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 Cedex, France
Received 3 November 2004; returned 15 December 2004; revised 3 January 2005; accepted 18 January 2005
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Abstract |
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Methods: ß-Lactamase genes were cloned, sequenced and expressed in Escherichia coli. Kinetic parameters were determined using purified enzymes.
Results: Metallo-ß-lactamases SLB-1 and SFB-1 were identified from S. livingstonensis and S. frigidimarina, respectively, sharing 65% amino acid identity and being distantly related to other Ambler class B ß-lactamases (40 and 36% amino acid identity with GIM-1 and IMP-1 from Pseudomonas aeruginosa, respectively). SLB-1 had an EDTA-inhibited and broad-spectrum ß-lactam hydrolysis profile, whereas SFB-1 did not hydrolyse cephalosporins, with activity being weakly inhibited by EDTA and dipicolinic acid.
Conclusions: This work provides further evidence that psychrophilic bacterial species may constitute a reservoir of ß-lactam resistance genes.
Keywords: metallo-ß-lactamases , S. livingstonensis , S. frigidimarina , carbapenems
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Introduction |
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Materials and methods |
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S. frigidimarina LMG19475strain was isolated from congelation ice recovered in the Prydz Bay, Antarctica,5 and S. livingstonensis NF22 (or LMG 19866T) strain was isolated from sediment collected in Johnson's Dock (Livingston Island, South Shetland Islands, Antarctica).5 Escherichia coli DH10B was used for cloning and transformation experiments.
Antimicrobial agents and MIC determinations
The agents and their sources have been described elsewhere.7 Antibiotic-containing discs (Bio-Rad, Marnes-la-Coquette, France) were used for routine antibiograms (www.sfm.asso.fr) in order to evaluate MICs of ß-lactams for the Shewanella strains. MICs of ß-lactams for E. coli strains harbouring recombinant plasmids were determined by an agar dilution technique on MuellerHinton (MH) plates with an inoculum of 104 cfu per spot.8 Plates were incubated at 35°C for 18 h. MICs of ß-lactams were determined alone or in combination with a fixed concentration of clavulanic acid (2 mg/L). MICs were interpreted according to the guidelines of the NCCLS.9 Cultures of Shewanella strains were performed on MH agar plates, since these species do not require higher NaCl concentrations as opposed to some other Shewanella species, and they were incubated at 16°C for 24 h because they do not grow at 35°C.1
Cloning experiments and analysis of recombinant plasmids
Genomic DNA from S. frigidimarina LMG19475and S. livingstonensis NF22 strains was extracted with a classical procedure used for Gram-negatives as described previously.8 These DNAs were subsequently restricted by BamHI and then ligated into BamHI-digested pBK-CMV phagemid (Stratagene, Amsterdam, The Netherlands). Recombinant plasmids were transformed into E. coli strain DH10B by electroporation (Gene Pulser II; Bio-Rad, Ivry-sur-Seine, France) and transformants were selected on Trypticase soy (TS) agar containing ampicillin (20 mg/L) and kanamycin (30 mg/L), giving rise to E. coli (pSFB-1) and E. coli (pSLB-1), respectively. Recombinant plasmids were purified with the Qiagen plasmid Midi kit (Qiagen, Courtaboeuf, France) and cloned DNA inserts of recombinant plasmids were sequenced on both strands, using an Applied Biosystems sequencer (ABI 3100). The nucleotide and deduced protein sequences were analysed with software available over the Internet (www.ncbi.nlm.nih.gov). A dendrogram of ß-lactamases SFB-1 and SLB-1 was derived from multiple alignment by a parsimony method using the phylogeny package PAUP (Phylogenetic Analysis Using Parsimony) version 3.0.10
Genetic support of the ß-lactamase genes
Extraction of plasmid DNA from S. frigidimarina and S. livingstonensis was attempted as previously described.11 Direct transfer of resistance markers into streptomycin-resistant E. coli DH10B was also attempted by electroporation, and transformants were selected on TS agar plates containing amoxicillin (20 mg/L).
Isoelectric focusing analysis
The ß-lactamase extracts from cultures of clinical isolates and purified enzymes were subjected to analytical isoelectric focusing (IEF) using an ampholine polyacrylamide gel with a pH range of 3.59.5 (Ampholine PAG plate; Amersham Pharmacia Biotech) for 90 min at 50 mA. The focused ß-lactamases were detected by overlaying the gel with a 1 mM nitrocefin solution (Calbiochem, Merck Eurolab SAS; Fontenay-sous-bois, France).
ß-Lactamase purification
Cultures of E. coli DH10B harbouring recombinant plasmid pSFB-1 or pSLB-1 were grown at 37°C in 4 L of TS broth containing amoxicillin (20 mg/L). Bacterial suspensions were pelleted, resuspended in 40 mL of 50 mM sodium phosphate buffer (pH 7.0), disrupted by sonification (three times at 30 W for 1 min using a Vibra Cell 75022 Phospholyser from Bioblock, Illkirch, France), and centrifuged for 1 h at 48000 g at 4°C. Suspensions were ultracentrifuged at 100 000 g for 1 h at 4°C, filtered through a 0.45 µm filter (Millipore), treated with DNAse (Roche, Meylan, France) for 1 h at room temperature and the supernatant dialysed overnight against 20 mM diethanolamine (pH 8.0). Extracts were loaded onto a pre-equilibrated Q-Sepharose column (Amersham Pharmacia Biotech). Enzymes were eluted with a gradient of NaCl and fractions containing the highest ß-lactamase activity using nitrocefin as substrate were dialysed overnight at 4°C against 50 mM phosphate buffer (pH 7.0).
Biochemical analysis of ß-lactamase
Partially purified ß-lactamase was used for kinetic measurements performed at 30°C in 50 mM sodium phosphate (pH 7.0) containing 50 µM ZnCl2. For comparison, some kinetic measurements were realized using 30 mM HEPES buffer (pH 7.5) containing 50 µM ZnCl2. The rates of hydrolysis were determined with a spectrophotometer, ULTROSPEC 2000 (Amersham Pharmacia Biotech). The wavelengths and extinction coefficients of ß-lactams have been described previously.12 The Km and Vmax values were determined by analysing ß-lactam hydrolysis under initial rate conditions using the EadieHoffstee linearization of the MichaelisMenten equation.13 The Km values are expressed in µM, and Vmax values are expressed relative to that of amoxicillin (Vmax=100).
Various concentrations of EDTA, dipicolinic acid and clavulanic acid were pre-incubated with the enzyme for 3 min at 30°C before testing the rate of cefalothin (100 µM) hydrolysis. The 50% inhibitory concentrations (IC50 s) of these inhibitors were determined as the concentrations that inhibited hydrolysis activity by 50%. Results are expressed in µM.
Specific activities of partially purified enzymes from cultures of E. coli DH10B (pSFB-1) and E. coli DH10B (pSLB-1) were determined as previously reported with 100 µM amoxicillin as substrate.13 One unit of enzyme activity was defined as the activity that hydrolysed 1 µmole of amoxicillin per min per mg of protein. The total protein content was measured with bovine serum albumin as the standard (Bio-Rad DC protein assay kit).
Nucleotide sequence accession numbers
The nucleotide sequences of the blaSFB-1 and blaSLB-1 genes have been deposited in the GenBank database under the accession numbers AY590119 and AY590118, respectively.
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Results and discussion |
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Cloning of BamHI-restricted DNA of S. livingstonensis NF22 and S. frigidimarina LMG19475into pBK-CMV cloning vector gave E. coli DH10B (pSLB-1) and E. coli DH10B (pSFB-1) recombinant strains, respectively. IEF analysis revealed that both E. coli DH10B (pSLB-1) and E. coli DH10B (pSFB-1) produced ß-lactamase activity with a pI of 8.8.
DNA and protein sequence analysis
Partial DNA sequence analysis of an 3 kb insert of pSLB-1 identified an open reading frame (ORF) of 750 bp encoding a 249-amino-acid pre-protein sharing consistent identity with Ambler class B ß-lactamases. The overall GC content of blaSLB-1 was 42%, which lies within the expected range of the G + C ratio of Shewanella genes.5
Similarly, sequence analysis of the 1902 bp insert of pSFB-1 identified an ORF of 735 bp encoding a 245-amino-acid pre-protein also corresponding to an Ambler class B ß-lactamase. The GC content of blaSFB-1 was 46%. Whereas no homology was identified upstream of either ß-lactamase gene, the sequences identified in their 3'-end shared 83% nucleotide identity, suggesting a similar genetic context.
SLB-1 and SFB-1 shared 65% amino acid identity and comparison of their amino acid sequences with that of class B ß-lactamase IMP-1 is shown in Figure 1. The highest percentages of identity were found with plasmid-encoded Ambler class B carbapenemases GIM-1 (40%) and IMP-1 (36%). Analysis of SFB-1 and SLB-1 sequences revealed that both ß-lactamases may be classified in the molecular subclass B1 of Ambler class B ß-lactamases.14 A dendrogram obtained by parsimony analysis showed that SLB-1 and SFB-1 both clustered nearer to most acquired carbapenem-hydrolysing ß-lactamases than to other known naturally occurring Ambler class B ß-lactamases (Figure 2).
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No plasmid was detected in either of the Shewanella strains, and direct transformation assays failed to transfer any ß-lactam resistance marker to E. coli, suggesting that their carbapenemase genes were chromosomally located.
Susceptibility testing
By disc diffusion testing, S. frigidimarina LMG19475and S. livingstonensis NF22 were fully susceptible to all tested antibiotics including aminoglycosides, chloramphenicol and quinolones, and MIC values of ß-lactams were low ( < 0.01 mg/L). Once expressed from a recombinant E. coli DH10B clone, ß-lactamase SLB-1 conferred a resistance phenotype consistent with that of an Ambler class B carbapenemase (Table 1). This recombinant strain was resistant to penicillins and ceftazidime and had a reduced susceptibility to carbapenems. However, E. coli DH10B (pSLB-1) remained fully susceptible to aztreonam and resistant to inhibitors such as clavulanic acid and tazobactam. By contrast, E. coli DH10B (pSFB-1) exhibited only a reduced susceptibility to several ß-lactams compared with E. coli DH10B (Table 1). E. coli DH10B (pSFB-1) was still susceptible to cefalothin, cefuroxime and ceftazidime, whereas it was of intermediate susceptibility to amoxicillin and ticarcillin. Nevertheless, MICs of carbapenems for E. coli DH10B expressing SFB-1 were reduced compared with those of E. coli DH10B.
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Purified enzyme extracts of SFB-1 and SLB-1 with high-level activity could not be obtained. Specific ß-lactamase activity of partially purified extracts from culture of E. coli DH10B (pSLB-1) was 14 mU/mg of protein, whereas that from culture of E. coli DH10B (pSFB-1) was 6 mU/mg of protein, using cefalothin as substrate. The purification factor of these partially purified ß-lactamases obtained from E. coli cultures was 25-fold. Their purity was estimated to be
90% by SDSPAGE analysis (data not shown). These values led us to perform kinetic determinations in conditions that did not allow determination of kcat. Thus, only Vmax values are given here. Use of a HEPES buffer did not significantly influence the kinetic data.
Catalytic efficiency values (Vmax/Km) of ß-lactamase SLB-1 revealed that penicillins were good substrates, like cefalothin, cefuroxime and cefoxitin that were hydrolysed at the same level compared with benzylpenicillin (Table 2). Despite detectable hydrolysis, kinetic parameters of ceftazidime and cefepime could not be determined accurately, due to too high Km values. The hydrolysis spectrum of SLB-1 included carbapenems, meropenem being significantly more hydrolysed than imipenem (Table 2). As for other metallo-ß-lactamases, the monobactam aztreonam was not hydrolysed by SLB-1. These kinetic parameters showed that SLB-1 was a member of functional group 3a of the Bush classification for metallo-ß-lactamases.15 This group includes most of the class B ß-lactamases responsible for intrinsic or acquired resistance to carbapenems.
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As a classical zinc-dependent protein, SLB-1 activity measured with amoxicillin as substrate was inhibited by EDTA (IC50=50 µM) and by dipicolinic acid (IC50=110 µM) but not by class A ß-lactamase inhibitors, such as clavulanate (IC50 > 1 mM). Surprisingly, SFB-1 had a peculiar behaviour since its activity was very weakly inhibited by EDTA (IC50=75 mM) or dipicolinic acid (IC50 > 500 µM), clavulanate remaining inactive.
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Conclusions |
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The ß-lactamase SLB-1 possesses typical kinetic parameters of a metallo-ß-lactamase. In contrast, SFB-1 is an unusual ß-lactamase, whereas a significant degree of identity was found between SFB-1 and SLB-1.
In addition to many carbapenemases identified in several flavobacterial species, in Legionella sp. and in Janthinobacterium lividum that constitute important sources of class B ß-lactamase genes, we report here novel metallo-enzymes from other environmental species, S. frigidimarina and S. livingstonensis. Identification of SFB-1 and SLB-1 that are probably chromosome-encoded adds to the diversity of class B enzymes, but they are distantly related to the plasmid-mediated class B ß-lactamases increasingly reported worldwide in clinically significant Gram-negative bacilli.20
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Acknowledgements |
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