Genetic Diversity of Carbapenem-Hydrolyzing Metallo-β-Lactamases from Chryseobacterium(Flavobacterium) indologenes

Bacterial strains.The bacterial strains used in this work are listed in Table 1. C. indologenes 002, 007, 008, and 009 were isolated from bronchoalveolar brush border, blood culture, rectal swab, and biliary liquid drainage, respectively, at the Hôpital de Bicêtre (Le Kremlin-Bicêtre, France) from 1997 to 1999. C. indologenes 003 and 004 were isolated from blood cultures at the Hôpital Robert Debré (Paris, France).C. indologenes 005 was from the Assistance Publique-Hôpitaux de Marseille (Marseille, France). C. indologenes 006 was isolated at the Hôpital de la Pitié Salpêtrière (Paris, France), and theC. indologenes reference strain CIP101026 was from the Pasteur Institute (Paris, France) strain collection. EachC. indologenes isolate was identified according to standard biochemical techniques (19, 29, 31), and their identification was checked at the Center for Bacterial Identification at the Pasteur Institute. These C. indologenes isolates were epidemiologically unrelated, as assessed by pulsed-field gel electrophoresis ofXbaI-restricted DNA (data not shown).

Escherichia coli DH10B and rifampin-resistant E. coli JM109 were used for cloning and conjugation, respectively (Table 1). All strains were stored at −70°C in Trypticase soy (TS) broth supplemented with 15% glycerol until testing.

Antimicrobial agents and MIC determinations.The antimicrobial agents used in this study have been described elsewhere (21). MICs were determined by an agar dilution technique with Mueller-Hinton agar (Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) and an inoculum of 10⁴ CFU per spot. The plates were incubated at 35°C for 18 h before MIC determinations (16).

Cloning experiments and analysis of recombinant plasmids.Genomic DNA was extracted as described previously (3). Fragments from Sau3AI partially digested genomic DNA fromC. indologenes CIP101026 were cloned into the pBK-CMV phagemid (Ozyme, Saint Quentin-en-Yvelines, France) and expressed in E. coli DH10B as described previously (3). Antibiotic-resistant colonies were selected on amoxicillin (30 μg/ml)- and kanamycin (30 μg/ml)-containing TS agar plates.

Recombinant plasmid DNA was obtained from 100-ml TS broth cultures grown overnight in the presence of amoxicillin (30 μg/ml) at 37°C. Plasmid DNAs were recovered by using Qiagen (Courtaboeuf, France) columns before restriction digest analyses.

Conjugation assays and plasmid content.Direct transfer of resistance markers into in vitro-obtained rifampin-resistant E. coli JM109 was attempted by liquid and solid conjugation assays (21). Transconjugants were selected on TS agar plates containing rifampin (200 μg/ml) and amoxicillin (30 μg/ml). Plasmid DNA extraction of C. indologenes isolates was attempted by using methods reported previously (2).

Hybridization experiments and PCR strategy.The electrophoresis gel containing SspI-restricted genomic DNAs of C. indologenes isolates was transferred on a nylon membrane (Hybond N⁺; Amersham Pharmacia Biotech, Les Ullis, France) by the Southern method (22), and the transferred DNAs were UV cross-linked (Stratalinker; Stratagene) (21). The probe made from a PCR-generated 695-bp internal fragment of the bla gene for IND-1 (bla_IND-1) was labeled with an ECL nonradioactive labeling and detection kit (Amersham Pharmacia Biotech).

In order to amplify IND-like β-lactamase genes from otherC. indologenes isolates, we designed primers based on the external regions ofbla_IND-2: primer 1, 5′-GGTTTGCATATCTATCTGCC-3′; and primer 2, 5′-ATCCAAAGAGAGGCTGGAGT-3′. PCR products were obtained for only four C. indologenes isolates. Thus, we designed other primers based on the conserved regions ofbla_IND-1 and bla_IND-2(primer 3, 5′-GCCCAGGTTAAAGATTTTGTAAT-3′; and primer 4, 5′-CATGGCCACCGCCTTTCCATTC-3′) to characterize the other IND-like β-lactamase genes (Fig. 1).

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Fig. 1.

Strategies of direct PCR and IPCR. Black bars represent the internal PCR product sequence obtained with primers 3 and 4. Grey bars represent known sequences of IPCR products. revA and revB (or revC and revD for C. indologenes 009) represent primers used for amplification of flanking regions. T3 and T7 primers were used for sequencing the IPCR products. Primers 5 and 6 were used for cloning the entire bla_IND-3 gene into pPCR-Script Cam SK(+) and into pBK-CMV. Primers 7 and 8 (same positions as primers 5 and 6, respectively) were used for cloning thebla_IND-4 gene. Double-headed arrows represent the entire bla_IND-related gene fragment.

After an initial denaturation step (5 min at 95°C), 42 cycles of amplification were performed as follows: denaturation at 95°C for 1 min, annealing at 53°C for 1 min, and DNA extension at 72°C for 1 min. A terminal DNA extension step was also performed at 72°C for 10 min. After sequencing of the PCR products, 580-bp-long sequences were characterized. In order to obtain the entire sequences of the β-lactamase genes, the inverse PCR (IPCR) technique was used (20). This technique enabled us to amplify, by PCR, flanking regions of a DNA fragment. Primers in inverted orientation with regard to the gene were used to amplify the restricted DNA fragment, which had been previously circularized by ligation (Fig. 1). Briefly, 4 μg of genomic DNA was restricted by AvaII (known to be absent from the bla_IND-1 and bla_IND-2coding sequences). Since an AvaII restriction site was present in another bla_IND-like gene (later identified as bla_IND-4),PstI was used in IPCR for C. indologenes 009. After heat inactivation of the restriction enzyme, T4 DNA ligase was added to the reaction mixture and incubation was performed at 4°C overnight. IPCR was performed using the degenerate primers revA (5′-CCAYGGGACRTCAAATAAGAC-3′) and revB (5′-CWGCMACYGAYCTKGGATATA-3′) (where Y was C or T, R was A or G, W was A or T, M was A or C, and K was G or T), designed by comparison ofbla_IND-1 and bla_IND-2sequences and exhibiting orientations opposite those of primers 3 and 4, respectively (Fig. 1). Since IPCR with revA and revB failed, primers revC (5′-CTTTGCCGTCAAAAACTCCG-3′) and revD (5′-GCTAATGTAGAACAATGGCC-3′) were used in IPCR forC. indologenes 009. A 40-cycle amplification protocol was used, each cycle consisting of a denaturation step at 95°C for 1 min, a primer annealing step at 53°C, and an elongation step at 72°C for 3 min; 3 U ofTaq polymerase was added per tube. The 1.4- or 1.7-kb IPCR products were purified, cloned into pPCR-Script Amp SK(+) as described by the manufacturer (Stratagene), and sequenced using T3 and T7 universal primers. DNA sequences adjacent to the AvaII (orPstI) restriction site in the cloned IPCR product were added to the PCR products obtained with primers 3 and 4 in order to deduce the entire bla_IND-like gene sequences from fiveC. indologenes isolates.

In order to establish a valid comparison of MICs of β-lactams forE. coli DH10B harboring eitherbla_IND-1, bla_IND-2,bla_IND-3, or bla_IND-4, PCR products of bla_IND-3 from C. indologenes 005, obtained using external primers 5 (5′-CCCAGCAAGTCCTAACTTTAATTAC-3′) and 6 (5′-CTAGTTACCTAGAGATAGCACG-3′), and ofbla_IND-4 from C. indologenes 009, obtained using external primers 7 (5′-TTATGAGGAAAAATGTTAGG-3′) and 8 (5′-GAACAGTTAATAGAAAAGCGGG-3′), were cloned first into pPCR-Script Cam SK(+) (Stratagene) and then into pBK-CMV and transformed back into E. coli DH10B by electroporation.

DNA sequencing and protein analysis.Once thebla_IND-like gene sequences were reconstituted using the above-described PCR strategy, a series of external primers (primers 1 and 2, 5 and 6, and 7 and 8) were used to obtain a PCR fragment for each C. indologenes strain as a template; the fragments were sequenced directly using an Applied Biosystems sequencer (ABI 373). Sequencing was also performed for the inserts of recombinant plasmids pSO-2, pSO3, and pSO4. The nucleotide and deduced protein sequences were analyzed with software available over the Internet from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) and from Pedro's BioMolecular Research Tools website (http://www.fmi.ch/biology/research_tools.htlm).

Multiple nucleotide or protein sequence alignments were carried out using the program ClustalW, available over the Internet from the University of Cambridge website (http://www2.ebi.ac.uk/clustalw). Among the Ambler class B β-lactamases, nine were compared to IND-like β-lactamases: GOB-1 and BlaB from C. meningosepticum(2, 26), VIM-1 and VIM-2 from Pseudomonas aeruginosa (12, 23), CphA from Aeromonas hydrophila (14), IMP-1 from Serratia marcescens (18), CcrA from Bacteroides fragilis (25), B-II from Bacillus cereus(13), and L-1 from Stenotrophomonas maltophilia(30).

β-Lactamase purification.A culture of E. coliDH10B(pSO-2) was grown overnight at 37°C in 6 liters of TS broth containing kanamycin (30 μg/ml) and amoxicillin (30 μg/ml). Bacterial suspensions were pelleted, resuspended in 60 ml of 100 mM phosphate buffer (pH 6.0), disrupted by sonification (three times at 50 W for 30 s each time using a Vibra Cell 75022 Phospholyser; Bioblock, Illkirch, France), and centrifuged for 1 h at 48,000 × g and 4°C. Nucleic acids were precipitated by the addition of 0.2 M spermin (7% [vol/vol]; Sigma, Saint-Quentin Fallavier, France) overnight at 4°C. This suspension was ultracentrifuged at 100,000 × g for 1 h at 4°C. Similar unpurified β-lactamase extracts were obtained from 10-ml cultures of C. indologenes isolates and subsequently resuspended in 0.5 ml of sodium phosphate buffer.

The β-lactamase extract from E. coli DH10B(pSO-2) was filtered through a 0.45-μm-pore-size filter (Millipore) prior to being loaded onto a preequilibrated S-Sepharose column (Amersham Pharmacia Biotech). The enzyme recovered in the flowthrough was dialyzed overnight at 4°C against 20 mM Tris-HCl buffer (pH 8.0). The enzyme fraction was then loaded onto a preequilibrated Q-Sepharose column (Amersham Pharmacia Biotech). The enzyme was eluted with a linear NaCl gradient (0 to 1 M) in Tris-HCl buffer (pH 8.0). The β-lactamase was eluted at a concentration of 350 mM NaCl. The fraction containing the β-lactamase activity was dialyzed overnight against 100 mM phosphate buffer (pH 7.0) containing 50 μM ZnCl₂. The specific activities of the β-lactamase extract and of the β-lactamase purified from E. coli DH10B(pSO-2) were compared using 100 μM imipenem as the substrate as described previously (2).

N-terminal sequencing and IEF analysis.In order to determine the site for cleavage of the mature protein for IND-2 β-lactamase, the purified enzyme was submitted to Edman analysis as described previously (2) but with the following modifications in the protein transfer technique. The buffer consisted of 10 mM 3-cyclohexylamino-propane sulfonic acid (CAPS) (pH 11)–10% (vol/vol) methanol, and the transfer was carried out for 30 min at 50 V.

A purified enzyme preparation from a culture of E. coliDH10B(pSO-2) and extracts from cultures of 10 C. indologenes isolates were subjected to analytical isoelectric focusing (IEF) on a pH 3.5 to 9.5 Ampholine polyacrylamide gel (Ampholine PAG plate; Amersham Pharmacia Biotech) for 90 min at 1,500 V, 50 mA, and 30 W. The focused β-lactamases were detected by overlaying the gel with a 0.2% (wt/vol) starch agar gel containing 1% (wt/vol) benzylpenicillin in 100 mM phosphate buffer (pH 7.0) as described previously (2, 14). The pI values were determined and compared to those of known β-lactamases.

Kinetic measurements and identification of relative molecular mass.The relative molecular mass of the β-lactamase IND-2 fromE. coli DH10B(pSO-2) was estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis. Purified enzyme and marker proteins were boiled for 5 min in a 1% SDS–3% β-mercaptoethanol solution and then subjected to electrophoresis on a 12% polyacrylamide gel (25 mA, 4 h) (3). Renaturation of β-lactamase activity after denaturing electrophoresis and revelation by overlaying the gel with 1 mM nitrocefin were performed as described previously (14).

Purified β-lactamase was used for kinetic measurements performed at 30°C with 100 mM phosphate buffer (pH 7.0) supplemented with 50 μM ZnCl₂ (1). The rates of hydrolysis were determined with an ULTROSPEC 2000 UV spectrophotometer (Amersham Pharmacia Biotech) and analyzed by computer with SWIFT II software (Amersham Pharmacia Biotech). K_m andk_cat values were determined by analyzing β-lactam hydrolysis under initial rate conditions using the Eadie-Hoffstee linearization of the Michaelis-Menten equation as described previously (7).

Various concentrations of EDTA or clavulanic acid were preincubated with the enzyme for 10 min at 30°C before the rate of imipenem hydrolysis was tested. Then, the 50% inhibitory concentration was determined for each inhibitor.

Induction experiments and specific activity.The inducibility of CHβL expression in C. indologenes clinical isolates was examined as described previously (22) with cefoxitin (5 μg/ml) or imipenem (1 μg/ml) as the inducer. Hydrolysis measurements were recorded with imipenem as the substrate. The total protein content was measured with bovine serum albumin as the standard (Bio-Rad DC protein assay kit).

Specific activities with benzylpenicillin, imipenem, or meropenem as substrates were determined for culture extracts from E. coliDH10B harboring either pSO-2 or pSO-4 in order to compare the efficacies of carbapenem hydrolysis by the β-lactamases IND-2 and IND-4.

Nucleotide sequence accession numbers.The IND-like nucleotide and deduced amino acid sequences reported in this work will appear in the GenBank/EMBL database under accessions no. AF219127 toAF219135.