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Antimicrobial Agents and Chemotherapy, January 2003, p. 19-26, Vol. 47, No. 1
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.1.19-26.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Integration of a Transposon Tn1-Encoded Inhibitor-Resistant ß-Lactamase Gene, blaTEM-67 from Proteus mirabilis, into the Escherichia coli Chromosome

Thierry Naas,1* Marie Zerbib,2 Delphine Girlich,1 and Patrice Nordmann1

Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique-Hôpitaux de Paris, Faculté de Médecine Paris-Sud, 94275 Le Kremlin-Bicêtre,1 Unité d'Hygiène, Hôpital Foch, 92151 Suresnes, France2

Received 20 February 2002/ Returned for modification 4 June 2002/ Accepted 21 September 2002


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ABSTRACT
 
Proteus mirabilis NEL-1 was isolated from a urine sample of a patient hospitalized in a long-term care facility. Strain NEL-1 produced a ß-lactamase with a pI of 5.2 conferring resistance to amoxicillin and amoxicillin-clavulanic acid. Sequencing of a PCR amplicon by using TEM-specific primers revealed a novel blaTEM gene, blaTEM-67. TEM-67 was an IRT-1-like TEM derivative related to TEM-65 (Lys39, Cys244) with an additional Leu21Ile amino acid substitution in the leader peptide. The biochemical properties of TEM-67 were equivalent to those described for TEM-65. Analysis of sequences surrounding blaTEM-67 revealed that it was located on a transposon, Tn1, which itself was located on a 48-kb non-self-transferable plasmid, pANG-1. Electroporation of plasmid pANG-1 into Escherichia coli DH10B resulted in the integration of blaTEM-67 into the chromosome, whereas it remained episomal in the P. mirabilis CIP103181 reference strain. Further characterization of pANG-1 revealed the presence of two identical sequences on both sides of Tn1 that contained an IS26 insertion sequence followed by a novel colicin gene, colZ, which had 20% amino acid identity with other colicin genes. The characterization of this novel TEM derivative provides further evidence for the large diversity of plasmid-encoded ß-lactamases produced in P. mirabilis and for their spread to other enterobacterial species through transposable-element-mediated events.


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INTRODUCTION
 
ß-Lactamase inhibitors, such as clavulanic acid, sulbactam, or tazobactam, are largely prescribed in association with amino- and ureidopenicillins for treating gram-negative infections (42). They are suicide inactivators of Ambler class A ß-lactamases (1, 42). Several mechanisms, however, allow Enterobacteriaceae to overcome the efficacy of these molecules, such as overproduction of a cephalosporinase or of narrow-spectrum class A enzymes, limited uptake of the antibiotics, production of OXA-type enzymes, and production of ß-lactamase inhibitor-resistant TEM or SHV derivatives (42).

Since 1992, when the first inhibitor-resistant TEM (IRT) ß-lactamase was identified, numerous IRT variants in Escherichia coli clinical isolates have been characterized, indicating a diversity of these ß-lactamase genes (16). DNA sequence analyses have shown that the IRT enzymes differ from the TEM-1 progenitor by as many as three amino acid substitutions located predominantly at Ambler positions Met69, Trp165, Arg244, Arg275, and/or Asn276 (4, 16, 41). The first report of IRTs in Enterobacteriacae other than E. coli was that of Lemozy et al., who identified an IRT enzyme in Klebsiella pneumoniae (17). IRT ß-lactamase-producing strains of K. pneumoniae may become epidemic (11). Bret et al. (3) described an IRT ß-lactamase derived from TEM-2 that was produced by a strain of Proteus mirabilis. Since then, several IRT derivatives have been found in P. mirabilis (2, 5). IRT enzymes have also been found in Citrobacter freundii (29).

We report on a novel plasmid-encoded IRT ß-lactamase from a P. mirabilis clinical strain that displayed resistance to amoxicillin and amoxicillin-clavulanic acid on a routine disk diffusion antibiogram. We have characterized its plasmid and transposable determinants. In addition, we have identified a putative novel colicin gene on the same plasmid.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. The bacterial strains were grown in Trypticase soy (TS) medium containing the appropriate antibiotic at 37°C under aerobic conditions. P. mirabilis NEL-1 was identified with the API-20E system (BioMérieux, Marcy-l'Etoile, France). Electrocompetent E. coli DH10B (Life Technologies, Eragny, France) and P. mirabilis CIP103181 (Institute Pasteur, Paris, France) were used as recipients in electroporation experiments. In vitro-obtained ciprofloxacin-resistant E. coli strain JM109 (33, 43) and in vitro-obtained nalidixic-acid resistant P. mirabilis CIP103181 were used for conjugation experiments. E. coli NCTC 50192 harboring 154-, 66-, 38, and 7-kb plasmids was used as a plasmid-containing reference strain (8). Plasmid pK19, which encodes resistance to kanamycin (36), and pPCRscriptCam, which encodes chloramphenicol resistance (Stratagene, Amsterdam, Netherlands), were used for subcloning experiments. Recombinant plasmid pPL-1 (33) was used as a control in electroporation experiments.

Antimicrobial agents and MIC determinations. Routine antibiograms were determined by the disk diffusion method on Mueller-Hinton agar (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France). The antimicrobial agents and their sources have been described elsewhere (33). MICs of selected ß-lactams were determined by an agar dilution technique on Mueller-Hinton plates with a Steers multiple inoculator and an inoculum of 104 CFU/spot (40). All plates were incubated at 37°C for 18 h. MICs of ß-lactams were determined alone or in combination with a fixed concentration of 2 µg of clavulanic acid per ml, 4 µg of tazobactam per ml, or 8 µg of sulbactam per ml. MIC results were interpreted according to NCCLS guidelines (28).

PCR analyses. The PCR amplification and the primers (TEM-F, TEM-B, SHV-F, SHV-B, CARB-A, CARB-B, OXA-1A, OXA-1B, OXA-2-A, and OXA2-B) used to search for ß-lactamase genes (blaTEM-like, blaSHV-like, blaCARB-like, blaOXA-1, and blaOXA-2) in P. mirabilis NEL-1 have been described previously (33, 35). Total DNA was prepared as previously described (24). For PCR experiments, 500 ng of total DNA of P. mirabilis NEL-1 was used in standard PCR mixtures (33, 37).

Pulsed-field gel electrophoresis. Agarose plugs of P. mirabilis strains and of E. coli recombinant clones were prepared according to the instructions of the manufacturer (Bio-Rad, Ivry-sur-Seine, France). DNAs were restricted with either XbaI or SfiI. Electrophoresis through a 1% agarose gel in 0.5x Tris-borate-EDTA buffer was performed with a contour-clamped homogeneous electric field DRIII apparatus (Bio-Rad). The migration conditions used were as follows: 14°C, 6 V/cm, 120° switch angle, and 24 h of run time, with two subsequent linear switch ramps of 5 to 15 s for 12 h followed by 30 to 50 s for an additional 12 h. A lambda DNA ladder (Bio-Rad) was used as a DNA molecular size marker. After migration, the gel was stained in a solution of 0.5 µg of ethidium bromide per ml.

Hybridization. Restricted and agarose gel-separated DNA fragments were transferred onto a Hybond N+ nylon membrane by using a vacuum blotting system (Amersham Pharmacia Biotech, Orsay, France) and subsequently cross-linked with an UV Stratalinker (Stratagene).

Hybridizations were performed as described by manufacturer, using the ECL nonradioactive labeling and detection kit (Amersham Pharmacia Biotech). The probes consisted of the 760-bp SacI-KpnI fragment from plasmid pTEM (pPCRscriptCam containing a 760-bp internal PCR fragment of TEM-1) for blaTEM enzymes, a PCR-generated Tn1-specific probe consisting of a 450-bp intragenic tnpA fragment, or a PCR-generated 550-bp specific probe for colZ.

Plasmid content and mating-out assays. Plasmid DNAs of P. mirabilis NEL-1 and recombinant E. coli clones were prepared with a Qiagen (Paris, France) plasmid DNA Maxi kit. Sizes of restricted plasmid fragments were estimated by comparison to the 1-kb DNA ladder molecular size standard (Life Technologies).

The extracted plasmid DNAs from P. mirabilis NEL-1 were subjected to electroporation into E. coli DH10B and P. mirabilis reference strain CIP103181 according to the instructions of the manufacturer (Gene Pulser II; Bio-Rad). Recombinant bacteria were plated onto TS agar plates containing 100 µg of amoxicillin per ml.

Direct transfer of resistance into in vitro-obtained ciprofloxacin-resistant E. coli JM109 and into nalidixic acid-resistant P. mirabilis CIP103181 was attempted by liquid and solid mating-out assays at 37°C. Bacteria were grown in exponential growth phase until an optical density at 600 nm of 0.6 was reached and were then mixed in a 1/2 donor-to-recipient ratio. Four hundred microliters of P. mirabilis NEL-1 and 800 µl of either of the recipient bacteria were incubated for 3 h at 37°C under good aeration and very gentle agitation. Subsequently, 600 µl was removed and spotted onto a nitrocellulose membrane placed onto a TS agar plate and incubated overnight at 37°C. To the remaining 600 µl, an equal volume of fresh TS medium was added prior to an overnight incubation at 37°C. Transconjugant selection was performed on TS agar plates containing either ciprofloxacin (3 µg/ml) and amoxicillin (100 µg/ml) for E. coli JM109 or nalidixic acid (100 µg/ml) and amoxicillin (100 µg/ml) for P. mirabilis CIP103181.

Cloning experiments and analysis of recombinant plasmids. Recombinant plasmid pMZ-1 was constructed by ligating a 9.5-kb HindIII fragment from plasmid pANG-1 into a HindIII-digested pK19 plasmid (36), and recombinant plasmid pMZ-2 was constructed by ligating a 4.5-kb HindIII-EcoRV fragment from pANG-1 into a SmaI-HindIII-restricted pK19 plasmid (36) (Fig. 1). Recombinant plasmid pMZ-3 was constructed by ligating a 5.3-kb EcoRV-HindIII fragment from pANG-1 into a SmaI-HindIII digested pK19 plasmid (36). The restriction enzymes and the ligase were from Amersham Pharmacia Biotech. Recombinant plasmid DNAs were prepared with Qiagen Maxi columns.



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  		FIG. 1. Plasmid pANG-1 and recombinant plasmids pMZ-1 to -3. (A) Restriction analysis of the natural plasmid pANG-1 (panel I) and Southern hybridization of that gel with a blaTEM intragenic probe (panel II). Lanes 1, undigested plasmid pANG-1; lanes 2, HincII-restricted plasmid; lanes 3, EcoRV-restricted plasmid; lanes 4, HindIII-restricted plasmid; lanes 5, HindIII-EcoRV-restricted plasmid; lanes 6, EcoRV-HincII-restricted plasmid. (B) Schematic map of plasmid pANG-1 encoding TEM-67 from P. mirabilis NEL-1. E and H, EcoRV and HindIII cleavage sites, respectively. The numbers within the bars representing the enzyme cleavage sites indicate the sizes of the fragments in kilobase pairs. EcoRV-HindIII double digestion is indicated with dotted lines (C) Schematic map of recombinant plasmids pMZ-1, pMZ-2, and pMZ-3. The thin line represents the cloned inserts from pANG-1, while the dotted lines indicate the pK19 or pPCRscriptCam vector sequence. The open boxes represent the genes, and the arrows indicate their translational orientation. (D) Structure of the colZ promoter region. The conserved regions (-35, -10, and +1) for RNA polymerase binding are represented, as is the ribosome binding site (RBS).

ß-Lactamase preparation and IEF analysis. A culture of E. coli DH10B(pMZ-1), grown overnight in 2 liters of TS broth, was pelleted and resuspended in 30 ml of 20 mM Tris HCl buffer (pH 7.5). After addition of lysozyme (1 mg/ml) and DNase 1 (1 µg/ml), the cells were incubated at 4°C with magnetic stirring for 30 min, prior to sonication (three times at 20 W for 60 s each time with a Vibra Cell 75022 Phospholyser [Bioblock, Illkirch, France]) and centrifugation at 48,000 x g for 1 h at 4°C. The supernatant was subsequently ultracentrifuged at 100,000 x g for 1 h at 4°C. The crude ß-lactamase extract was then dialyzed overnight against the same buffer.

The ß-lactamase extract was filtered through a 0.45-µm-pore-size filter (Millipore, Saint-Quentin-en-Yvelines, France) and ultrafiltrated with a Vivaspin 100,000 column (Sartorius, Gottingen, Germany) prior to being loaded onto a preequilibrated Q-Sepharose column (Amersham Pharmacia Biotech) in 20 mM Tris HCl buffer (pH 7.5). The ß-lactamase activity, as determined qualitatively for each fraction by using nitrocefin hydrolysis (Oxoid, Dardilly, France), was eluted with a linear NaCl gradient (0 to 1 M). The fractions containing the highest ß-lactamase activity were pooled and subsequently dialyzed overnight against 20 mM bis-Tris buffer (pH 6.4) prior to being loaded onto a preequilibrated Q-Sepharose column with the same buffer. The ß-lactamase activity was eluted with a linear NaCl gradient (0 to 400 mM). The fractions containing the highest ß-lactamase activity were pooled, dialyzed overnight against 50 mM phosphate buffer (pH 7.0), and concentrated with a Vivaspin 10,000 column (Sartorius). The protein content was measured by using the DC protein assay (Bio-Rad). The purity of the enzymatic preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with Coomassie blue staining (37).

The purified enzyme from E. coli DH10B(pMZ-1) and the crude ß-lactamase extracts prepared from 10-ml cultures of P. mirabilis NEL-1 and E. coli MZ1 (32, 33) were subjected to analytical isoelectrofocusing (IEF) as previously described. The pI values were determined by using pI 4 to 9.6 IEF standards (Bio-Rad) and were compared to those of known ß-lactamases (33, 35).

Kinetic measurements. All kinetic measurements were performed at 30°C in 100 mM sodium phosphate (pH 7.0) as described previously (33). The K[infi]m (expressed in micromolar), relative Vmax (expressed relative to that of benzylpenicillin, which was set at 100), and kcat values were determined with an Ultrospec 2000 spectrophotometer (Amersham Pharmacia Biotech) by analyzing ß-lactam hydrolysis under the initial rate conditions by using the Eadie-Hofstee linearization of the Michaelis-Menten equation as previously described (35). The 50% inhibitory concentrations (IC50) for clavulanic acid, sulbactam, and tazobactam were measured as described previously(35).

DNA sequencing and protein analysis. After PCR amplification, the DNA was purified with the Qiaquick PCR purification kit (Qiagen). The blaTEM-1 amplicon was sequenced on both strands by using laboratory-designed primers on an ABI 377 sequencer (Applied Biosystems, Les Ulis, France). The sequence was confirmed by sequencing of the 4.5-kb DNA insert from pMZ-2 and by sequencing of the 9.5-kb DNA insert from pMZ-1. Sequencing of part of plasmid pMZ-3 completed the sequence of colZ. The nucleotide sequence 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). Multiple-sequence alignment of deduced peptide sequences was carried out online at the website of the University of Cambridge by using the program ClustalW (http://www.ebi.uk/clustallW). The predictions of the leader peptide cleavage site (SignalP) and theoretical molecular weight and pI (Compute MW/pI) were performed by using software available at http://www.expasy.org/tools/.

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper will appear in the GenBank-EMBL-DDBJ nucleotide databases under accession number AF091113.


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RESULTS AND DISCUSSION
 
Clinical case. A 93-year-old woman resident at the long-term care facility of the Hôpital Bicêtre, Le Kremlin-Bicêtre, France (a suburban hospital of Paris), was treated between March and December 1997 with a series of antibiotics for repeated nosocomial urinary tract infections. Successively, she received amoxicillin, norfloxacin, amoxicillin-clavulanic acid, norfloxacin, nitrofurantoin, and ciprofloxacin. P. mirabilis NEL-1 was isolated in 1998, several weeks after she had received another cure with amoxicillin-clavulanic acid. A routine antibiogram indicated that P. mirabilis NEL-1 was characterized by resistance to amoxicillin and amoxicillin-clavulanic acid and susceptibility to all the other ß-lactams tested (penicillins and cephalosporins). This resistance pattern was confirmed by the MIC results (Table 1). P. mirabilis NEL-1 was susceptible to most antibiotics tested (gentamicin, tobramycin, netilmicin, amikacin, chloramphenicol, nalidixic acid, pefloxacin, ciprofloxacin, sulfamides, and trimethoprim) but was resistant to tetracycline, nitrofurantoin, and colistin (data not shown).


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  		TABLE 1. MICs of ß-lactams for P. mirabilis NEL-1, P. mirabilis MZ1 to -3, E. coli MZ1- to -9, E. coli DH10B(pMZ-1), and reference strains E. coli DH10B and P. mirabilis CIP103181

Cloning and sequencing of blaTEM-67 and protein sequence analysis. Preliminary PCR experiments indicated that P. mirabilis strain NEL-1 gave a positive signal with intragenic primers specific for blaTEM-like genes, whereas primers specific for blaSHV-like, blaCARB-like, blaOXA-1, and blaOXA-2 genes failed to produce positive PCR signals. Direct sequencing of the TEM intragenic PCR product showed 98% DNA identity with blaTEM-1.

Extraction of plasmid DNA from P. mirabilis NEL-1 revealed a 48-kb plasmid, present at a low copy number as estimated from the yields of several repeated plasmid DNA extractions. Hybridization experiments along with PCR experiments demonstrated that this plasmid, named pANG-1, carried two TEM ß-lactamase genes located on 9.8-and 13.7-kb EcoRV fragments (Fig. 1A). HindIII and HindIII-EcoRV digestions of plasmid pANG-1, however, revealed only one hybridization signal each at 9.6 and 4.5 kb, respectively (Fig. 1A and B). The 4.5-kb EcoRV-HindIII fragment from pANG-1 was cloned into pK19, resulting in pMZ-2, and was sequenced on both strands (Fig. 1C). Analysis of this insert for coding regions revealed an open reading frame of 897 bp encoding a 299-amino-acid preprotein of ca. 33 kDa. The DNA sequence of this gene corresponded to the sequenced PCR product. All of the differences with the blaTEM-1 prototype were confirmed. In fact, this gene was identical to the blaTEM-2-derived genes at positions 226, 317, 346, 436, 604, 682, and 925, which discriminate the blaTEM-1- and blaTEM-2-derived genes (12). Only one nucleotide change (C to A at position 317) results in the Gln39Lys amino acid substitution, while the six other nucleotide substitutions remain silent (12). The blaTEM-67 gene differed from the blaTEM-2 gene at two positions, leading to two point mutations. The first mutation consisted of a nucleotide change (C to T at position 929) which led to the amino acid substitution Arg244Cys. This substitution has been previously described for IRT-1 and is responsible for the IRT phenotype (2, 3). The second mutation consisted of a C-to-A change at position 327, which resulted in a Leu21Ile mutation. This mutation has not been reported in TEM derivatives (http://www.ncbi.nlm.nih.gov). Multiple-sequence alignment of deduced peptide sequences was carried out online at the website of the University of Cambridge by using the program ClustalW (http://www.ebi.uk/clustallW). The predictions of the leader peptide cleavage site (SignalP) and theoretical molecular weight and pI (Compute MW/pI) were performed by using software available at http://www.lahey.org/studies/temtable.stm). Position 21 of TEM-4, -9, -25, -48, -49, -53, -73, -74, -85, and -86 has a Leu21Phe mutation; however, this amino acid change is near the leader peptide cleavage site of the enzyme and was suggested to play no role in the enzymatic activity (32). A Leu-to-Ile change probably does not affect the activity of the enzyme either (Table 2). Excluding this position in the functionality of TEM-67, this enzyme resembled TEM-65. blaTEM-specific PCR using DNA from the two gel-extracted EcoRV bands that were blaTEM hybridization positive and subsequent sequencing of the PCR products revealed, in both cases, the blaTEM-67 sequence, suggesting that pANG-1 harbored two identical blaTEM genes.


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  		TABLE 2. Steady-state kinetic parameters of ß-lactamase TEM-67 compared to those of ß-lactamase TEM-2 and IRT ß-lactamase TEM-65a

Plasmid analysis and mating-out assays. A plasmid DNA preparation from P. mirabilis NEL-1 revealed the presence of a 48-kb plasmid, named pANG-1, that carried the blaTEM-67 gene (Fig. 1A and B). Plasmid pANG-1 was non-self-transferable to either P. mirabilis or E. coli by conjugation. However, E. coli and P. mirabilis electroporants were obtained. Transformation by electroporation into E. coli DH10B was relatively inefficient at 103 electroporants/µg of plasmid DNA, whereas a similar-size plasmid, pPL-1 (20-kb) (33), had an efficiency of 108 electroporants/µg of DNA. Electroporation efficacies in P. mirabilis CIP103181 were similar for both plasmids but remained relatively low (105/µg DNA). Nine E. coli electroporants, named E. coli MZ1 to MZ9, and three P. mirabilis electroporants, named P. mirabilis MZ1 to MZ3 were retained for further analysis.

The blaTEM-67 gene was the only antibiotic resistance marker transferred to either E. coli or P. mirabilis as seen on a routine antibiogram, suggesting that it is the only marker present on that plasmid (data not shown). MICs of ß-lactams for E. coli MZ1 to -9 and for P. mirabilis MZ1 to -3 were similar (Table 1). These MICs were lowered slightly after addition of clavulanic acid only. Once cloned onto a high-copy-number plasmid, the MICs of ß-lactams increased at least fourfold (Table 1).

Preparations of plasmid DNA from P. mirabilis MZ1 to -3 revealed the presence of the same 48-kb plasmid, pANG-1. However, despite several attempts, plasmid DNA extractions from E. coli MZ1 to -9 failed. PCR analysis using blaTEM-specific primers with whole-cell DNA of E. coli MZ1 to -9 confirmed the presence of blaTEM-67, thus suggesting a likely chromosomal integration of this gene.

Restriction analysis of pANG-1 revealed a complex genetic organization. By adding the sizes of the EcoRV restriction fragments, a ca. 48-kb plasmid is obtained, while with HindIII, only 24 kb is obtained. Therefore, each HindIII fragment may correspond to two identical-size fragments. Similar observation was made with the HindIII-EcoRV double digestion. HincII digestions revealed several bands that appeared with different intensities, suggesting the existence of several fragments with identical size. Based on these results, a plasmid map showing the different restriction patterns observed was constructed (Fig. 1A and B).

Biochemical properties of TEM-67. IEF analysis revealed that P. mirabilis NEL-1, P. mirabilis MZ1 to -3, and E. coli MZ1 to -9 displayed only one ß-lactamase activity with a pI of 5.2 (data not shown). This pI is very close to the theoretical pI of 5.22 for the mature TEM-67 protein and differs only slightly from the pI obtained for TEM-65 (5.4). Kinetic parameters of purified ß-lactamase TEM-67 (Fig. 2), obtained from an E. coli(pMZ-2) culture, showed hydrolytic activity primarily against benzylpenicillin and amoxicillin. The rates of hydrolysis of ticarcillin and cephalothin were lower (Table 2). Overall, the rates of hydrolysis of TEM-67 were similar to those obtained for TEM-65 (2). Inhibition studies indicated that the TEM-67 ß-lactamase activity had high IC50s for clavulanate, tazobactam, and sulbactam (17, 3.3, and 145 µM, respectively, compared to TEM-2 values of 0.09, 0.04, and 5 µM, respectively [2]). The values obtained for TEM-67 were equivalent within the experimental error to those obtained for TEM-65 (IC50s for clavulanate and tazobactam, 9 and 3 µM, respectively) (2).



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  		FIG. 2. Analysis of TEM-67 purification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Lane 1, crude ß-lactamase extract; lane 2, protein extract after first Q-Sepharose column; lane 3, purified protein. Molecular size markers were run in lane M, and their sizes are indicated on the right.

Analysis of the genetic environment of blaTEM-67. Sequencing of the blaTEM-67 flanking sequence revealed the presence of a Tn1 transposon backbone, as for blaTEM-2-derived genes (6, 12, 18). Indeed, blaTEM-1-like genes are located on Tn2 backbones, whereas blaTEM-2-like genes are inserted on Tn1 transposons (6, 12). Sequencing of Tn1 from both plasmids pMZ-1 and pMZ-2 revealed identical DNA sequences on both sides of the transposon (Fig. 1C).

On either side of Tn1, 112 bp of DNA sequence with no significant homology to any known DNA sequence preceded an IS26 element which had an 8-bp target site duplication. Three hundred base pairs downstream of the insertion sequence, a putative colicin gene was found. The presence of these large, identical, and symmetrical DNA sequences on both sides of transposon Tn1 may be the result of a fusion of two smaller and identical replicons. This fusion could be mediated by either of the two IS26 elements or by Tn1. Analysis of the restriction map of plasmid pANG-1 (Fig. 1B) shows that this symmetry around the Tn1 structure is also present. The presence of a second blaTEM-67 gene on pANG-1 favors a Tn1-mediated plasmid dimerization.

Analysis of blaTEM-67 inserted into the chromosome of E. coli. Pulsed-field gel electrophoresis analysis of XbaI- and SfiI-restricted DNAs of E. coli MZ1 to -9 revealed minor differences in their banding patterns compared to E. coli DH10B (Fig. 3A). Hybridization of the XbaI-digested DNA fragments with an internal probe for blaTEM-67 revealed a unique hybridizing fragment of ca. 48 kb in P. mirabilis NEL-1 (Fig. 3B), which reflects linearization of the plasmid and thus confirms the estimated size of the plasmid obtained after restriction analysis (Fig. 1A and B). One unique signal of different molecular size was observed for each of the E. coli electroporants, thus suggesting the random integration of blaTEM-67 into the chromosome of E. coli. Similar results were obtained with SfiI restriction, except that plasmid pANG-1 (Fig. 3B, lane P) remained undigested because no SfiI site is present on the plasmid. These results are further evidence of blaTEM-67 integration into the E. coli chromosome in a random manner, even though some preferred sites may exist, since strains 6 and 7 were indistinguishable. Hybridization of the XbaI-restricted pulsed-field gel electrophoresis gel with a Tn1-specific probe revealed the same signal as with the blaTEM-67 probe. These results indicate that in this process some of Tn1 may have been integrated as well. Similarly, when the SfiI-restricted part of the gel was hybridized with a colZ probe, the same hybridization signals were observed as with the blaTEM-67 probe for E. coli MZ4 and -5, suggesting that part of plasmid pANG-1 became integrated into the chromosome.



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  		FIG. 3. Pulsed-field gel electrophoresis and Southern hybridization. (A) Macrorestriction profiles of P. mirabilis NEL-1, E. coli MZ1 to -9, and E. coli DH10B isolates obtained after digestion with XbaI and SfiI. (B) Southern hybridization of the macrorestricted DNA with an internal probe for blaTEM-67. Lanes P, P. mirabilis NEL-1; lanes 1 to 9, E. coli MZ1 to -9, respectively; lanes E, E. coli DH10B. Molecular size markers were run in lanes M, and their sizes are indicated on the left.

Southern hybridization of HindIII-restricted whole-cell DNA of P. mirabilis MZ1 to -3 and P. mirabilis NEL-1, with a blaTEM-67-specific probe revealed a unique hybridization signal of 9.6 kb (Fig. 4), as expected for a HindIII restriction of plasmid pANG-1 (Fig. 1A and B). Southern hybridization of HindIII-restricted whole-cell DNA of E. coli MZ1 to -9 showed unique hybridization signals of different molecular size, except for E. coli MZ4 and -5, which displayed two hybridization signals (Fig. 4). The presence of fragments of different sizes favors random integration of blaTEM-67 into the E. coli chromosome.



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  		FIG.4. Southern hybridization, with an internal probe for blaTEM-67, of HindIII-restricted whole cell DNAs of P. mirabilis MZ1 to -3 (lanes 1 to 3, respectively), P. mirabilis CIP103181 (lane 4), P. mirabilis NEL-1 (lane 5), E. coli DH10B (lane 6), and E. coli MZ1 to -9 (lanes 7 to 15, respectively). Molecular size markers are indicated on the left.

The IS26 elements present on both sides of Tn1 (thus forming an IS26 composite transposon) and Tn1 itself may likely account for the insertion of blaTEM-67 into the E. coli chromosome. Of nine independent insertion events, almost all occurred at different insertion sites (Fig. 3 and 4), suggesting a weak target site preference. Both, Tn1- and IS26-mediated transpositional events have weak or almost no target site specificity (19). While Tn1 can mediate direct transposition or replicon fusion, IS26 gives rise exclusively to replicon fusion (cointegrates), in which the donor and target replicons are separated by two directly repeated insertion sequence copies (10, 13, 19, 22). Tn2680, a composite transposon made of a kanamycin resistance gene surrounded by two IS26 in direct repeats, was able to mediate transposition of the kanamycin gene through cointegration of the entire plasmid carrying Tn2680 and subsequent resolution by homologous recombination between two IS26 elements (13). Tn2000, a composite transposon made of two IS26 elements in opposite orientation surrounding a truncated class 1 integron, is thought to move through a similar mechanism (10, 26). Duplication of Tn1 in E. coli MZ4 and -5 may suggest that in these strains the integration could have been the result of a Tn1-mediated cointegration. The presence of only one copy of Tn1 in the remaining strains could indicate that the integration might have been the result of a direct Tn1 transposition. IS26-mediated events, or events due to additional elements located on regions of pANG-1 that have not been sequenced, cannot be ruled out. The mechanisms by which plasmid pANG-1 or part of it might have been integrated into the E. coli chromosome range from homologous and illegitimate recombination to insertion sequence- or transposon-mediated transposition or cointegration (10, 19). Further investigations, such as cloning and sequencing of the ends of the inserted DNA, will be necessary in order to discriminate between these possibilities.

Characterization of a colicin gene. On both sides of Tn1 and downstream of the IS26 elements, a putative colicin gene, named colZ, was found. This gene shared weak amino acid identities (20%) with known colicin genes. The N-terminal two-thirds of the protein had less than 10% amino acid sequence identity with other colicin genes, while the C-terminal end, which corresponds to the active site of this class of proteins, shared a higher degree of identity (40%) (Fig. 5).



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  		FIG. 5. Amino acid sequence alignment of the C-terminal end of ColZ with those of five known colicins: ColA, ColE1, ColK, and ColB from E. coli (23, 30, 34, 38) and ColU from Shigella boydii (39). Asterisks indicate conserved amino acids within the colicin proteins. Underlined amino acid sequence represent the 10 known alpha-helices as determined for ColA (27). Helices 8 and 9 are shown in grey.

Colicins are plasmid-encoded toxic exoproteins, also called bacteriocins, that are produced by colicinogenic strains of E. coli and some related species of the Enterobacteriaceae family (21). Colicin-producing cells produce a specific immunity protein that protects them from the action of their own toxin (15, 21, 30). To date, at least 23 colicin types that exert an inhibitory effect on sensitive bacteria of the same family and preferably on strains of the same species have been described in detail. Colicin polypeptide chains can be divided into separate functional domains, each of which is responsible for one step in the interaction between the colicin and a sensitive bacterium. The central domain of colicins is involved in the attachment of the colicin molecule to a specific outer membrane receptor protein, the N-terminal domain mediates translocation through the cell envelope, and the C-terminal domain exerts the lethal effect (27, 30).

These domains can be depicted in ColZ, and the bundle of eight amphipathic helices surrounding two hydrophobic helices (H8 and H9) are also present within the C-terminal domain of the protein. Sequence homology studies have separated pore-forming colicins into two groups, type A (colicins A, B, N, and U) and type E1 (colicins E1, 5, K, 10, Ia, and Ib) (21). ColZ belongs to group E1. Preliminary attempts failed to show any activity of ColZ towards P. mirabilis CIP103181 or E. coli DH10B. Analysis of the DNA sequence upstream of colZ gene revealed typical E. coli promoter sequences and a typical ribosomal binding site (Fig. 1C). Whether these sequences are functional will be further investigated, especially in respect to induction of colicin production (30). In addition, the presence of IS26 just upstream of colZ could also contribute to colZ gene expression, since this insertion sequence element is known to carry mobile promoter sequences for blaSHV-2a gene expression (25).

Conclusion. As the world's population grows older, more individuals in industrialized countries will stay for extended periods in long-term care facilities (20). Elderly people are at increased risk for developing infections, and antibiotics are among the most commonly used medication in long-term care facilities (7, 20, 31), turning these facilities into the ideal setting for evolution. In fact, several new TEM-derived extended-spectrum ß-lactamases and IRTs have been described from Enterobacteriaceae in long-term care facility patients (2, 9, 11, 16). P. mirabilis, which is often involved in contamination and colonization but rarely in severe infections, is the second most frequently isolated Enterobacteriaceae (7.7%), after E. coli, in French hospitals (9). Several TEM-2-related IRTs, TEM-44 (IRT-13), TEM-65 (IRT-16), TEM-73 (IRT-18-), TEM-74 (IRT-19) (2), and now TEM-67, have been identified in P. mirabilis. The frequency of TEM-2 in P. mirabilis is high, i.e., 32.7% of penicillinase-producing strains (2), thus explaining why most of the IRTs isolated in this species are TEM-2 related. Finally, as exemplified by our clinical case, frequent treatment of urinary tract infections, which are common infections in elderly patients, with amoxicillin-clavulanic acid may strongly select for IRT-expressing isolates of enterobacterial species, such as P. mirabilis, in which TEM ß-lactamases may evolve and then disseminate to other Enterobacteriaceae.

TEM ß-lactamases have been isolated in hospitals since the 1960s, and the number of variants is growing rapidly (to more then 100). For most of these variants, mutations occurred at key residues involved in extending the substrate profile. With time, TEM ß-lactamases may diversify at amino acid positions different from those directly involved in the catalytic activity of the enzyme. blaTEM-67 is located on a plasmid, pANG-1, with a rather complex and atypical genetic structure. This plasmid is very unstable and becomes integrated into the chromosome of E. coli. The reason for this integration, however, remains unclear. One could speculate that (i) the origin of replication is not functional in E. coli, (ii) the product of the colZ gene is toxic for E. coli when expressed at high copy number, or (iii) the presence of the two IS26 elements along with Tn1 is unstable in E. coli. Similar observations were made for plasmids from Enterobacteriaceae that, once transferred to P. aeruginosa, often cannot replicate and become chromosomally associated (14).


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ACKNOWLEDGMENTS
 
This work was funded by a grant (UPRES-EA) from the Ministères de l'Education Nationale et de la Recherche, Université Paris XI, Paris, France.

We are grateful to C. Bizet for the gift of the P. mirabilis CIP103181 reference strain.


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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: 33-1-45-21-36-24. Fax: 33-1-45-21-63-40. E-mail: thierry.naas{at}bct.ap-hop-paris.fr. Back


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Antimicrobial Agents and Chemotherapy, January 2003, p. 19-26, Vol. 47, No. 1
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.1.19-26.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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