., Fontana, R. et al. (1999). Cloning and characterization of blaVIM, a new integron-borne metallo-ß-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrobial Agents and Chemotherapy 43, 158490.
14 . Arakawa, Y., Marakami, M., Suzuki, K., Ito, H., Wacharotayankun, R., Ohsuka, S. et al. (1995). A novel integron-like element carrying the metallo ß-lactamase gene blaIMP. Antimicrobial Agents and Chemotherapy 39, 16125.[Abstract]
15
.
Bernardet, J. F., Segers, P., Vancanneyt, M., Berthe, F., Kersters, K. & Vandamme, P. (1996). Cutting a gordian knot: amended classification and description of the genus Flavobacterium, amended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. nov. (Basonym, Cytophaga aquatilis Strohl and Tait 1978). International Journal of Systematic Bacteriology 46, 12848.
16 . Carson, J., Schmidtke, L. M. & Munday, B. L. (1993). Cytophaga johnsonae: a putative skin pathogen of juvenile farmed barramunid, Later calcarifer Bloch. Journal of Fishery Diseases 16, 20918.
17 . Lednicka, D., Mergaert, J., Cnockaert, M. C. & Swings, J. (2000). Isolation and identification of cellulolytic bacteria involved in the degradation of natural cellulosic fibres. Systematic and Applied Microbiology 23, 2929.[ISI][Medline]
18
.
Poirel, L., Naas, T., Guibert, M., Chaibi, E. B., Labia, R. & Nordmann, P. (1999). Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum ß-lactamase encoded by an Escherichia coli integron gene. Antimicrobial Agents and Chemotherapy 43, 57381.
19 . National Committee for Clinical Laboratory Standards. (2001). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
20
.
Naas, T., Blot, M., Fitch, W. M. & Arber, W. (1994). Insertion sequence-related genetic variation in resting Escherichia coli K-12. Genetics 136, 72130.
21 . Nordmann, P. & Naas, T. (1994). Sequence analysis of PER-1 extended-spectrum beta-lactamase from Pseudomonas aeruginosa and comparison with class A ß-lactamases. Antimicrobial Agents and Chemotherapy 38, 10414.[Abstract]
22 . Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
23
.
Naas, T., Sougakoff, W., Casetta, A. & Nordmann, P. (1998). Molecular characterization of OXA-20, a novel class D ß-lactamase, and its integron from Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 42, 207483.
24 . Nielsen, H., Engelbrecht, J., Brunak, S. & Von Heijne, G. (1997). A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. International Journal of Neurology and Systematics 8, 58199.
25
.
Galleni, M., Lamotte-Brasseur, J., Rossolini, G. M., Spencer, J., Dideberg., O. & Frère, J.-M. (2001). Standard numbering scheme for class B beta-lactamases. Antimicrobial Agents and Chemotherapy 45, 6603.
26
.
Hunnicutt, D. W. & McBride, M. J. (2001). Cloning and characterization of the Flavobacterium johnsoniae gliding motility genes gldD and gldE. Journal of Bacteriology 183, 416775.
27
.
Rasmussen, B. A. & Bush, K. (1997). Carbapenem-hydrolyzing ß-lactamases. Antimicrobial Agents and Chemotherapy 41, 22332.
28 . Mazel, D., Coic, E., Blanchard, S., Saurin, W. & Marliere, P. (1997). A survey of polypeptide deformylase function throughout the eubacterial lineage. Journal of Molecular Biology 266, 93949.[CrossRef][ISI][Medline]
29 . Blattner, F. R., Burland, V., Plunkett, G., Sofia, H. J. & Daniels, D. L. (1993). Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 min. Nucleic Acids Research 21, 540817.[Abstract]
30
.
Overhage, J., Kresse, A. U., Priefert, H., Sommer, H., Krammer, G., Rabenhorst, J. et al. (1999). Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199. Applied and Environmental Microbiology 65, 95160.
31 . Carfi, A., Duée, E., Galleni, M., Frère, J.-M. & Dideberg, O. (1998). 1.85 Å resolution structure of the Zn(II) ß-lactamase from Bacillus cereus. Acta Crystallographica D 54, 31323.[CrossRef][ISI][Medline]
32 . Concha, N. O., Rasmussen, B. A., Bush, K. & Herzberg, O. (1996). Crystal structure of the wide-spectrum binuclear zinc ß-lactamase from Bacteroides fragilis. Structure 4, 82336.[ISI][Medline]
33 . Wang, Z., Fast, W., Valentine, A. M. & Benkovic, S. J. (1999). Metallo-ß-lactamase: structure and mechanism. Current Opinion in Chemical Biology 3, 61422.[CrossRef][ISI][Medline]