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Antimicrobial Agents and Chemotherapy, July 2000, p. 1878-1886, Vol. 44, No. 7
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing beta -Lactamases in Chryseobacterium meningosepticum

Samuel Bellais, Daniel Aubert, Thierry Naas, and Patrice Nordmann*

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre Cedex, France

Received 4 October 1999/Returned for modification 14 February 2000/Accepted 21 April 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Although the carbapenem-hydrolyzing beta -lactamase (CHbeta L) BlaB-1 is known to be in Chryseobacterium meningosepticum NCTC 10585, a second CHbeta L gene, blaGOB-1, was cloned from another C. meningosepticum clinical isolate (PINT). The G+C content of blaGOB-1 (36%) indicated the likely chromosomal origin of this gene. Its expression in Escherichia coli DH10B yields a mature CHbeta L with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In E. coli, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broad-spectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CHbeta Ls, sharing 18% with the closest, L-1 from Stenotrophomonas maltophilia, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CHbeta Ls (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like beta -lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated C. meningosepticum isolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like beta -lactamases was not found in two different isolates. C. meningosepticum is a bacterial species with two types of unrelated chromosome-borne class B CHbeta Ls that can be expressed in E. coli and, thus, may represent a clinical threat if spread in gram-negative aerobes.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Chryseobacterium meningosepticum is the most clinically important human pathogen among the Chryseobacterium and Flavobacterium genera. It is responsible for neonatal meningitis, with a mortality of up to 50% (17). C. meningosepticum is also found in pneumonia (J. Fujita, Y. Hata, and S. Irino, Letter, Lancet 335:544, 1990) and endocarditis (7, 52) in immunocompromised patients.

C. meningosepticum (formerly known as Flavobacterium meningosepticum) belonged to the Flavobacterium genus until 1994. Since then, it has been reclassified and belongs now to the Chryseobacterium genus, like Chryseobacterium indologenes and Chryseobacterium gleum (56).

C. meningosepticum is naturally resistant to most beta -lactams, including carbapenems (16). A carbapenem-hydrolyzing beta -lactamase, (CHbeta L) BlaB (BlaB-1), from C. meningosepticum NCTC 10585 (CIP 6058) has been described (46). This enzyme belongs to the Ambler class B metallo-beta -lactamase group (2), with a broad substrate profile, a relative molecular mass of 26 kDa, and a pI value of 8.5 (46). Recently, in the same species, Ambler class A extended-spectrum beta -lactamases have also been characterized (6, 45). These extended-spectrum beta -lactamases are inhibited by clavulanic acid, cefoxitin, moxalactam, and imipenem, and their substrate profile does not include carbapenems.

Metalloenzymes usually have a broad spectrum of hydrolysis, except for CphA-1 from Aeromonas hydrophila (31, 49), and are resistant to clinically available beta -lactamase inhibitors (8). Within the last few years, metallo-beta -lactamases IMP-1, VIM-1, and VIM-2 have been identified as chromosome, plasmid, and/or integron located in several pathogens, such as Acinetobacter baumannii (14), Alcaligenes xylosoxydans, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens (3, 21, 22, 26, 30, 36, 42). IMP-1 is widespread in Japan (50, 51). The origin of these CHbeta Ls remains, however, unknown.

Our preliminary experiment using isoelectric focusing (IEF) electrophoresis revealed a heterogeneity of pI values in C. meningosepticum isolates. Thus, characterization of the beta -lactamase content of C. meningosepticum initiated with the class A ESBLs was continued (6). We report the molecular and biochemical characterization of the CHbeta L GOB-1 that was weakly related to any class B CHbeta Ls, including BlaB-1. Additionally, sequence analysis of the CHbeta L genes of 10 C. meningosepticum isolates revealed that each isolate possessed a combination of both types of CHbeta Ls. A combination of two naturally occurring CHbeta L genes in the same bacterial species had not been reported previously.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Bacterial strains. C. meningosepticum PINT was isolated at the Raymond Poincaré hospital (Garches, France). C. meningosepticum AMA and GEO were isolated at the Bicêtre hospital (Le Kremlin-Bicêtre, France), both from tracheoalveolar aspirations. C. meningosepticum AB1572 and H01J100 were from Brita Bruun (11), and reference strains C. meningosepticum CIP 6057 (NCTC 10016), CIP 6058 (NCTC 10585), CIP 6059 (NCTC 10586), CIP 7830 (NCTC 11305), and CIP 79.5 (NCTC 11306) were from the Pasteur Institute (Paris, France). The C. meningosepticum isolates and reference strains were epidemiologically unrelated (data not shown).

Escherichia coli DH10B and rifampin-resistant E. coli JM109 were used for cloning and conjugation assays, respectively, and have been described previously (40, 41). C. meningosepticum isolates were identified as previously described (6, 39, 56). 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 (41). MICs were determined by an agar dilution technique on Mueller-Hinton agar (Sanofi-Diagnostics Pasteur) with an inoculum of 104 CFU per spot (34). The plates were incubated at 35°C for 18 h before MIC determinations were performed as previously described (34).

Cloning experiments, PCR amplifications, and recombinant plasmids. Genomic DNAs were extracted as described previously (35). Fragments from Sau3AI partially digested genomic DNA from C. meningosepticum PINT were cloned in pBK-CMV phagemid (Stratagene, Ozyme, Amsterdam, The Netherlands) (Table 1) and expressed in E. coli DH10B as previously described (35). Antibiotic-resistant colonies were selected onto amoxicillin (30 µg/ml) and kanamycin (30 µg/ml) containing TS agar plates.

                              
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  		TABLE 1.   Plasmids used in this study

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 columns (Qiagen, Courtaboeuf, France) before restriction digest analyses.

16S rDNA fragments were amplified by PCR using the universal 16S RNA primers 5'-AGAGTTTGATCHTGGYTYAGA-3' and 5'-ACGGYTACCTTGTTACGACTTC-3', where Y is C or T and H is A, C, or T (4), and genomic DNAs of C. meningosepticum isolates as the template. Primers used to amplify blaGOB-1-like genes were primer 1 (5'-GCTATGAGAAATTTTGCTACACTG-3') or primer 3 (5'-GGAGTGGTAAAAGATGAAATGTGC-3') and primer 2 (5'-TCATACTTATTTATCTTGGG-3') (Fig. 1).


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  		FIG. 1.   Nucleotide sequence of a 2,384-bp DNA fragment of recombinant plasmid pBS2 carrying blaGOB-1 and the 117 bp of the 3' end of the endo-beta-N-acetylglucosaminidase gene of C. meningosepticum PINT. The deduced amino acid sequences are given in a single-letter code. The start and stop codons of the blaGOB-1 gene and the stop codon of the endo-beta-N-acetylglucosaminidase gene are in bold. The vertical arrow indicates the peptide leader cleavage site in E. coli as determined by N-terminal sequencing. The putative -35 and -10 sequences of the putative promoter and ribosome binding site (RBS) for blaGOB-1 are underlined. Primers 1, 2, and 3 used to PCR amplify blaGOB-1-like genes from other C. meningosepticum isolates are indicated by an arrow.

In order to establish a comparison of MICs of beta -lactams for E. coli DH10B harboring either blaGOB-1 or blaBlaB-1, PCR products of blaGOB-1 from C. meningosepticum PINT were obtained using primers 2 and 3, and those for blaBlaB-1 from C. meningosepticum CIP 6058 were obtained using primers 4 (5'-GTGAATGTAGCAGAGTGTTAATG-3') and primer 5 (5'-GTTGTCTGGTTAAGCGTTCG-3') located at the 5' and the 3' end of blaBlaB-1 (Table 1) (46). Each PCR fragment was cloned into the same pPCR-Script CamSK vector (Stratagene) and electrotransformed into E. coli DH10B.

Conjugation assays, plasmid content, and Southern hybridization. Plasmid DNA extractions of C. meningosepticum isolates were attempted according to two different methods (18, 24). Direct transfer of resistance genes into in vitro-obtained rifampin-resistant E. coli JM109 was attempted by liquid and solid conjugation assays and by electroporation of the putative plasmid DNA suspension into E. coli DH10B (41). Transconjugants and electroporants were selected on TS agar plates containing either rifampin (200 µg/ml) and amoxicillin (30 µg/ml) or amoxicillin, respectively. Southern hybridizations were performed using a 0.8% electrophoresis gel containing unrestricted genomic DNAs of C. meningosepticum isolates and a PCR-prepared internal probe for blaGOB-1 (see below). Visualization was made using the ECL nonradioactive hybridization kit as described by the manufacturer (Amersham Pharmacia Biotech, Orsay, France).

DNA sequencing and protein analysis. Sequencing of the 2.4-kb cloned DNA fragment of recombinant plasmid pBS2, of 16S rDNA fragments, and of PCR products that contained blaBlaB-1-like and blaGOB-1-like genes was performed using an ABI 373 sequencer (Applied Biosystems, Foster City, Calif.). The nucleotide and deduced protein sequences were analyzed with software available over the Internet at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov.) and at Pedro's BioMolecular Research Tools website (http://www.fmi.ch/biology/research_tools.html.), and hydrophobicity analysis of the N-terminal region of the open reading frame (ORF) was performed as described (http://genome.cbs.dtu.dk./services/SignalP/ [25]). Multiple nucleotide or protein sequence alignments were carried out using the program ClustalW (http://www2.ebi.ac.uk/clustalw). A dendrogram of GOB-1 beta -lactamase was derived from the multiple sequence alignment by a parsimony method using the phylogeny package PAUP (Phylogenetic Analysis Using Parsimony) version 3.0 (53).

beta -Lactamase extraction. A culture of E. coli DH10B (pBS2) was grown overnight at 37°C in 4 liters of TS broth containing kanamycin (30 µg/ml) and amoxicillin (30 µg/ml). Bacterial suspensions were pelleted, resuspended in 40 ml of 20 mM Tris-HCl buffer (pH 8), disrupted by sonification (three times at 50 W for 30 s using a Vibra Cell 75022 Phospholyser [Bioblock, Illkirch, France]), and centrifuged for 1 h at 48,000 × g at 4°C. Nucleic acids were precipitated by addition of 0.2 M spermine (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, and the supernatant contained the beta -lactamase extract.

beta -Lactamase purification. The beta -lactamase extract from E. coli DH10B (pBS2) was filtered through a 0.45-µm-pore-size filter (Millipore, Saint-Quentin-en-Yvelines, France) prior its loading onto a preequilibrated Q-Sepharose column (Amersham Pharmacia Biotech). The enzyme which was recovered in the flowthrough was then dialyzed overnight at 4°C against 50 mM phosphate buffer, pH 7. The enzymatic fraction was then loaded onto a preequilibrated S-Sepharose column (Amersham Pharmacia Biotech). The enzyme was eluted by a linear NaCl gradient (0 to 1 M) in phosphate buffer (pH 7). The beta -lactamase was eluted at a concentration of 170 mM NaCl. The fraction containing the beta -lactamase activity was dialyzed overnight against 30 mM cacodylate buffer, pH 6.5, containing 50 µM ZnCl2. The specific activities of the beta -lactamase extract and of the purified beta -lactamase from E. coli DH10B (pBS2) were compared using 100 µM of imipenem as substrate as previously described (40).

N-terminal sequencing and isoelectric focusing. In order to determine the site for cleavage of the mature protein of GOB-1 beta -lactamase, the purified enzyme was submitted to an Edman analysis (19) at the laboratory for protein microsequencing at the Pasteur Institute, Paris, France. Purified enzyme and marker proteins were subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis (20 mA, 5 h, room temperature). Proteins were then electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore) by using the Mini Protean II transfer cell (8 by 7.3 cm) (Bio-Rad) in 50 mM Tris-50 mM borate buffer (pH 8.7) at room temperature (3.5 V/cm, overnight). The membrane was then rinsed in distilled water and stained with a solution made of 0.05% Coomassie brilliant blue R-250 in methanol and water (50:50 [vol/vol]) for 5 min. The membrane was then destained in methanol and water (50:40 [vol/vol]) and acetate and water (10:40 [vol/vol]). The protein band was then excised with a razor blade and allowed to air dry. The amino-terminal sequence of the beta -lactamase was determined with an automated Edman sequencer on a model 473A gas phase sequencer (Applied Biosystems).

The purified enzyme from a culture of E. coli DH10B (pBS2) and beta -lactamase extracts from cultures of 10 C. meningosepticum isolates were subjected to analytical IEF on an ampholine polyacrylamide gel with a pH of 3.5 to 9.5 (Ampholine PAG plate; Amersham Pharmacia Biotech) for 90 min at 1,500 V, 50 mA, and 30 W. The focused beta -lactamases were detected by overlaying the gel with 1 mM nitrocefin (Oxoid, Paris, France) or with an iodine-starch agar gel containing 0.5% (wt/vol) of imipenem (31) in 100 mM phosphate buffer (pH 7.0). These gels were preincubated with or without 100 mM EDTA (ethylenediaminetetraacetic acid) and with or without 5 mM clavulanic acid (38). The pI values were determined and compared to those of known beta -lactamases.

Kinetic measurements and relative molecular mass determination. Purified beta -lactamase was used for kinetic measurements performed at 30°C in 30 mM cacodylate (pH 6.5) supplemented with 50 µM ZnCl2 as described previously (46). The rates of hydrolysis were determined with a Pharmacia ULTROSPEC 2000 spectrophotometer and were computer analyzed using the SWIFT II software (Amersham Pharmacia Biotech).

Km and kcat values were determined by analyzing the beta -lactam hydrolysis under initial rate conditions by using the Eadie-Hoffstee linearization of the Michaelis-Menten equation as previously described (13, 41).

Various concentrations of EDTA or clavulanic acid were preincubated with the enzyme for 10 min at 30°C before testing the rate of imipenem hydrolysis. The 50% inhibitory concentration (IC50) of these inhibitors was then determined.

The relative molecular mass of the purified beta -lactamase was determined by gel filtration using a 1.6- by 47-cm column packed with Superdex 75 (Amersham Pharmacia Biotech) equilibrated and eluted with phosphate buffer (pH 7) containing 150 mM NaCl. Each elution peak was tested for beta -lactamase activity by using nitrocefin as substrate. The peak that showed the highest beta -lactamase activity was linearly plotted against the logarithm of the molecular masses of the standard proteins (Amersham Pharmacia Biotech) to determine the relative molecular mass of the purified beta -lactamase.

Nucleotide sequence accession numbers. The nucleotide and deduced beta -lactamase amino acid sequences reported in this work have been assigned to the GenBank and EMBL databases under the accession no. AF189290 to AF189305 and AF090141. The nucleotide sequences of the 16S rDNAs have been assigned to the accession no. AF207070 to AF207079.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cloning and sequence analysis of blaGOB-1. Partially Sau3AI-digested genomic DNA from C. meningosepticum PINT was cloned into the BamHI site of pBK-CMV. Three recombinant E. coli DH10B clones were obtained. One of them, harboring pBS2 (the smallest insert [2.4 kb]), was selected for further studies.

DNA sequence analysis of the 2,384-bp insert of pBS2 revealed an ORF of 873 bp, encoding a 290-amino-acid preprotein (Fig. 1). Putative -35 (TTGAAA) and -10 (TTTATT) promoter regions and a ribosome binding site (AAAACA) were found along with a putative ATG initiation codon at position 243 (Fig. 1).

The G+C content of this ORF was 36%, which lies close to the G+C ratio found for other C. meningosepticum genes recorded in the EMBL and GenBank sequence database (36.1 to 41.6%). The codon usage of this ORF was also similar to those calculated for the set of these C. meningosepticum genes (data not shown). From the sequencing data, one would expect the first 18 amino acids of this ORF, which contains numerous hydrophobic residues found by hydrophobicity analysis, to be the leader peptide (Fig. 1). This was indeed the case, since Edman analysis (nine cycles) determined the N-terminal sequence of the purified protein from a culture of E. coli DH10B (pBS2) cells as being QVVK. The cleavage site of the leader peptide was therefore deduced to be just after the alanine residue at position 18 (Fig. 1).

Further DNA sequence analysis of the downstream region of this ORF identified the 3' end terminal sequence of an endo-beta-N-acetylglucosaminidase F1 gene (Fig. 1) (54).

The mature protein (named GOB-1 for class B beta -lactamase of C. meningosepticum) expressed in E. coli DH10B cells had a relative molecular mass determined by gel filtration to be 28.2 kDa. His-101, Asp-103, His-162, and His-225 identified by biochemical analysis or by crystal structure analysis as interacting with a Zn2+ cofactor in Bacillus cereus 569H/9 enzyme or in CcrA were found in GOB-1 (Fig. 2) (10, 12). However, the histidine residue at position 99 found in most class B CHbeta Ls was changed for a glutamine residue in GOB-1 (Fig. 2). The comparison of GOB-1 with other class B beta -lactamases revealed only weak identity (Fig. 3). The highest percentage of identity was with L-1 from Stenotrophomonas maltophilia (18%) and only 11% with BlaB-1 from C. meningosepticum.


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  		FIG. 2.   Multiple-sequence alignment of amino acid sequence of GOB-1 from C. meningosepticum PINT isolate with those of eight class B CHbeta Ls. Sequence comparison was performed first by aligning the proteins by using the ClustalW program. Then, adjustments were made to reduce the number of gaps and to maintain alignment of the putative active residues of the active sites. The origins of metallo-beta -lactamases are as follows: CphA-1 from A. hydrophila AE036 (31), BII from B. cereus 5/B/6 (29), BlaB (BlaB-1) from C. meningosepticum CIP 6058 (NCTC 10585) (46), IND-1 from C. indologenes (5), VIM-1 from P. aeruginosa VR-143/97 (28), CcrA from B. fragilis TAL 3636 (44), IMP-1 from S. marcescens TN9106 (36), and L-1 from S. maltophilia IID1275 (57). Amino acids that were identical for at least five out of nine aligned amino acid sequences are shaded in grey. Stars refer to conserved amino acids identified by crystal structure determination as interacting in the binding to the Zn2+ cofactor or to the water molecule in the B. cereus 569H/9 enzyme or in CcrA (12, 20). The numbering scheme refers to the CcrA enzyme (44). Dashes indicate gaps introduced to optimize the alignment.


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  		FIG. 3.   Dendrogram obtained for nine representative CHbeta Ls calculated with ClustalW followed by adjustments to reduce the number of gaps and to maintain alignment of the residues identified in the active sites of some CHbeta Ls. Branch lengths are to scale and proportional to the number of amino acid changes. The percentages at the branching point (bold and underlined) refer to the number of times a particular node was found in 100 bootstrap replications (the stars indicate uncertainty of nodes with bootstrap values of less than 50%). The distance along the vertical axis has no significance. BlaB-1 (BlaB) and GOB-1 were from C. meningosepticum, IND-1 was from C. indologenes, CphA-1 was from A. hydrophila, L-1 was from S. maltophilia, BII was from B. cereus, VIM-1 and VIM-2 were from P. aeruginosa, CcrA was from B. fragilis, and IMP-1 was from S. marcescens. Percent amino acid identities to GOB-1 are indicated in parentheses.

beta -Lactam resistance phenotype and plasmid analysis. The MICs of beta -lactams for C. meningosepticum PINT showed that it was resistant to all tested beta -lactams except piperacillin, as previously reported (Table 2) (6, 16). Similar MICs (within a two-dilution range) were obtained for the C. meningosepticum isolates except for C. meningosepticum H01J100, for which MICs of all beta -lactams were lower (data not shown).

                              
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  		TABLE 2.   MICs of beta -lactams for C. meningosepticum PINT, E. coli DH10B (pBS3), E. coli DH10B (pBS4), and the E. coli DH10B reference strain

Recombinant plasmids pBS3 and pBS4 were constructed by cloning the ORF of blaGOB-1 and blaBlaB-1 in plasmid pPCR-Script Cam SK, respectively, without the putative promoter regions of these beta -lactamase genes (Table 1). E. coli DH10B (pBS3) showed a decreased susceptibility to all beta -lactams except to aztreonam (Table 2), thus indicating that blaGOB-1 was involved at least partially in the resistance to carbapenems of C. meningosepticum PINT. MICs of penicillins were higher against E. coli DH10B (pBS4) than those against E. coli DH10B (pBS3), while the opposite was found for cephalosporins (Table 2). Both recombinant E. coli strains remained fully susceptible to aztreonam. MICs of carbapenems were similar against E. coli DH10B (pBS3) and E. coli DH10B (pBS4), although the amino acid identity of GOB-1 and BlaB-1 was low.

Plasmid analysis and attempts to transfer the beta -lactam resistance markers from C. meningosepticum to E. coli failed, thus suggesting the likely chromosomal origin of blaGOB-1.

Biochemical properties of GOB-1. IEF analysis revealed that E. coli DH10B (pBS2) produced only one beta -lactamase activity with a pI value of 8.7. This pI value did not correspond to the pI value of 8.3 found for the carbapenem-hydrolyzing activity identified in C. meningosepticum PINT.

Specific activity prior to and after purification enabled us to determine the 400-fold purification factor for GOB-1 from E. coli DH10B (pBS2). The specific activity of the purified enzyme was 73.2 µmol · min-1 · mg of protein-1.

Kinetic parameters of GOB-1 revealed a broad spectrum of hydrolysis with a strong activity against meropenem, compared to that against imipenem (Table 3). GOB-1 beta -lactamase has a strong activity against amoxicillin, benzylpenicillin, piperacillin, and extended-spectrum cephalosporins. Hydrolysis of aztreonam was not detectable. The hydrolytic activity of GOB-1 beta -lactamase was inhibited by EDTA (IC50, 25 µM) but not by class A beta -lactamase inhibitors, such as clavulanic acid (IC50, >10 mM). GOB-1 was therefore classified the functional CHbeta L group 3a according to the Bush classification (9, 43).

                              
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  		TABLE 3.   Kinetic parameters of beta -lactam antibiotics for the purified carbapenem-hydrolyzing beta -lactamase GOB-1

Distribution of GOB-1-like and BlaB-like beta -lactamases and 16S rDNA sequencing. EDTA-inhibited activities obtained by comparison of pI values with or without EDTA were heterogeneous for the 10 C. meningosepticum isolates (Table 4). Only one EDTA-inhibited hydrolysis activity was detected for C. meningosepticum PINT, AMA, 7830, 79.5, CIP 6057, AB1572, and H01J100 isolates. Three isolates produced two EDTA-inhibited activities (Table 4). Additionally, clavulanic acid-inhibited beta -lactamase activities varied from one isolate to the other (Table 4). Southern hybridization experiments using nonrestricted genomic DNA of C. meningosepticum isolates and a PCR-amplified 731-bp fragment internal to blaGOB-1 as a probe yielded a hybridization signal that corresponded to the chromosomal band (data not shown), showing that each C. meningosepticum isolate possessed a chromosomally located blaGOB-1-like gene. PCR fragments of GOB-1- and BlaB-1-like genes of 10 C. meningosepticum isolates (except for a blaGOB-1-like gene from C. meningosepticum CIP 7830 that failed to yield a PCR-positive result) were sequenced on both strands. Sequences for entire blaBlaB-1-like genes and a partial portion of the blaGOB-1-like genes yielding 252 out of 290 amino acids were obtained using the designed PCR primers. The deduced amino acid sequences revealing heterogeneity among GOB-1-like beta -lactamases having 90 to 100% amino acid identity (Fig. 4).

                              
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  		TABLE 4.   pI values of beta -lactamase activity detected in C. meningosepticum isolates and in E. coli DH10B harboring pBS2 (GOB-1) or pBS4 (BlaB-1) and the corresponding GOB-1-like and BlaB-1-like sequences



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  		FIG. 4.   Amino acid comparison of the GOB-1-like beta -lactamases from nine C. meningosepticum isolates. Dashes indicate identical amino acids, and dots indicate undetermined sequences. GOB-1 was from C. meningosepticum PINT, CIP 6057, and AB1572, GOB-2 was from C. meningosepticum HO1J100, GOB-3 was from C. meningosepticum CIP 6059, GOB-4 was from C. meningosepticum GEO, GOB-5 was from C. meningosepticum CIP 6058, GOB-6 was from C. meningosepticum AMA, and GOB-7 was from C. meningosepticum CIP 79.5. Numbering is according to the GOB-1 sequence.

Alignment of the BlaB-1-like sequences of 10 C. meningosepticum isolates also revealed heterogeneity, with 86 to 100% amino acid identity (Fig. 5). The same GOB-1-like or BlaB-1-like sequences were found in several isolates, for example, GOB-1 in C. meningosepticum PINT, AB1572, and CIP 6057 and BlaB-1 in C. meningosepticum PINT and CIP 6058 (Table 4). However, within two given C. meningosepticum isolates, the same combination of GOB-1-like and BlaB-1-like beta -lactamases was not found (Table 4). 16S rDNA sequencing identified homogeneous sequences (from 96 to 99% identity) among the studied C. meningosepticum isolates (data not shown, accession numbers available).


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  		FIG. 5.   Amino acid comparison of the BlaB-1-like beta -lactamases from 10 C. meningosepticum isolates. Dashes indicate identical amino acids. BlaB-1 was from C. meningosepticum CIP 6058 and PINT, BlaB-2 was from C. meningosepticum AMA, BlaB-3 was from C. meningosepticum GEO and CIP 6059, BlaB-4 was from C. meningosepticum CIP 6057, BlaB-5 was from C. meningosepticum CIP 79.5, BlaB-6 was from C. meningosepticum CIP 7830, BlaB-7 was from C. meningosepticum AB1572, and BlaB-8 was from C. meningosepticum H01J100. Numbering is according to the BlaB-1 sequence.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

GOB-1 is a broad-spectrum class B beta -lactamase like the previously identified BlaB-1 (BlaB) in C. meningosepticum NCTC 10585 (CIP 6058). Comparison of their kinetic constants revealed that BlaB-1 hydrolyzed benzylpenicillin better than GOB-1. Additionally, a comparison of MICs of beta -lactams for E. coli expressing either GOB-1 or BlaB-1 revealed that GOB-1 hydrolyzed ceftazidime and cefoxitin more significantly than BlaB-1 does (hydrolysis constants of BlaB-1 for ceftazidime are not available [46]). GOB-1 beta -lactamase hydrolysis of meropenem was greater than that of imipenem. Imipenem is usually hydrolyzed better than meropenem by class B CHbeta Ls with two exceptions, the group 3a B. cereus II enzyme and the group 3b AsbM1 enzyme from A. hydrophila (43). GOB-1, like BlaB-1, conferred only a slight increase in the MICs of carbapenems once its gene was cloned on a multicopy plasmid and expressed in E. coli. Similar results have been found for the CHbeta Ls IMP-1, VIM-1, and VIM-2 (28, 36, 42). These results, together with data for kinetic constants of carbapenems, may indicate that an additional decrease of outer membrane permeability for carbapenems may explain the resistance to carbapenems observed for C. meningosepticum (32). In this regard, the pI value of 8.3 for the EDTA-inhibited beta -lactamase identified in C. meningosepticum PINT did not correspond to the pI value of 8.7 for GOB-1 expressed in E. coli DH10B. This result may be explained either by a pI value of 8.3 corresponding to BlaB-1 also found in C. meningosepticum PINT, by a weak or lack of expression of GOB-1, or to differences in leader peptide cleavage in E. coli and in C. meningosepticum. Such a difference in the N-terminal end of the mature protein of GOB-1 may lead to its low concentration in the periplasmic space in E. coli. Whatever the reason is, the low level of resistance to carbapenems conferred by GOB-1 in E. coli may explain its difficulty in being detected once expressed in enterobacterial clinical isolates. Studies of the pI values of the C. meningosepticum isolates revealed that GOB-1-like and BlaB-1-like beta -lactamases may not always be expressed since for some C. meningosepticum isolates, only one EDTA-inhibited beta -lactamase was evidenced by IEF gel electrophoresis although two CHbeta L genes had been identified (Table 4). However, since the pI values of GOB-1 and BlaB-1 were very close, one cannot exclude that they cannot be distinguished on the IEF gel.

Several variants of CHbeta Ls have been found in S. maltophilia, A. hydrophila, and Bacteroides fragilis. However, in these species, it was determined that variants from reference CHbeta Ls had 88 to 95% identity (31, 38, 43, 47, 48). To the best of our knowledge, it is the first time that two CHbeta Ls with only 11% amino acid identity were identified in the same bacterial species. The significance of this result remains to be determined. The regulation of these CHbeta Ls, if any, would be of interest as described for the A. hydrophila CHbeta L (1). It may be hypothesized that CHbeta Ls in C. meningosepticum may counteract the effect of antibiotics produced by this Chryseobacterium species (33, 37). Additionally, the presence of two CHbeta L genes in C. meningosepticum may be used as a tool for a PCR-based identification of this species.

The amino acid sequence of GOB-1 allowed its classification in the sequence-based subclass B3 of metallo-CHbeta Ls (43) along with L-1, the only other member of this subgroup, whereas BlaB-1 is a member of the subclass B1.

The primary structure of GOB-1 beta -lactamase keeps most of the conserved amino acid residues of class B beta -lactamases that act in the interaction with the Zn2+ cofactor or with the water molecule located in the active site, as shown for CcrA (58) or for L-1 (55): His-101, Asp-103, His-162, and His-225 (43). However, like CphA-1, GOB-1 lacks the His-99 residue, which is also involved in Zn2+ binding, but possesses instead a glutamine residue (asparagine in CphA) (31). Therefore, the absence of His-99 does not seem to be involved in narrowing the spectrum of GOB-1 (Fig. 2).

GOB-1 beta -lactamase, like L-1, lacks a cysteine residue at position 181 that is involved in the interaction with a Zn2+ ion. In L-1, Cys-181 function is replaced by a histidine residue located at position 104 (55). It could be the same for GOB-1, which possesses also a histidine residue at this same position.

While this work was in progress, two BlaB variants were reported in GenBank, BlaC from C. meningosepticum NCTC 10016 and BlaB-2 from C. meningosepticum 97/P/5443. We have also identified BlaC (BlaB-4) from the same C. meningosepticum NCTC 10016 isolate and BlaB-2 from another C. meningosepticum isolate (C. meningosepticum AMA).

Although some genetic variation was identified among BlaB-1-like and GOB-1-like sequences, none of the studied C. meningosepticum isolates could be assigned to a special C. meningosepticum subgroup. Indeed, the C. meningosepticum isolates had 96 to 99% identity, according to the results of 16S rDNA sequencing.

Time will tell if gram-negative aerobes, such as C. meningosepticum, may be a reservoir for diffusion of CHbeta L genes to opportunistic pathogens. P. aeruginosa and Acinetobacter spp. that share low natural permeability towards most beta -lactams are good candidates for expressing these carbapenem resistance genes. Finally, since C. meningosepticum CHbeta Ls provide only a low level of resistance to carbapenems once they are expressed in E. coli, their routine detection in gram-negative clinical pathogens shall be performed at best with PCR-based methods previously described for blaIMP-1 detection among American and Japanese isolates (23, 52).


    ACKNOWLEDGMENTS

This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche, Université Paris XI, Faculté de Médecine Paris Sud (grant UPRES, JE-2227), and the French network "Les beta -lactamases: de l'observation clinique à la structure," France.

We thank E. Ronco and B. Bruun for the gift of some C. meningosepticum isolates and L. Poirel for precious advice.


    FOOTNOTES

* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France. Phone: 33 1 45 21 36 32. Fax: 33 1 45 21 63 40. E-mail: nordmann.patrice{at}bct.ap-hop-paris.fr.


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