Molecular and biochemical characterization of a

Advance Access published January 6, 2003

Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkg069

Molecular and biochemical characterization of a carbapenem-hydrolysing β-lactamase from Flavobacterium johnsoniae Thierry Naas, Samuel Bellais and Patrice Nordmann* 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

Keywords: class B β-lactamase, Flavobacterium johnsoniae, carbapenem

Introduction Metallo-β-lactamases (Ambler class B) are zinc-dependent enzymes that possess the unusual property of hydrolysing most β-lactams, including carbapenems.1 Several metallo-βlactamases play a role in intrinsic resistance to β-lactams of bacterial species, such as the L-1 enzyme from Stenotrophomonas maltophilia,2 Bc-II from Bacillus cereus,3 CcrA from Bacteroides fragilis (a definite subspecies of B. fragilis),4,5 CphA from Aeromonas hydrophila,6 FEZ-1 from Legionella gormanii,7 THIN-B from Janthinobacterium lividum,8 like enzymes from Chryseobacterium meningosepticum (GOB-1-like and BlaB-like enzymes) and Chryseobacterium indologenes (IND-1-like enzymes).9–12 Additionally, metalloenzymes (IMP and VIM series) of unknown origin are emerging worldwide as a source of acquired carbapenem resistance in Gram-negative bacteria.1,13,14 Thus, research on identification of metallo-β-lactamases is driven by the need for universal metallo-enzyme inhibitors and the identification of the reservoir of acquired metallo-β-lactamases.

Following an extensive phylogenetic study, the taxonomy of the Cytophaga–Flavobacterium–Bacteroides 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 β-lactamase from F. johnsoniae, in the process identifying a novel member of the highly divergent subclass B1 of class B enzymes.

Materials and methods Bacterial strains and culture 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

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*Corresponding author. Tel: +33-1-45-21-36-32; Fax: 33-1-45-21-63-40; E-mail: [email protected] ...................................................................................................................................................................................................................................................................

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Flavobacterium johnsoniae CIP100931 is resistant to most β-lactam antibiotics and has a decreased susceptibility to carbapenems. A β-lactamase gene was cloned and expressed in Escherichia coli DH10B. The purified β-lactamase, JOHN-1, with a pI value of 9.0 and with a determined relative molecular mass of ∼27 kDa was found to be a monomeric zinc-dependent enzyme that hydrolyses penicillins, narrow- and expanded-spectrum cephalosporins, carbapenems, but not monobactams. Sequence analysis revealed that JOHN-1 is a molecular class B β-lactamase that is most closely related to BlaB from Chryseobacterium meningosepticum and IND-1 from Chryseobacterium indologenes (47% and 41% amino acid identity, respectively). JOHN-1 is a new member of the highly divergent subclass B1 lineage of metallo-enzymes. Although F. johnsoniae and Chryseobacterium spp. are phylogenetically related bacteria, this report further underlines the heterogeneity of class B β-lactamases that are naturally produced by environmental Gram-negative aerobes and that are now recognized as the most important reservoir for these β-lactamase genes.

T. Naas et al. Trypticase soy (TS) broth (Becton Dickinson, Le-Pont-deClaix, France) or on TS-containing agar for 48 h at 30°C in an aerobic atmosphere.

GenBank and EMBL databases under the accession no. AY028464.

β-Lactamase purification 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 β-lactams were determined by an agar dilution technique using Mueller–Hinton agar (Sanofi-Diagnostics Pasteur, Marnes-La-Coquette, France) with an inoculum of 104 cfu per spot.19

DNA techniques

IEF analysis and determination of relative molecular mass Purified enzyme from E. coli DH10B (pJOHN-1) and a β-lactamase-containing crude extract from a 100 mL culture of F. johnsoniae CIP100931 were subjected to analytical isoelectric focusing (IEF) analysis on a pH 3.5–9.5 ampholinecontaining polyacrylamide gel (Ampholine PAG plate, Amersham Pharmacia Biotech), as described previously.18 The relative molecular mass of the purified β-lactamase was estimated using the same gel filtration column as described for β-lactamase purification.9

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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 pBKCMV 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 β-lactamase amino acid sequence reported in this work have been assigned to the

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 Tris–HCl 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 Tris–HCl (pH 8.0). This extract was loaded onto a pre-equilibrated Q-Sepharose column (Amersham Pharmacia Biotech) in the same Tris–HCl 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 β-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 β-lactamase activity were pooled, dialysed overnight against 150 mM NaCl and concentrated with a Vivaspin 10 000 column (Sartorius, Göttingen, Germany). The concentrated β-lactamase extract was loaded onto a 1.6 × 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 β-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 β-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 SDS–PAGE analysis.22

Metallo-β-lactamase from F. johnsoniae

Kinetic parameters of JOHN-1 β-lactamase Kinetic data using the JOHN-1 β-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.

Results and discussion Cloning, DNA and protein sequence analysis

Susceptibility testing MICs of β-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 β-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.9–11 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. When cloned on a plasmid vector and expressed in E. coli DH10B, blaJOHN-1 confers a similar degree of resistance to β-lactams to that observed for the class B β-lactamases of C. indologenes (IND-1) and C. meningosepticum (BlaB and GOB-1) when similarly expressed in E. coli (Table 1).9–12 The moderate levels of resistance could be due to a weak functionality of promoters of these β-lactamase genes in E. coli, because they do not have promoters that closely resemble typical consensus promoter sequences for E. coli.

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Twelve ampicillin-resistant E. coli DH10B clones harbouring recombinant plasmids with inserts of 6–10 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 Ser–Leu–Gly) 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 (33–38%).15 The deduced protein shares significant identity with Ambler class B β-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 Captocytophaga– Flavobacterium–Bacteroides branch of eubacteria.26 A comparison of the amino acid sequence of JOHN-1 β-lactamase with those of other class B β-lactamases revealed 47% and 41% identity with the most closely related enzymes, BlaB-1 from C. meningosepticum and IND-1 from C. indologenes, respectively (Figure 2). JOHN-1 β-lactamase is more distantly related to other metallo-enzymes involved in intrinsic carbapenem resistance and to the metallo-enzymes that are responsible for acquired resistance, such as VIM-1 and IMP-1 (Figure 2). JOHN-1 β-lactamase is most closely related to metallo-enzymes BlaB of C. meningosepticum and IND of C. indologenes, and this relationship parallels the taxonomic positions of these Chryseobacterium species with that of F. johnsoniae.9,10,15 The six conserved amino acid residues involved in Zn2+ and/or water molecule binding

(His-116, His-118, Asp-120, His-196, Cys-221 and His-263) (class B β-lactamase numbering25) by metallo-β-lactamases are found in the JOHN-1 sequence (Figure 1). Accordingly, JOHN-1 can be classified in subclass B1 of the structural classification of metallo-β-lactamases (Figure 2).27 Analysis of the flanking sequences of blaJOHN-1 revealed ORFs that shared identity with chromosomally encoded genes of other bacterial species. An ORF located downstream of blaJOHN-1 (nucleotides 1214–2888) in the same orientation encodes a 558-amino-acid pre-protein that displays 33% amino acid identity with the primosomal replication factor of Bacillus subtilis.28 Downstream from this second ORF, two other ORFs, encoding proteins of 271 and 252 amino acids, were identified. They share 61% and 33% identity with the pseudouridine synthetase of E. coli29 and with a transcription regulator of Pseudomonas spp.,30 respectively. These findings indicated a likely chromosomal location of the blaJOHN-1 gene (data not shown). 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 PCRprepared 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).

T. Naas et al.

Biochemical properties of β-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 β-lactamase activity in that strain (data not shown). IEF analysis showed that E. coli DH10B (pJOHN-1) produces a β-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 β-lactamase (9.1). The specific activity of the purified JOHN-1 β-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 SDS–PAGE 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-β-lactamases, except L-1 from S. maltophilia, JOHN-1 is a monomeric enzyme.

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Figure 1. Multiple sequence alignment of the amino acid sequence of the JOHN-1 β-lactamase from F. johnsoniae CIP100931 with those of 12 other metallo-β-lactamases. The class B β-lactamase numbering scheme is indicated above the sequences.25 The origins of the metallo-β-lactamases are as follows: BlaB-1 from C. meningosepticum CIP 6058,9 IND-1 from C. indologenes 001,10 VIM-1 from Pseudomonas aeruginosa VT-143/97,13 Bc-II from B. cereus 5/B/6,3 CcrA from B. fragilis,5 IMP-1 from Serratia marcescens TN9106,14 CphA from A. hydrophila AE036,6 SfhI from Serratia fonticola (GenBank accession no. AF197943), L-1 from S. maltophilia IID1275,2 THIN-B from J. lividum,8 GOB-1 from C. meningosepticum PINT,9 FEZ-1 from L. gormanii ATCC 33297T.7 Dashes indicate gaps introduced to optimize the alignment. The vertical arrow indicates the putative cleavage site of the peptide secretion leader sequence of JOHN-1. Amino acids that may be involved in binding Zn2+ and/or a water molecule within the putative catalytic site of the enzymes are shaded in grey.25

Metallo-β-lactamase from F. johnsoniae Table 1. MICs of β-lactams for F. johnsoniae CIP100931, E. coli DH10B (pJOHN-1) and reference strain E. coli DH10B MICs (mg/L) β-Lactam

Kinetic analysis of JOHN-1 revealed that this enzyme has a broad substrate profile that includes carbapenems, as is the case for most metallo-β-lactamases.1 Like other metallo-βlactamases, its substrate profile does not include the monobactam aztreonam.27 The overall catalytic activity of JOHN-1 for all β-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-βlactamases. A comparison of MIC values of β-lactams for F. johnsoniae CIP100931 (Table 1) and the kinetic parameters of JOHN-1 (Table 2) indicates that expression of the JOHN-1 β-lactamase does not explain the entire β-lactam resistance profile of this strain. For example, F. johnsoniae CIP100931 is resistant to ceftazidime, cefepime and aztreonam, whereas the JOHN-1 β-lactamase has poor or no catalytic activity with these compounds. Other β-lactam resistance mechanisms such as impermeability, efflux and penicillin-binding affinity may contribute to the β-lactam resistance phenotype observed for F. johnsoniae CIP100931.

E. coli DH10B (pJOHN-1)

E. coli DH10B

32 128 0.25 16 128 256 128 64 64 32 256 128 2 8

>512 >512 128 8 256 16 4 0.25 1 128 32 0.06 1 0.25

1 1 64 1 2 0.06 0.06 0.06 0.06 4 0.12 0.06