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Antimicrobial Agents and Chemotherapy, April 2007, p. 1365-1372, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01152-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Chromosome-Encoded Narrow-Spectrum Ambler Class A ß-Lactamase GIL-1 from Citrobacter gillenii{triangledown}

Thierry Naas,* Daniel Aubert, Ayla Özcan, and Patrice Nordmann

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

Received 14 September 2006/ Returned for modification 13 October 2006/ Accepted 8 January 2007


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ABSTRACT
 
A novel ß-lactamase gene was cloned from the whole-cell DNA of an enterobacterial Citrobacter gillenii reference strain that displayed a weak narrow-spectrum ß-lactam-resistant phenotype and was expressed in Escherichia coli. It encoded a clavulanic acid-inhibited Ambler class A ß-lactamase, GIL-1, with a pI value of 7.5 and a molecular mass of ca. 29 kDa. GIL-1 had the highest percent amino acid sequence identity with TEM-1 and SHV-1, 77%, and 67%, respectively, and only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (C. koseri), CdiA1 (C. diversus), and SED-1 (C. sedlaki), respectively. The substrate profile of the purified GIL-1 was similar to that of ß-lactamases TEM-1 and SHV-1. The blaGIL-1 gene was chromosomally located, as revealed by I-CeuI experiments, and was constitutively expressed at a low level in C. gillenii. No gene homologous to the regulatory ampR genes of chromosomal class C ß-lactamases was found upstream of the blaGIL-1 gene, which fits the noninducibility of ß-lactamase expression in C. gillenii. Rapid amplification of DNA 5' ends analysis of the promoter region revealed putative promoter sequences that diverge from what has been identified as the consensus sequence in E. coli. The blaGIL-1 gene was part of a 5.5-kb DNA fragment bracketed by a 9-bp duplication and inserted between the D-lactate dehydrogenase gene and the ydbH genes; this DNA fragment was absent in other Citrobacter species. This work further illustrates the heterogeneity of ß-lactamases in Citrobacter spp., which may indicate that the variability of Citrobacter species is greater than expected.


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INTRODUCTION
 
On the basis of DNA relatedness and biochemical studies, the genus Citrobacter has been divided into 11 genomospecies, which are found in water, soil, food, and the intestinal tracts of animals and humans (3, 4, 5, 11, 12). Some species can be opportunistic pathogens in the hospital environment, most often causing urinary tract, respiratory tract, and superficial wound infections (10). In Citrobacter freundii, C. youngae, C. murliniae, C. braakii, and C. werkmanii, resistance to ß-lactams has been associated with the production of a chromosomally encoded and inducible class C ß-lactamase that presents at most 5% amino acid divergence (3, 18, 22). However, several Citrobacter species do not produce a class C ß-lactamase but do produce a class A penicillinase: CKO-1 from C. koseri (22, 28); CdiA from C. amalonaticus (19, 20, 28); and SED-1 from C. sedlakii, C. farmeri, and C. rodentium. (21, 22). Whereas CdiA and SED-1 share 95% amino acid sequence identity and are inducible, CKO-1 shares only 40% amino acid sequence identity with these two enzymes and is constitutively expressed (13, 21, 28).

Citrobacter gillenii was formerly called Citrobacter genomospecies 10 (4, 5). Known sources of its isolation are human stool, human urine, human blood, animal stool, and the environment. There is insufficient information to speculate on the clinical significance of C. gillenii. The type strain, C. gillenii CDC 4693-86 (ATCC 51117 and CCUG 30796), was isolated from human stools (4).

On the basis of 16S RNA relatedness, Citrobacter species may fall into two main 16S rRNA clusters (30). The first cluster contains the penicillinase-producing type species (C. farmeri, C. amalonaticus, C. sedlakii, C. rodentium, and C. koseri [C. diversus]); and the second cluster includes the cephalosporinase-producing species C. freundii, C. youngae, C. braakii, C. werkmanii, C. murliniae, and C. gillenii. Interestingly, C. gillenii branched off the C. freundii cluster, together with the species of the genus Kluyvera (3, 18, 22, 28). Avison and colleagues have suggested that ß-lactamase gene-specific PCR might be suitable for differentiating Citrobacter spp., especially those that are difficult to differentiate biochemically (3, 28). While the sequences of most Citrobacter ß-lactamase genes are known, only that of C. gillenii remains to be determined.

The aim of the present work was to characterize the ß-lactamases produced by a C. gillenii reference strain and to compare them with those of known chromosome- and plasmid-encoded ß-lactamases. The ß-lactamase was cloned, sequenced, and expressed in Escherichia coli. The genetic location of the gene was determined, and the enzymatic properties of the purified ß-lactamase were analyzed.


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MATERIALS AND METHODS
 
Bacterial strains. C. gillenii CIP 106783T, C. gillenii 4653.86, C. brakii CIP 104554T, C. werkmanii CIP 104555T, C. murliniae CIP 104556T, C. freundii P478, C. koseri CIP 105177, and E. coli CIP 54-8 type strains were from the Institut Pasteur (Institut Pasteur Strain Collection, Paris, France). Escherichia coli DH10B (Life Technologies, Eragny, France) was used as the host strain for electroporation and cloning experiments (18). The plasmid vector pK19, which carries a kanamycin resistance marker, was used for the cloning experiments (25).

Antimicrobial agents and MIC determinations. Routine antibiograms were determined by the disk diffusion method on Mueller-Hinton agar (Bio-Rad, Marnes-La-Coquette, France), and the susceptibility breakpoints were determined as described previously (23) and interpreted as recommended by the Clinical and Laboratory Standards Institute (formerly NCCLS) (8). All plates were incubated at 37°C for 18 h. The MICs of the ß-lactams were determined with the ß-lactam alone or the ß-lactam in combination with a fixed concentration of 2 µg/ml of clavulanic acid or 4 µg/ml of tazobactam.

Cloning experiments and analysis of recombinant plasmids. All enzymes for DNA manipulations were used according to the recommendations of the supplier (Amersham Biosciences, Orsay, France). Unless otherwise specified, standard molecular techniques were used (26). Whole-cell DNAs were extracted as described previously (17). Cloning resulted from the ligation of either HindIII-, BamHI-, or EcoRI-digested fragments from the genomic DNA from C. gillenii into the HindIII-, BamHI-, or EcoRI-restricted pK19 vector, respectively (25). Recombinant plasmids were transformed by electroporation (Gene Pulser II; Bio-Rad, Ivry-sur-Seine, France) into E. coli DH10B (Life Technologies). Antibiotic-resistant colonies were selected on Trypticase soy agar plates containing amoxicillin (50 µg/ml) and kanamycin (30 µg/ml).

Recombinant plasmid DNAs were extracted with a plasmid DNA maxi kit (QIAGEN, Courtaboeuf, France) and analyzed by restriction endonuclease digestions (Amersham Biosciences) and agarose gel electrophoresis (Life Technologies).

Plasmid content, mating out, and I-CeuI experiments. Extraction of plasmid DNA from C. gilleniii CIP 106783T and direct transfer of resistance into nalidixic acid-resistant E. coli DH10B were attempted as reported previously (23, 24).

To search for a possible chromosomal location of GIL-1 genetic support, we used the endonuclease I-CeuI (New England Biolabs, Hertfordshire, United Kingdom), which digests a 26-bp sequence in the rrn genes for the 23S large-subunit rRNA (14). After digestion, the separation of the resulting fragments was performed on a CHEF-DRII apparatus, as reported previously (14). The sizes of the I-CeuI-generated fragments were determined by comparison with those from the chromosomal DNA of Saccharomyces cerevisiae (Bio-Rad). After immobilization on Hybond N+ (Amersham Biosciences), the I-CeuI-generated fragments were hybridized with two different probes: a 16S rRNA probe specific for C. gillenii generated by PCR with the universal primers described previously (2) and a blaGIL-1-specific intragenic probe generated by PCR with primers GIL-1F and GIL-1R (Table 1). Labeling, hybridization, and revelation were performed as recommended by the manufacturer by using an enhanced chemiluminescence nonradioactive labeling and detection kit (Amersham Biosciences).


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  		TABLE 1. Primers used in this study

Mapping of the blaGIL-1 transcription start site. Reverse transcription and rapid amplification of cDNA ends (RACE) were performed with a 5'-RACE system (version 2.0; Invitrogen). Five micrograms of the total RNAs extracted from C. gillenii CIP 106783T and E. coli(pGIL-1) with an RNeasy Maxi kit (QIAGEN) were used to determine the blaGIL-1 transcription initiation site. Each 5'-RACE experiment was done in duplicate.

After a reverse transcription step with gene-specific primer GIL-GSP1 (Table 1) and reverse transcriptase, the cDNA was tailed with cytosines by using the terminal deoxynucleotidyltransferase and was subsequently amplified with another gene-specific primer, GIL-GSP2, combined with an oligo(dG) adapter primer provided with the kit. This PCR product was used as the template for a nested PCR assay with another adapter and primer GIL-GSP3 (Table 1). The PCR products obtained were sequenced. The transcription initiation site was determined as the first nucleotide following the sequence of the adapter primer.

PCR approach of genetic environment. PCR experiments were performed as described (26) on an ABI 2700 thermocycler (Applied Biosystems) using laboratory-designed primers (Table 1). Two microliters of supernatant from the whole-cell DNA extract was used as the template. PCR experiments were performed with 35 cycles consisting of 45 s of denaturation at 94°C, 45 s of annealing at 57°C, and 60 s of extension at 72°C.

In order to determine whether the genetic environment of blaGIL-1 is also present in other Citrobacter species, primers specific for the lactate dehydrogenase (LDH) gene and for the ydhB gene were used (Table 1). Similarly, C. gillenii was investigated for the absence of the blaAmpC gene by using primers specific for regions located in the frdD and blc genes, which are located on each side of the ampC/ampR operon in the AmpC-containing species C. freundii, C. braackii, C. murlinae, and C. werkmanii (3, 18). In case of negative PCR results, the quality of the extracted DNA was tested by amplifying either the blaAmpC gene (from AmpC-expressing Citrobacter) or the blaCKO-1 gene from C. koseri.

DNA sequencing and protein analysis. Both strands of the cloned DNA fragment of recombinant plasmid pGIL-1 were sequenced with an automated Applied Biosystems sequencer (ABI PRISM 3100). The nucleotide and the deduced protein sequences were analyzed by using software available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). The prediction of the leader peptide cleavage site was performed at the following website: http://www.cbs.dtu.dk/services/SignalP/. Dendrograms were derived from the multiple-sequence alignment by a parsimony method with the phylogeny package PAUP (Phylogenetic Analysis Using Parsimony), version 3.0 (27).

Overexpression and ß-lactamase purification. Recombinant plasmid pET-GIL-1, used for GIL-1 overexpression, was constructed as follows: a 848-bp fragment generated by PCR with primers containing the NdeI and the BamHI restriction sites (Table 1) was cloned into pPCRBluntII-TOPO plasmid (Invitrogen, Life Technologies, Cergy-Pontoise, France), according to the instructions of the manufacturer, resulting in plasmid pTOPO-GIL-1. The insert of the latter plasmid was removed with NdeI-BamHI and cloned into an NdeI/BamHI-restricted pET-9a expression vector, resulting in pET-GIL-1 (Stratagene, Amsterdam, The Netherlands).

A culture of E. coli DH10B(pET-GIL-1) was grown to an optical density at 600 nm of 0.6 and then induced with isopropyl-ß-D-thiogalactopyranoside (0.6 mM) for an additional 4 h at 37°C in 2 liters of Trypticase soy broth containing amoxicillin (100 µg/ml), as described previously (23). The ß-lactamase extract was obtained after sonification, as described previously (24). It was dialyzed overnight against 20 mM Tris-HCl buffer (pH 8.0). It was loaded onto a preequilibrated Q-Sepharose column (Amersham Pharmacia Biotech) in 20 mM Tris-HCl buffer (pH 8.0). The ß-lactamase activities, as determined qualitatively for each fraction by using nitrocefin hydrolysis (Oxoid, Dardilly, France), were recovered in the flowthrough and dialyzed overnight against 50 mM sodium phosphate buffer (pH 6.0). The ß-lactamase was then loaded onto an S-Sepharose column preequilibrated with the same buffer and eluted with a linear NaCl gradient (0 to 1 M). The fractions containing the highest ß-lactamase activity were pooled and dialyzed overnight against 50 mM phosphate buffer (pH 6.0) prior to concentration 10-fold (Vivaspin; molecular weight cutoff, 10,000; Sartorius, Göttingen, Germany). The protein content was measured by the Bio-Rad DC protein assay, and the specific activities of the crude extract and the purified ß-lactamase were determined as reported previously (24) with 100 µM cephalothin as the substrate. One unit of enzyme activity was defined as the amount of enzyme that hydrolyzes 1 µmol of substrate per min. The purities of the enzymes were estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (24).

N-terminal sequencing. To determine the cleavage site of the mature protein of the identified ß-lactamase, the purified enzyme was submitted to an Edman sequence analysis. Purified enzyme and marker proteins were subjected to SDS-PAGE analysis (23, 24). The proteins were then electrotransferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Guyancourt, France) by using a Mini Protean II transfer cell (8 by 7.3 cm; Bio-Rad) in 50 mM Tris-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 0.1% Coomassie brillant blue R-250 in methanol and water (50:40 [vol/vol]). The protein band was then excised with a razor blade and allowed to air dry. The N-terminal sequence of the mature ß-lactamase was determined with an automated Edman sequencer on a model 473A gas-phase sequencer (Applied Biosystems).

Biochemical properties. Crude ß-lactamase extracts and purified enzyme, obtained as described previously (18, 24) from 10-ml cultures of C. gillenii CIP 106783T, E. coli DH10B(pGIL-1), and E. coli DH10B(pET-GIL-1), were subjected to analytical isoelectrofocusing on an Ampholine-containing polyacrylamide gel with a pH range 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 ß-lactamases were detected by overlaying the gel with 1 mM nitrocefin (Oxoid). The pI values were determined and compared to those of known ß-lactamases (24).

The purified GIL-1 ß-lactamase was used for kinetic measurements, performed at 30°C in 100 mM sodium phosphate (pH 7.0). The rates of hydrolysis were determined with a spectrophotometer (ULTROSPEC 2000; Amersham Pharmacia Biotech). The wavelengths and absorption coefficients of ß-lactams have been referenced previously (23, 24).

Kinetic parameters (maximal velocity [Vmax] and the Michaelis-Menten constant [Km]) were determined by recording the initial rates of hydrolysis at different substrate concentrations and by analyzing the results with the regression analysis program SWIFT-II (Amersham Biosciences). Km values, defined as the substrate concentration ([S]) for half-maximal velocity, were determined by using the Eadie-Hoffstee linearization {V = (Vmax – V)Km/[S]} of the Michaelis-Menten equation {V = Vmax [S]/(Km + [S])}. kcat values were determined by dividing the Vmax (µM s–1) for each substrate by the concentration of purified enzyme (25.43 µM). In the cases of low Km values, Ki values were determined with cephalothin as the substrate.

Various concentrations of clavulanic acid, tazobactam, cefoxitin, and imipenem were preincubated with the enzyme for 3 min at 30°C before the rate of cephalothin (100 µM) hydrolysis was tested. The 50% inhibitory concentrations (IC50s) of these inhibitors were determined as the concentrations that inhibited hydrolytic activity by 50%.

Induction studies were performed with cultures of C. gillenii CIP 106783T, with 0.5 to 2 µg/ml cefoxitin or 0.06 to 0.24 µg/ml imipenem as ß-lactam inducers (24), and with 100 µM cephalothin as the substrate.

Nucleotide sequence accession number. The nucleotide sequences reported in this paper have been assigned to the GenBank nucleotide database under the accession numbers DQ991234 to DQ991238.


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RESULTS AND DISCUSSION
 
Cloning of the ß-lactamase gene from C. gillenii. Several E. coli transformants were obtained in each cloning experiment by selection on medium supplemented with kanamycin and amoxicillin. The smallest recombinant plasmid expressing resistance to amoxicillin, pGIL-1, which had a 7-kb BamHI insert (Fig. 1), was retained for further analysis.


Figure 1
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  		FIG. 1. (A) Schematic representation of recombinant clone pGIL-1 containing the blaGIL-1-coding region from C. gillenii CIP 106783T. The orientations of the genes are indicated with arrows, and a solid line indicates the cloning vector. Plasmid-located promoter pLacUV5 is indicated by an arrow. (B) Structure of the promoter of the blaGIL-1 gene. The +1 sign indicates the mRNA transcription start site, as determined by the 5'-RACE experiment. The deduced –10 and –35 regions of the promoter, the putative ribosomal binding site (RBS), and the translational initiation codon (ATG) are represented in boldface and capital letters. (C) Deduced amino acid sequence of GIL-1. The vertical arrow indicates the cleavage site of the leader peptide. The numbering is according to Ambler et al. (1). Structural elements characteristic of class A ß-lactamases are underlined.

Susceptibility testing. C. gillenii CIP 106783T was of intermediate susceptibility to amoxicillin and ticarcillin and was fully susceptible to the other ß-lactam antibiotics tested (Table 2). The ß-lactam resistance phenotype of C. gillenii CIP 106783T was suggestive of a narrow-spectrum penicillinase. The recombinant clone E. coli(pGIL-1) expressed a higher level of resistance, including resistance or intermediate susceptibility to piperacillin and cefuroxime.


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  		TABLE 2. MICs of ß-lactams for the C. gillenii CIP 106783T reference strain, E. coli DH10B(pGIL-1), and the E. coli DH10B reference strain

The MICs of cefotaxime, ceftriaxone, cefpirome, ceftazidime, cefepime, and aztreonam increased but were still in the susceptibility range (Table 2). Addition of clavulanic acid and tazobactam strongly lowered the MICs of the ß-lactams (Table 2). These results indicate that the ß-lactamase from C. gillenii CIP 106783T is a narrow-spectrum penicillinase with a slight extension of its spectrum toward the expanded-spectrum cephalosporins once it is expressed from a high-copy-number plasmid in E. coli. The discrepancy between the ß-lactam resistance profiles observed in the parental strain and in E. coli(pGIL-1) may be due to the lower level of expression of the enzyme once it is expressed from a chromosomal position versus the high level of expression when it is cloned on a high-copy-number plasmid in E. coli.

Sequence analysis of the ß-lactamase gene from C. gillenii. The 7-kb DNA insert was sequenced, and an open reading frame (ORF) of 858 bp was identified (Fig. 1). The G+C content of this ORF was 57%, which is near the range of the G+C contents of Citrobacter sp. genes (50 to 55%). Characteristic elements of Ambler class A and serine ß-lactamases were identified (Fig. 1C) within the deduced protein sequence of this ORF (286 amino acids), designated GIL-1 (1, 15).

The class A ß-lactamase identified, GIL-1, had the highest percent amino acid sequence identity with TEM-1, PLA-1, ORN-1 (29), and SHV-1 (77%, 74%, 72%, and 67% identity, respectively) but only 46%, 31%, and 32% amino acid sequence identity with CKO-1 (22, 28), Cdiv1 (19, 20), and SED-1 (21), respectively (Fig. 2).


Figure 2
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  		FIG. 2. Phylogenetic tree construction according to parsimony analysis (27) and on the basis of class A ß-lactamases. Branch lengths are to scale and are proportional to the number of nucleotides or amino acid changes. The amino acid sequences of the different ß-lactamases were recovered from GenBank databases. The percent amino acid identity of each ß-lactamase with GIL-1 is indicated into parentheses.

Mapping of blaGIL-1 transcription start site. By using 5'-RACE experiments, the site of the initiation of transcription of the blaGIL-1 gene was mapped in C. gillenii CIP 106783T to be 113 bp upstream of the translational start codon (Fig. 1B). Upstream of this transcriptional start site, a putative –35 promoter sequence (TAGACT) was found, and this was separated by 16 bp from a putative –10 promoter sequence (TACTAT) (Fig. 1B). This putative promoter sequence was divergent from the E. coli consensus promoter sequence.

Biochemical properties of GIL-1. The specific activity of purified ß-lactamase GIL-1, measured with 100 µM cephalothin as the substrate, was 502 U·mg of protein–1, with a 95-fold purification factor. Its purity was estimated to be >95% by SDS-PAGE analysis (data not shown).

Isoelectrofocusing analysis showed that cultures of C. gillenii CIP 106783T and E. coli DH10B(pCG-1) gave a single and identical ß-lactamase with a pI value of 7.5. The cleavage site of the leader peptide was calculated to be between an alanine residue and a histidine residue (between the AFA and HP motifs [Fig. 1C]). This cleavage site position was confirmed by an Edman sequence analysis, which determined the N-terminal end of the mature GIL-1 ß-lactamase to be the motif HPTTL. The relative molecular mass of GIL-1, determined by gel filtration of the purified enzyme, was ca. 29 kDa (data not shown).

The kinetic parameters of the GIL-1 ß-lactamase obtained with the purified enzyme showed that the enzyme had strong activities against penicillins and narrow-spectrum cephalosporins (Table 3). Surprisingly, the kcat values of cefpirome and cefepime were high, thus leading to relative kcat/Km values similar to those for cephalothin and cefuroxime, respectively. Significant hydrolytic activity against cefotaxime was also observed, whereas almost no activity against ceftazidime, aztreonam, and imipenem was detectable and no hydrolysis of cefoxitin was detected.


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  		TABLE 3. Kinetic parameters of purified ß-lactamase GIL-1a

Inhibition studies showed that the IC50 values of clavulanic acid and tazobactam were low, being 9 nM, and 40 nM, respectively. On the basis of its kinetic parameters, GIL-1 is a narrow-spectrum ß-lactamase of the group 2b ß-lactamases of the Bush-Medeiros-Jacoby classification (7).

Induction studies failed to detect inducible ß-lactamase expression with cultures of C. gillenii CIP 106783T and E. coli(pGIL-1). This result was consistent with the absence of a LysR-type regulator gene located upstream of the blaGIL-1 gene. This chromosomally encoded class A ß-lactamase was not inducible like the CKO-1 ß-lactamase of C. koseri (28) and as opposed to the naturally occurring class A enzymes of C. diversus and C. sedlaki (13, 20, 21).

Genetic environment and origin of blaGIL-1. Plasmid detection, mating-out assays, and electroporation attempts failed to detect plasmids or a plasmid-encoded ß-lactam resistance marker, suggesting a chromosomal location of the cloned gene. This result was confirmed with the results of the I-CeuI experiments, which clearly indicated its chromosomal location (Fig. 3).


Figure 3
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  		FIG. 3. Localization of blaGIL-1 in I-CeuI-generated fragments of C. gillenii CIP 106783T separated by PFGE. Lane A, I-CeuI restriction pattern of E. coli DH10B, used as a control; lane B, I-CeuI restriction pattern of C. gillenii CIP 106783T; lane C, hybridization of the I-CeuI restriction pattern of C. gillenii CIP 106783T with a probe specific for the 16S rRNA gene; lane D, hybridization of the I-CeuI restriction pattern of C. gillenii CIP 106783T with a probe specific for the blaGIL-1 gene.

Analysis of the surrounding DNA sequence of blaGIL-1 revealed that the gene was present on a 5.5-kb DNA fragment inserted between the LDH and the ydbH genes (16) (Fig. 4A). This 5.5-kb DNA fragment was not identified in reference strains of the other Citrobacter species, such as C. koseri, C. freundii, C. murliniae, C. werkmanii, and C. braakii, as illustrated in Fig. 4A. Detailed analysis of the DNA sequence of each side of the 5.5-kb insert revealed a 9-bp duplication that might be reminiscent of the insertion event (Fig. 4A). Similarly, the ampC-ampR genes located between the blc and sugE genes of C. freundii, C. murliniae, C. werkmanii, and C. braackii were not identified from C. gillenii CIP 106783T or C. koseri (Fig. 4B) (28). Again, a 12-bp imperfect duplication was found on both sides of ampC/ampR of C. murliniae. In C. gillenii, only one 12-bp sequence was present. These data suggest that ß-lactamase gene acquisition by these bacterial species may have been the result of different recombination events during the evolutionary process.


Figure 4
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  		FIG. 4. (A) Schematic representation of the DNA sequence present between the LDH and ydbH genes in C. gillenii CIP 106783T (CG), C. braakii CIP 104554T (CB), and C. freundii P478 (CF). Boxes represent genes, and their orientations are indicated with arrows. Gray triangles represent the DNA sequences that are absent in C. freundii or C. braakii. A 9-bp duplication present on each side of the inserted 5.5-kb DNA sequence is also indicated with shading in the lower part. The small arrows above the genes represent the primers used for PCR amplification. The DNA sequences on both sides of the putative insertion site are provided: CGL, the sequence derived from the left boundary (ldh primer); CGR, the sequence derived from the right boundary (ydbH primer). Identical positions are indicated by asterisks. (B) Schematic representation of the DNA sequence present between the fumarate reductase frdD and the lipocalin blc genes in C. gillenii CIP 106783T (CG), C. koseri CIP 105177 (CB), and C. murliniae CIP 104556T. Boxes represent genes, and their orientations are indicated with arrows. A 12-bp imperfect duplication present on each side of the ampC/ampR operon is shaded in the lower part. The small arrows above the genes represent the primers used for PCR amplification. The DNA sequences on both sides of the putative insertion site are provided: CML, the sequence derived from the left boundary (frd primer); CMR the sequence derived from the right boundary (blc primer). Identical positions are indicated by asterisks.

In addition to the blaGIL-1 gene, several ORFs were identified on the same 5.5-kb fragment (Fig. 1A). Upstream of the blaGIL-1 gene, three ORFs were identified that encoded putative proteins displaying weak amino acid sequence identity with hypothetical proteins of other gram-negative species. The putative product of ORF-CG3 is made of two domains; the N-terminal part shared 26% amino acid identity with a hypothetical protein from Agrobacterium tumefaciens, and the C-terminal part shared 48% amino acid identity with a hypothetical protein from Ralstonia solanacearum. The putative product of ORF-CG2 shares 32% amino acid identity with a putative chitisonase of Xanthomonas campestris, and that of ORF-CG1 is unrelated to any protein sequence in the databases. Downstream of the blaGIL-1 gene, ORF-CG4, which encodes a putative 80-amino-acid protein, displayed 67% amino acid identity (over 43 amino acids) with that of a putative protein that was previously identified on an E. coli plasmid, pO157 (6).

Conclusion. This work identified a novel chromosomally encoded class A ß-lactamase from an enterobacterial species. The catalytic properties of GIL-1, along with the IC50 values of clavulanate and tazobactam, were similar to the values of the class A ß-lactamases, notably, TEM-1, thus suggesting that the GIL-1 ß-lactamase may be classified into group 2b of the ß-lactamase classification of Bush et al. (7).

This work illustrates the heterogeneity of ß-lactamases among Citrobacter spp., which may indicate that the variability of the genus Citrobacter is greater than expected. The finding of an unrelated ß-lactamase in C. gillenii further supports the possibility that ß-lactamase gene sequencing might represent a tractable tool for the typing of Citrobacter spp. The GIL-1 ß-lactamase displayed 77% and 67% amino acid sequence identity with TEM-1 and SHV-1, respectively, thus further supporting the idea that TEM and SHV might be of enterobacterial origin. To our knowledge, 77% is the highest percentage of identity found between TEM-1 and a class A chromosomal ß-lactamase.

This work underlines the fact that ß-lactamase genes may be present in enterobacterial species, even though its presence may not be suspected by analysis of the ß-lactam resistance profile. Furthermore, it adds to the knowledge on the diversity of the naturally occurring ß-lactamases in members of the family Enterobacteriaceae.

Recent work by Warren et al. indicated that Citrobacter species may be divided into two main 16S RNA clusters (30) (Fig. 5). The bottom cluster includes C. freundii, C. youngae, C. braakii, C. werkmanii, C. murliniae, and C. gillenii. All these species except C. gilleni possess an inducible AmpC phenotype. The second cluster contains C. farmeri, C. amalonaticus, C. sedlakii, and C. rodentium, of which C. koseri (C. diversus) branches off. These strains have each been shown to possess one inducible class A ß-lactamase, all of which, except for that of C. koseri, share significant sequence homology (22); that of C. koseri possesses a class A ß-lactamase that is not inducible and that shares only 40% sequence homology with the other enzymes. Most interestingly, C. gillenii branches off from the other Citrobacter species, along with the Kluyvera species, which possess class A ß-lactamase genes at the origin of plasmid-mediated CTX-M-type ß-lactamases (9) (Fig. 5). However, GIL-1 shares only 35% amino acid sequence identity with the CTX-M enzymes, as exemplified by TOHO-1 (CTX-M-44) (Fig. 2).


Figure 5
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  		FIG. 5. Phylogenetic tree construction according to parsimony (27), based on 16S rRNA sequence comparison. Branch lengths are to scale and are proportional to the number of nucleotide changes. The nucleotide sequences were from Warren et al. (30).


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ACKNOWLEDGMENTS
 
This work was funded by a grant from the Ministère de l'Education Nationale et de la Recherche (grant UPRES-EA3539), Université Paris XI; by the Assistance Publique-Hôpitaux de Paris, Paris, France; and by the European Community (6th PCRD, grant LSHMCT-2003-503-335).


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FOOTNOTES
 
* Corresponding author. Mailing address: Service de Bactériologie-Virologie, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre Cédex, France. Phone: 33 1 45 21 29 86. Fax: 33 1 45 21 63 40. E-mail: thierry.naas{at}bct.ap-hop-paris.fr Back

{triangledown} Published ahead of print on 22 January 2007. Back


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Antimicrobial Agents and Chemotherapy, April 2007, p. 1365-1372, Vol. 51, No. 4
0066-4804/07/$08.00+0     doi:10.1128/AAC.01152-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jacoby, G. A. (2009). AmpC {beta}-Lactamases. Clin. Microbiol. Rev. 22: 161-182 [Abstract] [Full Text]  

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